Methods and compositions relating to polyphenol compositions

Abstract

Described herein are compositions and methods related to one or more polyphenol molecules; one or more biomolecules or active agents; and at least one of: at least one multivalent ion; and at least one endothelium permeabilization agent.

Description

Attorney Docket No: 002806-000156WOPTMETHODS AND COMPOSITIONS RELATING TO POLYPHENOL COMPOSITIONSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit under 35 U. S. C. § 119 of U. S. Provisional Application No. 63 / 755,667 filed February 7, 2025, the contents of which are incorporated herein by reference in their entirety.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing that has been submitted in XML format via Patent Center and is hereby incorporated by reference in its entirety. Said XML copy, created on January 26, 2026, is named ■i002806-000156WOPT_SL.xinf’ and is 169.059 bytes in size.TECHNICAL FIELD

[0003] The technology described herein relates to compositions that can bind to cells, e.g., mammalian cells to alter or modulate the activity the cells, and / or to delivery active agents.BACKGROUND

[0004] Functionalization of living cells - the addition of biomolecules that provide new or modulated activity' to the cells - has proven difficult. Methods of adding biomolecules to the cell surface often destroy or deactivate the biomolecule, damage or alter the cell in undesired way, or are time-consuming and specific to a particular biomolecule. Additionally, attempts to add biomolecules to a cell surface often result in internalization of the biomolecules (e.g., in Xu et al. EPPSCI 2018 87:165-196). A platform that permits rapid functionalization with a variety' of biomolecules will have wide-ranging therapeutic applications, both in using cells to deliver therapeutic molecules, and in controlling cellular activity via functionalization to utilize therapeutic activity of the cells themselves.SUMMARY

[0005] Described herein is the a composition that pennits a wide array of chemically divergent biomolecules to be readily adhered to mammalian cells without: 1) denaturing the biomolecules, 2) subjecting the biomolecules to phagocytosis or other internalization processes, or 3) damaging the mammalian cell. The compositions provide surprising improvements over prior art systems, e.g., without the multivalent ion levels described herein.

[0006] In one aspect of any of the embodiments, described herein is a composition comprising:a) one or more polyphenol molecules;b) one or more biomolecules or active agents; andc) at least one of:i) at least one multivalent ion: andii) at least one endothelium permeabilization agent.1Attorney Docket No: 002806-000156WOPTIll some embodiments of any of the aspects, the composition comprises: a) one or more polyphenol molecules; b) one or more biomolecules or active agents; and c) at least one multivalent ion. In some embodiments of any of the aspects, the composition comprises: a) one or more polyphenol molecules; b) one or more biomolecules or active agents; and c) at least one endothelium permeabilization agent. In some embodiments of any of the aspects, the composition comprises: a) one or more polyphenol molecules; b) one or more biomolecules or active agents; c) at least one multivalent ion; and d) at least one endothelium permeabilization agent.

[0007] In some embodiments of any of the aspects, the at least one multivalent ion is provided as FeCl₃. In some embodiments of any of the aspects, the at least one multivalent ion comprises Fe(III).

[0008] In some embodiments of any of the aspects, the at least one multivalent ion is at a concentration of no more than 1 mM. In some embodiments of any of the aspects, the at least one multivalent ion is at a concentration of at least 2 mM. In some embodiments of any of the aspects, the at least one multivalent ion is at a concentration of 2 mM to 37 mM. In some embodiments of any of the aspects, the multivalent ion is at a concentration of 5 mM to 20 mM. In some embodiments of any of the aspects, the polyphenol is at a concentration of at least 5 μM. In some embodiments of any of the aspects, the polyphenol is at a concentration of at least 10 μM. In some embodiments of any of the aspects, the polyphenol is at a concentration of at least 10 μM and the at least one multivalent ion is at a concentration of no more than 1 mM.

[0009] In some embodiments of any of the aspects, the one or more polyphenols collectively comprise at least one galloyl moiety and / or at least one catechol moiety’. In some embodiments of any of the aspects, the one or more polyphenols collectively comprise at least one galloyl moiety’ and at least one catechol moiety. In some embodiments of any of tire aspects, the one or more polyphenols each comprise at least one galloyl moiety’ and at least one catechol moiety. In some embodiments of any of the aspects, tire polyphenol is tannic acid.

[0010] In some embodiments of any of the aspects, the stoichiometric ratio of polyphenol molecules to biomolecules is 570 or less relative polyphenol. In some embodiments of any of the aspects, the stoichiometric ratio of tannic acid molecules to biomolecules is 190 to 570. In some embodiments of any of the aspects, the stoichiometric ratio of tannic acid molecules to biomolecules is 190.

[0011] In some embodiments of any of the aspects, the biomolecule and / or active agent is a nucleic acid, protein, a viral particle, a viral vector, a lipid nanoparticle, a polymer, alkaloid, polysaccharide, anthocyanin, lipid, antiviral drug, antibiotic, chemotherapeutic, or combination thereof. In some embodiments of any of the aspects, the biomolecule and / or active agent comprises or is a protein. In some embodiments of any of the aspects, the biomolecule and / or active agent is ovalbumin, serum albumin, interleukin-4, an antibody or antibody reagent, cholera toxin subunit B, biotin, cytokine, or lectin. In some embodiments of any of the aspects, the antibody or antibody2Attorney Docket No: 002806-000156WOPTreagent is specific for an immune checkpoint protein. In some embodiments of any of the aspects, the antibody or antibody reagent is specific for PD1 or PD-L1. In some embodiments of any of the aspects, the biomolecule and / or active agent is a viral particle to viral vector. In some embodiments of any of the aspects, the viral particle or viral vector is an adeno-associated virus vector. In some embodiments of any of the aspects, the adeno-associated virus vector is AAV9 or AAV6.

[0012] In some embodiments of any of the aspects, the at least one endothelium permeabilization agent is selected from the group consisting of: a Vascular Endothelial Growth Factor (VEGF) polypeptide: histamine; bradykinin: and serotonin. In some embodiments of any of the aspects, the at least one endothelium permeabilization agent is a Vascular Endothelial Growth Factor (VEGF) polypeptide.

[0013] In one aspect of any of the embodiments, described herein is a functionalized mammalian cell comprising at least one composition of as described herein adhered to the surface of the cell. In one aspect of any of the embodiments, described herein is a mammalian cell comprising at least one composition of as described herein adhered to the surface of the cell.

[0014] In some embodiments of any of the aspects, the cell is a hematopoietic cell. In some embodiments of any of the aspects, the cell is an erythrocyte, B cell. T cell, monocyte, macrophage, neutrophil or natural killer cell.

[0015] In some embodiments of any of the aspects, the biomolecule and / or active agent is an antibody or antibody reagent specific for an immune checkpoint protein and the cell is a macrophage. In some embodiments of any of the aspects, the biomolecule and / or active agent is an antibody or antibody reagent, cytokine, antiviral drug, antibiotic, viral particle, viral vector, or siRNA and the cell is an erythrocyte. In some embodiments of any of the aspects, the biomolecule and / or active agent is an antibody or antibody reagent, siRNA, or chemotherapeutic and the cell is a natural killer cell. In some embodiments of any of the aspects, the biomolecule and / or active agent is cytokine and the cell is a T cell. In some embodiments of any of the aspects, the biomolecule and / or active agent is an antiinflammatory drug and the cell is a neutrophil.

[0016] In some embodiments of any of the aspects, compositions as described herein, collectively comprising 10 to 1 trillion biomolecules are adhered to the surface of the cell.

[0017] In one aspect of any of the embodiments, described herein is a method of functionalizing a mammalian cell, the method comprising: contacting a mammalian cell with the composition described herein; whereby the combination adheres to the surface of the cell.

[0018] In one aspect of any of the embodiments, described herein is a method of administering a biomolecule and / or active agent to a patient in need of treatment with the biomolecule and / or active agent, the method comprising administering the composition as described herein or the cell as described herein to the patient. In one aspect of any of the embodiments, described herein is a cell as described herein for use in a method of administering a biomolecule and / or active agent to a patient3Attorney Docket No: 002806-000156WOPTill need of treatment with the biomolcculc, the method comprising administering the cell to the patient. In one aspect of any of die embodiments, described herein is a composition as described herein for use in a method of administering a biomolecule and / or active agent to a patient in need of treatment with the biomolecule, the method comprising administering the composition to the patient.

[0019] In some embodiments of any of the aspects, the cell is autologous to the patient. In some embodiments of any of the aspects, the administration is via intravenous injection and a plurality of the biomolecule administered to the patient is delivered to the lungs. In some embodiments of any of the aspects, the administration is via intravenous injection in a vein in a limb and a plurality of the biomolecule administered to the patient is delivered to the lungs. In some embodiments of any of the aspects, the administration is via injection to the carotid artery and a plurality of the biomolecule administered to the patient is delivered to the brain.

[0020] In some embodiments of any of the aspects, the cell is an erythrocyte and a plurality of the biomolecule and / or active agent administered to the patient is delivered to the lungs. In some embodiments of any of the aspects, the cell is a macrophage and a plurality of the biomolecule and / or active agent administered to the patient is delivered to the brain, a tumor, or a site of inflammation or autoimmune inflammation. In some embodiments of any of the aspects, the cell is a natural killer cell and a plurality of the biomolecule and / or active agent administered to the patient is delivered to a tumor. In some embodiments of any of the aspects, the cell is a T cell and a plurality of the biomolecule and / or active agent administered to the patient is delivered to a tumor. In some embodiments of any of the aspects, the cell is a neutrophil and a plurality of the biomolecule and / or active agent administered to the patient is delivered to the lungs or a site of inflammation.

[0021] In one aspect of any of the embodiments, described herein is a method of administering a viral vector and / or reducing the immune clearance of viral vectors, the method comprising administering the composition as described herein or the cell as described herein, wherein the biomolecule and / or active agent is a viral vector or viral particle.

[0022] In one aspect of any of the embodiments, described herein is a method of gene therapy comprising administering the composition as described herein or the cell as described herein to the patient, wherein the biomolecule and / or active agent comprises a nucleic acid sequence, e.g.. a nucleic acid sequence suitable for or configured for gene therapy. In some embodiments of any of the aspects, the gene therapy target is primarily in the lungs. In some embodiments of any of the aspects, the gene therapy target is primarily in the brain. In some embodiments of any of the aspects, wherein the biomolecule and / or active agent is a viral vector or viral particle.

[0023] In some embodiments of any of the aspects, the cell is a red blood cell. In some embodiments of any of the aspects, the viral vector or viral particle is an AAV viral vector or AAV viral particle. In some embodiments of any of the aspects, the AAV is AAV9 or AAV6. In some4Attorney Docket No: 002806-000156WOPTembodiments of any of the aspects, the AAV is AAV9. In some embodiments of any of the aspects, the AAV is AAV6.BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Fig. 1: Schematic description of RBC hitchhiking. RBCs with surface-loaded AAV and VEGF are administered via intravenous or intraarterial routes, after which they enter peripheral vessels in target organs (shown in the schematic: lung capillaries after intravenous injection). As the RBC complex squeezes through the capillaries, AAV and VEGF are dislodged through contact and shear forces. VEGF permeabilizes endothelium by disrupting cellular junctions, allowing AAV infiltration and transduction in deeper tissues. TA: tannic acid.

[0025] Figs. 2A-2B: Formulation process of RBC hitchhiking. (Fig. 2A) Schematic description of MARVEL preparation through one-pot synthesis. (Fig. 2B) SEM images of i) bare RBC, ii) RBC loaded with AAV. and iii) TA / Fe / AAV complex. Scale bars (B) i) and ii): 1 pm, and iii): 100 nm.

[0026] Figs. 3A-3I: RBC / AAV complex preparation and function characterizations in vitro and in vivo. (Fig. 3 A) The number of AAV loaded per RBC (n=3), (Fig. 3B) cumulative release of AAV in a mild shaking condition (n=3). and (Fig. 3C) shear-induced release of AAV were measured by PCR (n=3). (Fig. 3D) Confocal images of EA.hy926 cultured in a microfluidic chip were taken after a stream of AF647-labeled AAV was given. (Fig. 3E) AF647 signals from the labeled AAV were quantified from the confocal images. (Fig. 3F) In vitro transduction of EA.hy926 cells by RBC / AAV treated in a static or flow condition. (Fig. 3G) In vivo biodistribution of AAV 1 hour after intravenous injection was measured with PCR (n=3). No statistically significant difference between free AAV and RBC / AAV was observed in the liver, kidney, spleen, and heart accumulation. (Fig. 3H) Percent distribution of AAV among the major organs. (Fig. 31) Lung-to-liver ratio of AAV accumulation. Statistical analysis was performed with (Figs. 3E, 31) Student’s t-test, (Figs. 3G) two-way ANOVA, followed by Sidak’s multiple comparisons, or (Figs. 3F) one-way ANOVA, followed by Tukey’s multiple comparisons (**p<0.01, ***p<0.001, and ****p<0.0001).

[0027] Figs. 4A-4E: MARVEL preparation and functional characterization in vitro and in vivo. (Fig. 4A) VEGF loading on RBC was measured with ELISA. (Fig. 4B) Confocal images of primary human brain endothelial cells 6 hours after a 30-minute incubation with 20 ng / mL of VEGF with or without TA and Fe. No treatment (NC) serves as the negative control (NC). 24 hours of incubation with 100 ng / mL VEGF was used for the positive control (PS). Scale bar: 50 pm. (Fig. 4C) The amount of VEGF in the lungs 4 hours after intravenous injection was measured with ELISA. The percentages of transduced (Fig. 4D) endothelial and (Fig. 4E) epithelial cells characterized by GFP+ signals in the lungs were measured with flow cytometry 30 days after intravenous injection. Statistical analysis was performed with (Fig. 4C) two-way ANOVA, followed by Sidak’s multiple comparisons,5Attorney Docket No: 002806-000156WOPTor (Figs. 4B, 4D-4F) one-way ANOVA, followed by Tukey’s multiple comparisons (*p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001).

[0028] Figs. 5A-5F: in situ drug loading on RBC in whole blood and lung-specific targeting. (Fig. 5A) Loading of fluorescent polystyrene beads (PS) with or without TA and Fe on RBC in whole blood in a static condition, measured with a plate reader (Fig. 5B) The binding of AAV to RBC in whole blood under static condition was confirmed via flow cytometry. (Fig. 5C) Biodistribution of AAV 1 hour after intravenous injection of AAV alone or mixed with TA and Fe into the tail vein, and (D) the corresponding lung-to-liver ratio. The percentages of transduced (Fig. 5E) endothelial and (Fig. 5F) epithelial cells characterized by GFP+ signals in the lungs were measured with flow cytometry 30 days after intravenous injection. Statistical analysis was performed with (Figs. 5B, 5D) Students’ t-test, (Fig. 5C) two-way ANOVA, followed by Sidak’s multiple comparisons, or (Figs. 5E, 5F) one-way ANOVA. followed by Tukey’s multiple comparisons (*p<0.05, ***p<0.001. and ****p<0.0001).

[0029] Figs. 6A-6C: Formulation optimization to minimize hemolysis and aggregation. (Fig, 6A) Aggregation between Fe and TA was measured by absorbance at 350 nm. Aggregation was prevented at Fe concentrations below 1 mM. where the pH was maintained neutral. (Fig. 6B) Hemolysis was measured by absorbance at 450 nm of the supernatant of mixtures of RBC, TA, and Fe at different concentrations. The red dot indicates the condition with which the final formulation is made. (Fig. 6C) Aggregation was assessed under a light microscope by mixing RBC, TA, and Fe at different concentrations.

[0030] Figs. 7A-7B: Setup for the in vitro binding study with flow. (Fig. 7A) A peristaltic pump, consisting of a stepper motor and a power system, was used to continuously flow either PBS or cell culture media into a microfluidic chip. EA.hy926 human endothelial cell line was seeded at the surface of the chip as a model endothelium, on top of which AAV-loaded RBC is flown. (Fig. 7B) The microfluidic chip was monitored with a microscope to visually confirm the flow of RBC on the model endothelial cells.

[0031] Fig. 8: Flow enhances transduction in die model endothelial cell line, EA.hy926, by the AAVs. The fold changes in transduction between the static and flow conditions were measured 2 weeks after treating AAV encoding luciferase (AAV6-CMV-luc) to EA.hy926 in microfluidic chips. Luminescence was measured using a plate reader. Statistical anal sis was performed with a student’s t-test. *p<0.05.

[0032] Figs. 9A-9B: Transgene expression in the lungs. (Fig. 9A) Lungs of mice treated with RBC / AAV using GFP-encoded AAV (AAV6-CMV-GFP) were harvested three weeks after intravenous injection via the tail vein for cryoscctioning and fluorescence imaging. (Fig. 9B) Transgene expression in the lungs treated with free AAV or RBC / AAV, encoding GFP, was measured6Attorney Docket No: 002806-000156WOPT12 weeks after injection via ex vivo IVIS imaging. Statistical analysis was performed with a Student's t-test. *p<0.05.

[0033] Figs. 10A-10E: Transgene expression in the brain via carotid arterial injection of AAV. (Fig. 10A) Timeline of the experiment. 1.51e+10 vg of AAV9 encoding dsRED (AAV9-CMV-dsRED) was intraarterially injected via the carotid artery. Mice were sacrificed three weeks after the injection for ex vivo IVIS imaging. (Fig. 10B) Transgene expression in the brain was quantified by measuring the dsRED signals from the brain using IVIS. (Fig. IOC) Percent distribution of expressed transgene signals (dsRED) from organs. (Fig. 10D) Corresponding percentages of brain signals from the total body and (Fig. 10E) brain-to-liver ratios. Statistical analysis was performed with a student’s t-test. *p<0.05.

[0034] Fig. 11: Number of AAV loaded per RBC by the added amount of VEGF during MARVEL formulation measured by PCR. Statistical analysis was performed with a one-way ANOVA.

[0035] Fig. 12: FITC-BSA loading on RBC. FITC-BSA loading was measured using a plate reader with a standard curve prepared with a mixture of FITC-BSA and TA. The loading trend showed a dose-dependent increase.

[0036] Figs. 13A-13C: Morphological analysis of primary human brain endothelial cells after VEGF treatment. NC, negative control. PC, positive control. No treatment was done on NC. For PC, 100 ng / mL of VEGF was treated for 24 hours. For the free VEGF and TA+Fe groups, 20 ng / mL of VEGF was treated for 6 hours. The resulting cells w ere stained for confocal imaging. (Fig. 13 A) The cell surface area. (Fig. 13B) perimeter, and (Fig. 13C) Feret’s diameter were measured with Fiji software. Statistical analysis was performed with a one-way ANOVA, followed by Tukey's multiple comparisons test. *p<0.05, ***p<0.001. ****p<0.0001.

[0037] Fig. 14: Transwell setup using EA.hy926-coated insert for AAV transmigration across the endothelial layer. The transmigrated AAV in the bottom chamber after 48 hours of incubation was quantified with PCR. Statistical analy sis w as performed with a one-way ANOVA, follow ed by Tukey’s multiple comparisons test. *p<0.05, **p<0.01.

[0038] Figs. 15A-15C: In vivo safety study. IL-6 and TNF-a concentrations (Fig. 15A) in the lungs 4 hours after injection and (Fig. 15B) in the serum 15, 30, 45, and 60 minutes after injection were measured with ELISA (n=4). (Fig. 15C) Fold body weight change compared to the weights measured on DO before treatment (n=5). Overall, the data indicate the safety of the formulation. Statistical analyses for Fig. 15A were done with one-way ANOVA, followed by Tukey's multiple comparisons test. For Fig. 15B, one-way ANOVA was performed by comparing values at each time point to the baseline value.

[0039] Figs. 16A-16C: Model drug loading on RBC in whole blood. (Fig. 16A) PS bead loading setup for evaluating the binding to RBC in flow. The mouse blood flow w as given with a syringe7Attorney Docket No: 002806-000156WOPTpump at a 10 μL / sec rate into a 10 cm tubing with an inner diameter of 0.508 mm connected via a 25 gauge needle. PS bead was pulse injected into different length positions (2, 4, 6, and 8 cm points) into the tubing. Samples from the outlet were collected every second (Fig. 16B) and run on a flow cytometer to assess the binding. (Fig. 16C) Loading of Cy7-labeled lipid nanoparticle (LNP) on RBC with or without TA and Fe in whole blood in a static condition, measured with a flow cytometer. Statistical analysis was performed with a student’s t-test. ***p<0.001

[0040] Fig. 17A-17B: Characterization of AAV, TA, and Fe complex. (Fig. 17A) Representative flow plots of AAV binding on RBC in whole blood with or without the addition of TA and Fe. (Fig.17B) The sizes were measured with dynamic light scattering (DLS) at different TA-to-Fe ratios. The current study used a TA-to-Fe ratio 1:0.3, showing 344.4 nm of hydrodynamic size.

[0041] Fig. 18 depicts a gating scheme.

[0042] Fig. 19: Drug flux into tissue (A and C) and cumulative area under the curve (B and D) are plotted against time at a fixed C„ usingof 1 and 2. A of 1 and 3, and of 1 and 2. Higher drug flux and consequent higher drug accumulation are achieved with higherand

[0043] Figs. 20A-20C depicts a drug influx model.

[0044] Figs. 21-29 depict the performance of compositions described herein with the multivalent ions indicated, x-axis is tannic acid concentration (200 pM (column 1), diluted from left to right by 1 / 2, lowest concentration 0 pM (column 12)). y-axis is ion concentration (5 mM (row A), diluted from top to bottom by 1 / 2, lowest concentration 0 mM (row H)).

[0045] Fig. 30 depicts images of biodistribution 1 horn after IgG (tagged with Alexa Fluor 647) was intravenously injected into mice either as free IgG or IgG (100 pg) + tannic acid (TA) (100 pg) + Iron (FeCl3) (30 pg).

[0046] Fig. 31 depicts a graph of biodistribution 1 hour after IgG (tagged with Alexa Fluor 647) was intravenously injected into mice either as free IgG or IgG (100 pg) + tannic acid (TA) (100 pg) + Iron (FeCh) (30 pg).

[0047] Fig. 32 depicts graphs of biodistribution 1 hour after IgG (tagged with Alexa Fluor 647) was intravenously injected into mice either as free IgG or IgG (100 pg) + tannic acid (TA) (100 pg) + Iron (FeCl3) (30 pg).

[0048] Fig. 33 depicts a graph of IgG serum levels 1 hour after IgG (tagged with Alexa Fluor 647) was intravenously injected into mice either as free IgG or IgG (100 pg) + tannic acid (TA) (100 pg) + Iron (FeCla) (30 pg).

[0049] Fig. 34 depicts an image of lungs from mice 1 hour after they were intravenously injected into the tail vein with 100 pg of AF647-labeled mouse IgG. Lungs from mice treated with IgG+TA+Fe appear visibly darker compared to those treated with free IgG.8Attorney Docket No: 002806-000156WOPT

[0050] Fig. 35 depicts fluorescence microscopy of lungs from mice 1 hour after they were intravenously injected into the tail vein with 100 pg of AF647-labeled mouse IgG.

[0051] Fig. 36 depicts the results of TA / metal screening for strontium. The top table depicts hemolysis (absorbance at 540 nm). Higher numbers indicate more lysis which is not desired. The bottom left image of a plate shows agglutination. The bottom-right-most well is the negative control, which is the red blood cells without anything added. Pellets which are as similar as possible to the control are preferred. Larger pellets were considered agglutinated RBCs. The table on the bottom right shows the highest performing concentrations of TA and strontium.

[0052] Fig. 37 depicts the results of TA / metal screening for iron. The top table depicts hemolysis (absorbance at 540 nm). Higher numbers indicate more lysis which is not desired. The bottom left image of a plate shows agglutination. The bottom-right-most well is the negative control, which is the red blood cells without anything added. Pellets which are as similar as possible to the control are preferred. Larger pellets were considered agglutinated RBCs. The table on the bottom right shows the highest performing concentrations of TA and iron.

[0053] Fig. 38 depicts the results of TA / metal screening for nickel. The top table depicts hemolysis (absorbance at 540 nm). Higher numbers indicate more lysis which is not desired. The bottom left image of a plate shows agglutination. The bottom-right-most well is the negative control, which is the red blood cells without anything added. Pellets which are as similar as possible to the control are preferred. Larger pellets were considered agglutinated RBCs. The table on the bottom right shows the highest performing concentrations of TA and nickel.

[0054] Fig. 39 depicts the results of TA / metal screening for titanium. The top table depicts hemolysis (absorbance at 540 nm). Higher numbers indicate more lysis which is not desired. The bottom left image of a plate shows agglutination. The bottom-right-most well is the negative control, which is the red blood cells without anything added. Pellets which are as similar as possible to the control are preferred. Larger pellets were considered agglutinated RBCs. The table on the bottom right shows the highest performing concentrations of TA and titanium.

[0055] Fig. 40 depicts the results of TA / metal screening for gold. The top table depicts hemolysis (absorbance at 540 nm). Higher numbers indicate more lysis which is not desired. The bottom left image of a plate shows agglutination. The bottom-right-most well is the negative control, which is the red blood cells without anything added. Pellets which are as similar as possible to the control are preferred. Larger pellets were considered agglutinated RBCs. The table on the bottom right shows the highest performing concentrations of TA and gold.

[0056] Fig. 41 depicts the results of TA / metal screening for aluminum. The top table depicts hemolysis (absorbance at 540 nm). Higher numbers indicate more lysis which is not desired. The bottom left image of a plate shows agglutination. The bottom-right-most well is the negative control, which is the red blood cells without anything added. Pellets which are as similar as possible to the9Attorney Docket No: 002806-000156WOPTcontrol arc preferred. Larger pellets were considered agglutinated RBCs. The table on the bottom right shows the highest performing concentrations of TA and aluminum.

[0057] Fig. 42 depicts the results of TA / metal screening for zinc. The top table depicts hemolysis (absorbance at 540 nm). Higher numbers indicate more lysis which is not desired. The bottom left image of a plate shows agglutination. The bottom-right-most well is the negative control, which is the red blood cells without anything added. Pellets which are as similar as possible to the control are preferred. Larger pellets were considered agglutinated RBCs. The table on the bottom right shows the highest performing concentrations of TA and zinc.

[0058] Fig. 43 depicts the results of TA / metal screening for manganese. The top table depicts hemolysis (absorbance at 540 nm). Higher numbers indicate more lysis which is not desired. The bottom left image of a plate shows agglutination. The bottom-right-most well is the negative control, which is the red blood cells without anything added. Pellets which are as similar as possible to the control are preferred. Larger pellets were considered agglutinated RBCs. The table on the bottom right shows the highest performing concentrations of TA and manganese.

[0059] Fig. 44 depicts the results of TA / metal screening for gadolinium. The top table depicts hemolysis (absorbance at 540 nm). Higher numbers indicate more lysis which is not desired. The bottom left image of a plate shows agglutination. The bottom-right-most well is the negative control, which is the red blood cells without anything added. Pellets which are as similar as possible to the control are preferred. Larger pellets were considered agglutinated RBCs. The table on the bottom right shows the highest performing concentrations of TA and gadolinium.DETAILED DESCRIPTION

[0060] The inventors have developed a platform for adhering diverse biomolecules to the surface of mammalian cells. Unlike prior work in this area that resulted in internalization or denaturation of the biomolecules, the present methods and compositions provide surprising effectiveness. The compositions described herein do not: 1) denature or inactivate the biomolecules, 2) result in phagocytosis or other internalization of the biomolecules by the mammalian cell, or 3) damage to the mammalian cell. The inclusion of a multivalent ion provides surprising advantages over compositions lacking the multivalent ion.

[0061] Accordingly, in one aspect of any of the embodiments, described herein is a composition comprising: a) one or more polyphenol molecules: b) one or more biomolecules and / or active agents: and c) at least one of: i) at least one multivalent ion; and ii) at least one endothelium permeabilization agent.10Attorney Docket No: 002806-000156WOPT

[0062] In some embodiments of any of the aspects, a composition described herein comprising: a) one or more polyphenol molecules; b) one or more biomolecules and / or active agents; and at least one multivalent ion.

[0063] In some embodiments of any of the aspects, a composition described herein comprising: a) one or more polyphenol molecules; b) one or more biomolecules and / or active agents; and c) at least one endothelium permeabilization agent.

[0064] In some embodiments of any of the aspects, a composition described herein comprising: a) one or more polyphenol molecules; b) one or more biomolecules and / or active agents; c) at least one multivalent ion; and d) at least one endothelium permeabilization agent.

[0065] In some embodiments of any of the aspects, the composition is a nanocomplex. As used herein, “nanocomplex” refers to complexes of molecules, the complex having a size of about 0.1 nm to about 1000 nm. Further, the nanocomplex can be of any shape or form. e.g., spherical, rod, elliptical, cylindrical, capsule, or disc. In some embodiments of any of the aspects, the nanoparticle is of size from about 0.1 nm to about 100 nm, from about 0.1 nm to about 10 nm, or from about 1 nm to about 10 nm. In some embodiments of any of the aspects, the nanoparticle is of size from 0.1 nm to 100 nm, from 0.1 nm to 10 nm, or from 1 nm to 10 nm. In some embodiments of any of the aspects, the nanoparticle is less about 10 nm or smaller in size. In some embodiments of any of the aspects, the nanoparticle is 10 nm or smaller in size.

[0066] In some embodiments of any of the aspects, the composition is a functionalizing nanocomplex. A nanocomplex is functionalizing when it is capable of providing a new or modulated function or activity to a cell when it is adhered to the cell surface. That is, a nanocomplex is functionalizing when it comprises a molecule or moiety that can has a biological function or activity, or which can modulate the biological function or activity of a cell.

[0067] As used herein, “polyphenol” refers to a molecule comprising multiple phenol structural units. In some embodiments of any of the aspects, polyphenol is defined according to the WBSSH definition. In some embodiments of any of the aspects, the polyphenol comprises at least one galloyl moiety. In some embodiments of any of the aspects, the polyphenol comprises at least one catechol moiety. In some embodiments of any of the aspects, the polyphenol comprises at least one galloyl moiety and one catechol moiety. In some embodiments of any of the aspects, each polyphenol molecule comprises at least one galloyl moiety. In some embodiments of any of the aspects, each polyphenol molecule comprises at least one catechol moiety. In some embodiments of any of the aspects, each polyphenol molecule comprises at least one galloyl moiety and one catechol moiety. In some embodiments of any of the aspects, multiple different polyphenols can be present in the functionalizing nanocomplex. In such embodiments, the polyphenols can collective comprise at least one galloyl moiety and / or at least one catechol moiety.11Attorney Docket No: 002806-000156WOPT

[0068] As used herein, “galloyl’’ refers a gallic acid moiety or group found in a larger molecule. Gallic acid is depicted in Structure I.Structure I

[0069] As used herein, “catechol” refers a moiety or group having the formula of Structure II, found in a larger molecule.

[0070] Suitable polyphenols include but are not limited to tannins, gallic acid esters, proanthocyanins, and hydrolyzable tannins. In some embodiments of any of the aspects, the polyphenol is tannic acid. Tannic acid (1, 2,3,4, 6-penta-O-{3,4-dihydroxy-5-[(3, 4.5-trihydroxybenzoyl)oxy]benzoyl}-D-glucopyranose) can comprise quercitannic acid or gallotannic acid. In some embodiments of any of the aspects, the polyphenols of the functionalizing nanocomplex comprise, consist of, or consist essentially of tannic acid.12Attorney Docket No: 002806-000156WOPTStructure III (Tannic Acid)

[0071] In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of at least 1 pM In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of at least 6 pM. In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of at least 5 μM. In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of at least 10 pM.

[0072] In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of 0.5 pM to 1.5 pM. In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of 0.1 pM to 5 pM. In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of 0.1 pM to 1.5 pM. In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of 0.5 pM to 5 pM.

[0073] In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of at least 0.1 pM. In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of at least 0.5 μM.

[0074] The compositions described herein comprise biomolecules and / or active agents.

[0075] As used herein, the term “active agent” refers to an agent which, when released in vivo, possesses the desired biological activity, for example, therapeutic, diagnostic and / or prophylactic properties in vivo. Exemplary pharmaceutically active agents include, but are not limited to, those found in Harrison’s Principles of Internal Medicine, 19th Edition, Eds. T. R. Harrison et al. McGraw- 13Attorney Docket No: 002806-000156WOPTHill N. Y., NY; Physicians Desk Reference, 71st Edition, 2017, Oradell New Jersey, Medical Economics Co.; Pharmacological Basis of Therapeutics, 8th Edition, Goodman and Gilman, 2017; the current edition of the United States Pharmacopeia, Hie National Formulary; current edition of Goodman and Oilman's The Pharmacological Basis of Therapeutics; and current edition of The Merck Index, the complete content of all of which are herein incorporated in its entirety.

[0076] As used herein, “biomolecule” refers to any organic molecule that is part of or from a living organism. In some embodiments of any of the aspects, the biomolecule and / or active agent is an entity which is normally not present or not present at the levels being administered and / or provided to a cell, tissue or subject. A biomolecule and / or active agent can be selected from a group comprising: chemicals; small organic or inorganic molecules; signaling molecules; nucleic acid sequences; nucleic acid analogues; proteins: peptides; enzymes; aptamers; peptidomimetic, peptide derivative, peptide analogs, antibodies; intrabodies; biological macromolecules, extracts made from biological materials such as bacteria, plants, fungi, or animal cells or tissues; naturally occurring or synthetic compositions or functional fragments thereof. In some embodiments, the agent is any chemical, entity or moiety, including without limitation synthetic and naturally -occurring non-proteinaceous entities. Suitable biomolecules and / or active agents can include, but are not limited to: a nucleic acid (e.g.. a vector, inhibitory nucleic acid, siRNA, etc), protein, viral particle, viral vector, a lipid nanoparticle, a polymer, alkaloid, polysaccharide, anthocyanin, lipid, antiviral drug, antibiotic, chemotherapeutic, or combination thereof. In some embodiments, the biomolecule and / or active agent and / or active agents comprises a protein or is a protein. In some embodiments of any of the aspects, the protein is ovalbumin, serum albumin, interleukin-4, an antibody or antibody reagent, cholera toxin subunit B, biotin, cytokine, or lectin.

[0077] In some embodiments of any of the aspects, the antibody or antibody reagent is specific for, e.g.. binds specifically to, an immune checkpoint protein. In some embodiments of any of the aspects, the antibody or antibody reagent is specific for, e.g. binds specifically to, Programmed Cell Death Protein 1 (PD-1) or Programmed Cell Death Ligand 1 (PD-L1). Such antibodies and antibody reagents are known in the art, e.g.., Shiravand et al. Curr Oncol 29:3044-3060 (2022); Borgeaud et al. Cancer Treatment Reviews 120: 102614 (2023); each of which are incorporated by reference herein in their entireties. Further examples of such agents are provided elsewhere herein.

[0078] A nucleic acid molecule, as described herein, can be a vector, an expression vector, an inhibitory nucleic acid, an aptamer, a template molecule or cassette (e.g., for gene editing), or a targeting molecule (e.g., for CRISPR-Cas technologies), or any other nucleic acid molecule. The nucleic acid molecule can be RNA. DNA. or synthetic or modified versions thereof.

[0079] In some embodiments of any of the aspects, a composition described herein comprises at least one multivalent ion.14Attorney Docket No: 002806-000156WOPT

[0080] In some embodiments of any of the aspects, a composition described herein comprises at least one multivalent ion which is selected from the group consisting of: Al, Gd(III), Au(III), Fe(III), Mn (II), Ni(II), Sr, Ti(IV), or Zn.

[0081] In some embodiments of any of the aspects, the at least one multivalent ion comprises Al. In some embodiments of any of the aspects, the at least one multivalent ion consists of Al. In some embodiments of any of the aspects, the at least one multivalent ion consists essentially of Al.

[0082] In some embodiments of any of the aspects, the at least one multivalent ion comprises Gd(III). In some embodiments of any of the aspects, the at least one multivalent ion consists of Gd(III). In some embodiments of any of the aspects, the at least one multivalent ion consists essentially of Gd(III).

[0083] In some embodiments of any of the aspects, the at least one multivalent ion comprises Au(III). In some embodiments of any of the aspects, the at least one multivalent ion consists of Au(III). In some embodiments of any of the aspects, the at least one multivalent ion consists essentially of Au(III).

[0084] In some embodiments of any of the aspects, the at least one multivalent ion comprises Mn (II). In some embodiments of any of the aspects, the at least one multivalent ion consists of Mn (II). In some embodiments of any of the aspects, the at least one multivalent ion consists essentially of Mn (II).

[0085] In some embodiments of any of the aspects, the at least one multivalent ion comprises Ni(II). In some embodiments of any of the aspects, the at least one multivalent ion consists of Ni(II). In some embodiments of any of the aspects, the at least one multivalent ion consists essentially of Ni(II).

[0086] In some embodiments of any of the aspects, the at least one multivalent ion comprises Sr. In some embodiments of any of the aspects, the at least one multivalent ion consists of Sr. In some embodiments of any of the aspects, the at least one multivalent ion consists essentially of Sr.

[0087] In some embodiments of any of the aspects, the at least one multivalent ion comprises Zn. In some embodiments of any of the aspects, the at least one multivalent ion consists of Zn. In some embodiments of any of the aspects, the at least one multivalent ion consists essentially of Zn.

[0088] In some embodiments of any of the aspects, the at least one multivalent ion comprises Ti(IV). In some embodiments of any of the aspects, the at least one multivalent ion consists of Ti(IV). In some embodiments of any of the aspects, the at least one multivalent ion consists essentially of Ti(IV).

[0089] In some embodiments of any of the aspects, the at least one multivalent ion comprises Fe(III). In some embodiments of any of the aspects, the at least one multivalent ion consists essentially of Fe(III). In some embodiments of any of the aspects, the at least one multivalent ion consists of Fe(III).15Attorney Docket No: 002806-000156WOPT

[0090] In some embodiments of any of the aspects, the at least one multivalent ion is present as A1C13, GdCh, AuCk FeCk MnCh, NiCl2, SrCl2, [CH3CH(O-)CO2NH4]2Ti(OH)2,orZnCl2

[0091] In some embodiments of any of the aspects, the at least one multivalent ion is present as FeCk In some embodiments of any of the aspects, the Fe(III) is present as FeCk

[0092] In some embodiments of any of the aspects, the at least one multivalent ion is at a concentration of no more than 1 mM. In some embodiments of any of the aspects, the at least one multivalent ion is at a concentration of 1 pM to 1 mM. In some embodiments of any of the aspects, the at least one multivalent ion is at a concentration of 10 pM to 1 mM. In some embodiments of any of the aspects, the at least one multivalent ion is at a concentration of 50 pM to 1 mM. In some embodiments of any of the aspects, the at least one multivalent ion is at a concentration of 100 pM to 1 mM. In some embodiments of any of the aspects, the at least one multivalent ion is at a concentration of no more than 2 mM. In some embodiments of any of the aspects, the at least one multivalent ion is at a concentration of 2 pM to 2 mM. In some embodiments of any of the aspects, the at least one multivalent ion is at a concentration of 20 pM to 2 mM. In some embodiments of any of the aspects, the at least one multivalent ion is at a concentration of 200 pM to 2 mM. In some embodiments of any of the aspects, the at least one multivalent ion is at a concentration of 2 mM to 37 mM. In some embodiments of any of the aspects, the at least one multivalent ion is at a concentration of 5 mM to 20 mM.

[0093] In some embodiments of any of the aspects, the at least one multivalent ion is at a concentration of 0.01 mM to 10 mM. In some embodiments of any of the aspects, the at least one multivalent ion is at a concentration of 0.05 mM to 10 mM. In some embodiments of any of the aspects, the at least one multivalent ion is at a concentration of 0.1 mM to 10 mM. In some embodiments of any of the aspects, the at least one multivalent ion is at a concentration of 0.01 mM to 5 mM. In some embodiments of any of the aspects, the at least one multivalent ion is at a concentration of 0.01 mM to 1 mM. In some embodiments of any of the aspects, the at least one multivalent ion is at a concentration of 0.01 mM to 0.5 mM. In some embodiments of any of the aspects, the at least one multivalent ion is at a concentration of 0.01 mM to 0.1 mM. In some embodiments of any of the aspects, the at least one multivalent ion is at a concentration of 0.1 mM to 5 mM.

[0094] In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of 0.5 pM to 1.5 pM and the at least one multivalent ion is at a concentration of 0.01 mM to 10 mM. In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of 0.5 pM to 1.5 pM and the at least one multivalent ion is at a concentration of 0.05 mM to 10 mM. In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of 0.5 pM to 1.5 pM and the at least one multivalent ion is at a concentration of 0.1 mM to 10 mM. In some embodiments of any of the aspects, the at least one polyphenol molecule is at16Attorney Docket No: 002806-000156WOPTa concentration of 0.5 pM to 1.5 pM and the at least one multivalent ion is at a concentration of 0.01 mM to 0.5 mM. In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of 0.5 pM to 1.5 pM and the at least one multivalent ion is at a concentration of 0.05 mM to 10 mM. In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of 0.5 pM to 1.5 pM and the at least one multivalent ion is at a concentration of 0.01 mM to 0.5 mM. In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of 0.5 pM to 1.5 pM and the at least one multivalent ion is at a concentration of 0.05 mM to 0.5 mM. In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of 0.5 pM to 1.5 pM and the at least one multivalent ion is at a concentration of 0.01 mM to 0.1 mM. In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of 0.5 pM to 1.5 pM and the at least one multivalent ion is at a concentration of 0.05 mM to 0.5 mM.

[0095] In some embodiments of any of the aspects, a composition described herein comprises at least one endothelium permeabilization agent. As used herein, “endothelium permeabilization agent” refers to an agent which can increase the rate and / or amount of at least one material entering or crossing the endothelium, e.g. by at least 10% or more. e.g. by 10% or more. 50% or more. 70% or more, 80% or more, 90% or more, 95% or more, or 98 % or more. In some embodiments, the endothelium is vascular endothelium. In some embodiments of any of the aspects, the at least one material is the biomaterial and / or active agent of the composition described herein. The efficacy of an endothelium permeabilization agent, e.g. its ability to increase endothelium permeability' to the at least one material can be determined, e.g. by measuring the rate or amount of material crossing the endothelium. Such methods are described herein.

[0096] Endothelium permeabilization agents are known in the art and can include, but are not limited to, a Vascular Endothelial Growth Factor (VEGF) polypeptide; histamine; bradykinin; and serotonin. In some embodiments of any of the aspects, the at least one endothelium permeabilization agent is selected from the group consisting of: a Vascular Endothelial Growth Factor (VEGF) polypeptide; histamine; bradykinin; and serotonin. In some embodiments of any of the aspects, the at least one endothelium permeabilization agent comprises at least two agents selected from the group consisting of: a Vascular Endothelial Growth Factor (VEGF) polypeptide; histamine; bradykinin; and serotonin. In some embodiments of any of the aspects, the at least one endothelium permeabilization agent comprises a Vascular Endothelial Growth Factor (VEGF) polypeptide. In some embodiments of any of the aspects, the at least one endothelium permeabilization agent consists essentially of a Vascular Endothelial Growth Factor (VEGF) polypeptide. In some embodiments of any of the aspects, the at least one endothelium permeabilization agent consists of a Vascular Endothelial Growth Factor (VEGF) polypeptide.17Attorney Docket No: 002806-000156WOPT

[0097] As used herein, “Vascular Endothelial Growth Factor” or “VEGF” refers to the platelet-derived growth factor family of cysteine-knot growth factors that stimulate the formation of blood vessels. VEGF proteins regulate vasculogenesis and angiogenesis (the growth of blood vessels from pre-existing vasculature. VEGF includes VEGF-A, VEGF-B, VEGF-C, VEGF-D, and VEGF-E.

[0098] In some embodiments of any of the aspects, the VEGF is a VEGF-A, VEGF-B, VEGF-C, VEGF-D, or VEGF-E. In some embodiments of any of the aspects, the VEGF is a VEGF-A, VEGF-C, VEGF-D, or VEGF-E. In some embodiments of any of the aspects, the VEGF is a VEGF-A, or VEGF-E. In some embodiments of any of the aspects, the VEGF is a VEGF-A. In some embodiments of any of the aspects, the VEGF is a VEGF-E. VEGF sequences are known in the art.

[0099] As used herein, “VEGF A” refers to a cysteine-knot growth factor that promotes vasculogenesis and endothelium permeability. A number of isoforms of VEGF are known, including VEGF165. The sequences of VEGF are known in the art for a number of species, e.g.. human VEGF (NBCI Gene ID: 7422. polypeptide sequences NP_001020537.2 (SEQ ID NO: 1). NP_001020538.2 (SEQ ID NO: 2), NP_001020539.2 (SEQ ID NO: 3), NP_001020540.2 (SEQ ID NO: 4),NP 001020541.2 (SEQ ID NO: 5), NP_001028928.1 (SEQ ID NO: 6), NP_001165093.1 (SEQ ID NO: 7), NP_001165094.1 (SEQ ID NO: 8), NP_001165095.1 (SEQ ID NO: 9), NP_001165096.1 (SEQ ID NO: 10), NP_001165097.1 (SEQ ID NO: 11). NP_001165098.1 (SEQ ID NO: 12), NP 001165099.1 (SEQ ID NO: 13), NP 001165100.1 (SEQ ID NO: 14), NP 001165101.1 (SEQ ID NO: 15), NP 001191313.1 (SEQ ID NO: 16), NP 001191314.1 (SEQ ID NO: 17), NP 001273973.1 (SEQ ID NO: 18), NP 001303939.1 (SEQ ID NO: 19), and NP 003367.4 (SEQ ID NO: 20) and mRNA sequences NM 001025366.3 (SEQ ID NO: 21), NM 001025367.3 (SEQ ID NO: 22), NM_001025368.3 (SEQ ID NO: 23), NM_001025369.3 (SEQ ID NO: 24), NM_001025370.3 (SEQ ID NO: 25), NM_001033756.3 (SEQ ID NO: 26), NM_001171622.2 (SEQ ID NO: 27), NM_001171623.2 (SEQ ID NO: 28), NM_001171624.2 (SEQ ID NO: 29), NM_001171625.2 (SEQ ID NO: 30), NM_001171626.2 (SEQ ID NO: 31), NM_001171627.2 (SEQ ID NO: 32), NM 001171628.2 (SEQ ID NO: 33), NM_001171629.2 (SEQ ID NO: 34), NM_001171630.2 (SEQ ID NO: 35), NM_001204384.2 (SEQ ID NO: 36), NM_001204385.2 (SEQ ID NO: 37), NM_001287044.2 (SEQ ID NO: 38), NM_001317010.1 (SEQ ID NO: 39), NM_003376.6 (SEQ ID NO: 40)) and murine VEGF (NBCI Gene ID: 22339, polypeptide sequences NP 001020421.2 (SEQ ID NO: 41), NP 001020428.2 (SEQ ID NO: 42), NP_001103736.1 (SEQ ID NO: 43).NP_001103737.1 (SEQ ID NO: 44), NP_001103738.1 (SEQ ID NO: 45). NP 001273985.1 (SEQ ID NO: 46), NP 001273986.1 (SEQ ID NO: 47), NP_001273987.1 (SEQ ID NO: 48), NP_001303970.1 (SEQ ID NO: 49), and NP 033531.3 (SEQ ID NO: 50) and mRNA sequences NM 001025250.3 (SEQ ID NO: 51), NM 001025257.3 (SEQ ID NO: 52), NM 001110266.1 (SEQ ID NO: 53), NM 001110267.1 (SEQ ID NO: 54). NM 001110268.1 (SEQ ID NO: 55), NM 001287056.1 (SEQ ID NO: 56). NM 001287057.1 (SEQ ID NO: 57). NM 001287058.1 (SEQ ID NO: 58).18Attorney Docket No: 002806-000156WOPTNM 001317041.1 (SEQ ID NO: 59), and NM 009505.4 (SEQ ID NO: 60)); wherein the sequences are those associated with the foreign database entries as of February 5, 2025. The sequence of VEGF165 is known in tire art as well, e.g., human VEGF165mRNA is provided in NCBI as AF486837.1 (SEQ ID NO: 61). The structure and function of VEGF is known in the art.

[0100] In some embodiments of any of the aspects, the VEGF is a human VEGF polypeptide. In some embodiments of any of the aspects, the VEGF comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% to the full-length of a human VEGF. In some embodiments of any of the aspects, the VEGF comprises a sequence having at least 80%, at least 85%. at least 90%, at least 95%, at least 98%, or at least 99% to the full-length of a human VEGF-A. In some embodiments of any of the aspects, the VEGF comprises a sequence having at least 80%, at least 85%, at least 90%. at least 95%, at least 98%, or at least 99% to the full-length of a human VEGF165. In some embodiments of any of the aspects, the VEGF comprises human VEGF165.

[0101] The biomolecule(s) and / or active agent(s), polyphenol(s), and other elements (e.g., at least one of a multivalent ion and an endothelium permeabilization agent) of a composition described herein are in combination with each other to form the composition. As used herein, “in combination with” refers to two or more substances being present in the same formulation in any molecular or physical arrangement, e g., bound to each other, complexed, etc. A nanocomplex comprises two or more molecular structures that are linked by a direct or indirect covalent or non-covalent bond. Non-covalent interactions include, but are not limited to, electrostatic interactions, hydrogen bonding interactions, van der Waals interactions, dipole-dipole interactions, π-π stacking, magnetic interactions, and metal coordination. In some embodiments of any of the aspects, the biomolecule(s) and / or active agent(s) are complexed with the polyphenol(s) via non-covalent bonds. In some embodiments of any of the aspects, the biomolecule(s) and / or active agent(s) are complexed with the polyphenol(s) via covalent bonds. In some embodiments of any of the aspects, the biomolecule (s) and / or active agent(s) and / or the polyphenol(s) are complexed with the at least one multivalent ion via non-covalent bonds. In some embodiments of any of the aspects, the biomolecule (s) and / or active agent(s) and / or the polyphenol(s) are complexed with the at least one endothelium permeabilization agent via non-covalent bonds. In some embodiments of any of the aspects, the biomolecule (s) and / or active agent(s) and / or the polyphenol(s) are complexed with the at least one endothelium permeabilization agent via covalent bonds.

[0102] In some embodiments of any of the aspects, a nanocomplex can comprise 2 or more. 3 or more, 4 or more, or 5 or more different polyphenols. In some embodiments of any of the aspects, a nanocomplex can comprise 2 or more, 3 or more, 4 or more, or 5 or more different biomolecules and / or active agents. In some embodiments of any of the aspects, a nanocomplex can comprise 2 or more. 3 or more. 4 or more, or 5 or more different multivalent ions. In some embodiments of any of19Attorney Docket No: 002806-000156WOPTthe aspects, a nanocomplex can comprise 2 or more, 3 or more, 4 or more, or 5 or more different endothelium permeabilization agents.

[0103] In some embodiments of any of the aspects, a composition described herein comprises a) a polyphenol molecule, b) one or more biomolecule or active agents, and c) Fe(III). In some embodiments of any of the aspects, a composition described herein comprises a) a polyphenol molecule, b) one or more antibodies or antibody reagents, and c) Fe(III).

[0104] In some embodiments of any of the aspects, a composition described herein comprises a) a polyphenol molecule comprising at least one galloyl moiety and at least one catechol moiety, b) one or more biomolecule or active agents, and c) Fe(III). In some embodiments of any of the aspects, a composition described herein comprises a) a polyphenol molecule comprising at least one galloyl moiety and at least one catechol moiety, b) one or more antibodies or antibody reagents, and c) Fe(III).

[0105] In some embodiments of any of the aspects, a composition described herein comprises a) tannic acid, b) one or more biomolecule or active agents, and c) Fe(III). In some embodiments of any of the aspects, a composition described herein comprises a) tannic acid, b) one or more antibodies or antibody reagents, and c) Fe(III).

[0106] As described in the examples herein, the ratio of polyphenol molecules to biomolecules and / or active agents can influence the formation of the nanocomplexes. In some embodiments of any of the aspects, the stoichiometric ratio of polyphenol molecules to biomolecules and / or active agents is about 570:1 or less relative polyphenol molecule. In some embodiments of any of the aspects, the stoichiometric ratio of polyphenol molecules to biomolecules and / or active agents is 570: 1 or less relative polyphenol molecule. In some embodiments of any of the aspects, the stoichiometric ratio of polyphenol molecules to biomolecules and / or active agents is about 190: 1 or more relative polyphenol molecule. In some embodiments of any of the aspects, the stoichiometric ratio of polyphenol molecules to biomolecules and / or active agents is 190:1 or more relative poly phenol molecule. In some embodiments of any of the aspects, the stoichiometric ratio of polyphenol molecules to biomolecules and / or active agents is about 570:1 to about 190:1. In some embodiments of any of the aspects, the stoichiometric ratio of polyphenol molecules to biomolecules and / or active agents is 570:1 to 190:1.

[0107] In some embodiments of any of the aspects, the stoichiometric ratio of polyphenol molecules to biomolecules and / or active agents is 100:1 or more relative polyphenol molecule. In some embodiments of any of the aspects, the stoichiometric ratio of polyphenol molecules to biomolecules and / or active agents is 150:1 or more relative polyphenol molecule. In some embodiments of any of the aspects, the stoichiometric ratio of polyphenol molecules to biomolecules and / or active agents is 200:1 or more relative polyphenol molecule. In some embodiments of any of the aspects, the stoichiometric ratio of polyphenol molecules to biomolecules and / or active agents is20Attorney Docket No: 002806-000156WOPT250:1 or more relative polyphenol molecule. In some embodiments of any of the aspects, the stoichiometric ratio of polyphenol molecules to biomolecules and / or active agents is 300: 1 or more relative polyphenol molecule.

[0108] In some embodiments of any of the aspects, the stoichiometric ratio of polyphenol molecules to biomolecules and / or active agents is 500:1 to 100:1. In some embodiments of any of the aspects, the stoichiometric ratio of polyphenol molecules to biomolecules and / or active agents is 500:1 to 150:1. In some embodiments of any of the aspects, the stoichiometric ratio of polyphenol molecules to biomolecules and / or active agents is 500:1 to 200:1. In some embodiments of any of the aspects, the stoichiometric ratio of polyphenol molecules to biomolecules and / or active agents is 500:1 to 250:1. In some embodiments of any of the aspects, the stoichiometric ratio of polyphenol molecules to biomolecules and / or active agents is 500:1 to 300:1.

[0109] In some embodiments of any of the aspects, the stoichiometric ratio of polyphenol molecules to biomolecules and / or active agents is 600:1 to 100:1. In some embodiments of any of the aspects, the stoichiometric ratio of polyphenol molecules to biomolecules and / or active agents is 600:1 to 150:1. In some embodiments of any of the aspects, the stoichiometric ratio of polyphenol molecules to biomolecules and / or active agents is 600:1 to 200:1. In some embodiments of any of the aspects, the stoichiometric ratio of polyphenol molecules to biomolecules and / or active agents is 600:1 to 250:1. In some embodiments of any of the aspects, the stoichiometric ratio of polyphenol molecules to biomolecules and / or active agents is 600:1 to 300:1.

[0110] The combination of multivalent ion concentration and polyphenol molecule concentration can have a critical effect on providing effective formation of a nanocomplex while avoiding damage to the cell the composition will adhere to. This effect is illustrated in Figs. 6A-6C.

[0111] In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of at least 10 μM and the at least one multivalent ion is at a concentration of no more than 1 mM. In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of at least 10 μM and the at least one multivalent ion is at a concentration of 1 μM to 1 mM. In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of at least 10 μM and the at least one multivalent ion is at a concentration of 10 μM to 1 mM. In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of at least 10 μM and the at least one multivalent ion is at a concentration of 100 μM to 1 mM.

[0112] In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of at least 10 μM and the at least one multivalent ion is at a concentration of no more than 2 mM. In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of at least 10 μM and the at least one multivalent ion is at a concentration of 1 μM to 2 mM. In some embodiments of any of the aspects, the at least one polyphenol molecule is at a21Attorney Docket No: 002806-000156WOPTconcentration of at least 10 μM and the at least one multivalent ion is at a concentration of 10 μM to 2 mM. In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of at least 10 μM and the at least one multivalent ion is at a concentration of 100 μM to 2 mM.

[0113] In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of at least 5 μM and the at least one multivalent ion is at a concentration of no more than 1 mM. In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of at least 5 μM and the at least one multivalent ion is at a concentration of 1 μM to 1 mM. In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of at least 5 μM and the at least one multivalent ion is at a concentration of 10 μM to 1 mM. In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of at least 5 μM and the at least one multivalent ion is at a concentration of 100 μM to 1 mM.

[0114] In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of at least 5 μM and the at least one multivalent ion is at a concentration of no more than 2 mM. In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of at least 5 μM and the at least one multivalent ion is at a concentration of 1 μM to 2 mM. In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of at least 5 μM and the at least one multivalent ion is at a concentration of 10 μM to 2 mM. In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of at least 5 μM and the at least one multivalent ion is at a concentration of 100 μM to 2 mM.

[0115] In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of at least 6 μM and the at least one multivalent ion is at a concentration of no more than 1 mM. In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of at least 6 μM and the at least one multivalent ion is at a concentration of 1 μM to 1 mM. In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of at least 6 μM and the at least one multivalent ion is at a concentration of 10 μM to 1 mM. In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of at least 6 μM and the at least one multivalent ion is at a concentration of 100 μM to 1 mM.

[0116] In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of at least 6 μM and the at least one multivalent ion is at a concentration of no more than 2 mM. In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of at least 6 μM and the at least one multivalent ion is at a concentration of 1 μM to 2 mM. In some embodiments of any of the aspects, the at least one polyphenol molecule is at a22Attorney Docket No: 002806-000156WOPTconcentration of at least 6 μM and the at least one multivalent ion is at a concentration of 10 μM to 2 mM. In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of at least 6 μM and the at least one multivalent ion is at a concentration of 100 μM to 2 mM.

[0117] In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of 0.5 to 1.5 μM and the strontium ion is at a concentration of 0.1 to 10 mM.

[0118] In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of 0.5 to 1.5 μM and the iron ion is at a concentration of 0.05 to 0.5 mM.

[0119] In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of 0.5 to 1.5 μM and the nickel ion is at a concentration of 0.05 to 0.5 mM.

[0120] In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of 0.5 to 1.0 μM and the titanium ion is at a concentration of 0.05 to 0.1 mM.

[0121] In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of 0.5 to 1.5 μM and the gold ion is at a concentration of 0.01 to 0.1 mM.

[0122] In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of 0.5 to 1.5 μM and the aluminum ion is at a concentration of 0.01 to 0.5 mM.

[0123] In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of 0.5 to 1.0 μM and the zinc ion is at a concentration of 0.01 to 0.05 mM.

[0124] In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of 0.5 to 1.5 μM and the manganese ion is at a concentration of 0.05 to 1.5 mM.

[0125] In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of 0.5 to 1.5 μM and the gadolinium ion is at a concentration of 0.05 to 0.5 mM.

[0126] In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of at least 10 μM and the at least one multivalent ion is at a concentration of no more than 1 mM; and the stoichiometric ratio of polyphenol molecules to biomolecules and / or active agents is 500:1 to 100:1. In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of at least 10 μM and the at least one multivalent ion is at a concentration of no more than 1 mM; and the stoichiometric ratio of polyphenol molecules to biomolecules and / or active agents is 500:1 to 150:1. In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of at least 10 μM and the at least one multivalent ion is at a concentration of no more than 1 mM; and the stoichiometric ratio of polyphenol molecules to biomolecules and / or active agents is 500:1 to 200:1. In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of at least 10 μM and the at least one multivalent ion is at a concentration of no more than 1 mM; and the stoichiometric ratio of polyphenol molecules to biomolecules and / or active agents is 500:1 to 250:1. In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of at least 10 μM and the at least23Attorney Docket No: 002806-000156WOPTone multivalent ion is at a concentration of no more than 1 mM; and the stoichiometric ratio of polyphenol molecules to biomolecules and / or active agents is 500:1 to 300:1.

[0127] In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of at least 10 pM and the at least one multivalent ion is at a concentration of no more than 1 mM; and the stoichiometric ratio of polyphenol molecules to biomolecules and / or active agents is 600:1 to 100:1. In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of at least 10 μM and the at least one multivalent ion is at a concentration of no more than 1 mM; and the stoichiometric ratio of polyphenol molecules to biomolecules and / or active agents is 600:1 to 150:1. In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of at least 10 μM and the at least one multivalent ion is at a concentration of no more than 1 mM; and the stoichiometric ratio of polyphenol molecules to biomolecules and / or active agents is 600:1 to 200:1. In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of at least 10 μM and the at least one multivalent ion is at a concentration of no more than 1 mM; and the stoichiometric ratio of polyphenol molecules to biomolecules and / or active agents is 600:1 to 250:1. In some embodiments of any of the aspects, the at least one polyphenol molecule is at a concentration of at least 10 μM and the at least one multivalent ion is at a concentration of no more than 1 mM; and the stoichiometric ratio of polyphenol molecules to biomolecules and / or active agents is 600:1 to 300:1.

[0128] The compositions described herein can adhere to or associate with the surface of a mammalian cell, e.g., unlike most prior art molecules which cannot adhere or which are rapidly internalized. As used herein, “adhere” refers to the ability of the nanocomplex to attach to. cling to, stick to, or remain in association with the surface of the cell. Adhesion can comprise covalent and / or non-covalent interactions. In some embodiments of any of the aspects, a composition described herein can adhere to a mammalian cell surface for at least 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 48 hours, at least 2 days, at least 3 days, at least 1 week, or at least 2 weeks, e.g., before dissociating from the cell, being internalized by the cell, or degrading. In some embodiments of any of the aspects, a composition described herein can adhere to a mammalian cell surface for at least 24 hours, e.g., before dissociating from the cell, being internalized by the cell, or degrading. In some embodiments of any of the aspects, a composition described herein can adhere to a mammalian cell surface for at least 48 hours, e.g., before dissociating from the cell, being internalized by the cell, or degrading. In some embodiments of any of the aspects, at least 20%, at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of a composition described herein can adhere to a mammalian cell surface for at least 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 2 days, at least 3 days, at least 1 week, or at least 2 weeks.24Attorney Docket No: 002806-000156WOPT

[0129] In one aspect of any of the embodiments, provided herein is a cell comprising at least one composition as described herein, adhered to the surface of the cell. In some embodiments of any of the aspects, the cell is a mammalian cell. As used herein, “mammalian" or “mammal’' refers to refers to any animal that falls within a taxonomic classification of mammals. Mammals can refer to humans or non-human primates. Mammals can refer to livestock or pets including, for example, dogs, cats, rodents (including rabbits, mice, black rats, hamsters) and the like. Mammals may refer to agricultural animals including, for example, cows, sheep, pigs, horses and the like. In some embodiments, the cell is a primate cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a dog or cat cell. In some embodiments, the cell is a murine cell. In some embodiments, the cell is autologous to a patient.

[0130] The methods and compositions described herein are contemplated for use with all mammalian cell types. In some embodiments of any of the aspects, the cell is a hematopoietic cell. In some embodiments of any of tire aspects, the cell is an erythrocyte (red blood cell). B cell, T cell, monocyte, macrophage, neutrophil, or natural killer (NK) cell.

[0131] Particular combinations of biomolecules and / or active agents and cell types are contemplated herein, including:Biomolecule and / or active agent and / or active agent Cell TypeAn antibody or antibody reagent specific for an immune checkpoint MacrophageproteinAn antibody or antibody reagent, an antibody or antibody reagent Erythrocytespecific for an immune checkpoint protein, a cytokine, antiviral drug,viral vector, antibiotic, or siRNAAn antibody or antibody reagent, an antibody or antibody reagent Natural killer cell specific for an immune checkpoint protein siRNA, orchemotherapeuticCytokine T cellAnti-inflammatory drug Neutrophil

[0132] In some embodiments of any of the aspects, the cell is functionalized when the composition is adhered to the cell surface.

[0133] In some embodiments of any of the aspects, compositions described herein, collectively comprising 10 to 1 trillion biomolecules and / or active agents, are adhered to the surface of the cell. In some embodiments of any of the aspects, compositions described herein, collectively comprising 1 to 10 trillion biomolecules and / or active agents are adhered to the surface of the cell. In some embodiments of any of the aspects, compositions described herein, collectively comprising 100 to 1 trillion biomolecules and / or active agents are adhered to the surface of the cell. In some embodiments25Attorney Docket No: 002806-000156WOPTof any of the aspects, compositions described herein, collectively comprising 1,000 to 1 trillion biomolecules and / or active agents are adhered to the surface of the cell. In some embodiments of any of the aspects, compositions described herein, collectively comprising 10 to 100,00 biomolecules and / or active agents are adhered to the surface of the cell.

[0134] In some embodiments of any of the aspects, described herein is a population of cells as described herein, comprising a least two subpopulations of cells, each subpopulation comprising a different composition described herein. For example, a population of cells can comprise a first subpopulation comprising an adhered composition comprising VEGF and a second subpopulation comprising an adhered composition not comprising VEGF.

[0135] The compositions described herein can be assembled and adhered to a cell, e.g., a mammalian cell, according to the protocols provided in the Examples and the following method. In one aspect of any the embodiments, provided herein is a method of functionalizing a cell, the method comprising: a) combining one or more polyphenol molecules, one or more biomolecules and / or active agents one or more polyphenol molecules; one or more biomolecules or active agents; and at least one of: at least one multivalent ion; and at least one endothelium permeabilization agent; and b) contacting a cell with the combination resulting from step a; whereby composition described herein forms and adheres to the surface of the cell. In some embodiments of any the aspects, the combining step occurs before the contacting step. In some embodiments of any the aspects, the combining step occurs at least 30 seconds, at least 1 minute, at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 30 minutes, at least 45 minutes, at least 60 minutes or more before the contacting step.

[0136] In one aspect of any the embodiments, provided herein is a method of functionalizing a cell, the method comprising: contacting a cell with a composition described herein; whereby the composition described herein adheres to the surface of the cell.

[0137] In some embodiments of any of the aspects, the contacting occurs ex vivo. In some embodiments of any of the aspects, the contacting occurs in vivo. In some embodiments of any of the aspects, the contacting occurs in the blood of a patient. In some embodiments of any of the aspects, the contacting occurs in the vasculature of a patient.

[0138] In one aspect of any of the embodiments, described herein is a method of administering a biomolecule and / or active agent and / or active agent to a patient in need of treatment with the biomolecule and / or active agent and / or active agent, the method comprising administering a composition or cell comprising a composition as described herein to the patient. In one aspect of any of the embodiments, described herein is a method of administering a biomolecule and / or active agent and / or active agent to a patient in need of treatment with the biomolecule and / or active agent and / or active agent, the method comprising administering a composition as described herein to the patient. In one aspect of any of the embodiments, described herein is a method of administering a biomolecule26Attorney Docket No: 002806-000156WOPTand / or active agent and / or active agent to a patient in need of treatment with the biomolecule and / or active agent and / or active agent, the method comprising administering a cell comprising a composition as described herein to the patient.

[0139] In some embodiments of any of the aspects, the cell is autologous to the patient.

[0140] Particular cell types tend to accumulate to, migrate to, or travel through specific tissues and sites in the body. Accordingly, depending on the location that the user desires to deliver the biomolecule and / or active agent and / or active agent to (e.g., where a site of disease is located), particular cell types may be preferred. For example, in some embodiments of any of the aspects, the cell is an ery throcyte and a plurality' of the biomolecule and / or active agent administered to the patient is delivered to the lungs. In some embodiments of any of the aspects, the cell is a macrophage and a plurality' of the biomolecule and / or active agent administered to the patient is delivered to the brain, a tumor, or a site of inflammation or autoimmune inflammation. In some embodiments of any of the aspects, the cell is a natural killer cell and a plurality of the biomolecule and / or active agent administered to the patient is delivered to a tumor. In some embodiments of any of the aspects, the cell is a T cell and a plurality of the biomolecule and / or active agent administered to the patient is delivered to a tumor. In some embodiments of any of the aspects, the cell is a neutrophil and a plurality' of the biomolecule and / or active agent administered to the patient is delivered to the lungs or a site of inflammation.

[0141] In some embodiments of any of the aspects, a composition as described herein, e.g., a composition or cell comprising a composition as described herein, can further comprise a pharmaceutically acceptable carrier. As used herein, the terms "pharmaceutically' acceptable", "physiologically tolerable" and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a mammal without the production of undesirable physiological effects such as nausea, dizziness, gastric upset and the like. A pharmaceutically acceptable carrier will not promote the raising of an immune response to an agent with which it is admixed, unless so desired. The preparation of a pharmacological composition that contains active ingredients dissolved or dispersed therein is well understood in the art and need not be limited based on formulation. Typically, such compositions are prepared as injectable either as liquid solutions or suspensions, however, solid forms suitable for solution, or suspensions, in liquid prior to use can also be prepared. The preparation can also be emulsified or presented as a liposome composition. The active ingredient can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods described herein. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol or the like and combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like which enhance the effectiveness of the active27Attorney Docket No: 002806-000156WOPTingredient. The therapeutic composition of the present disclosure can include pharmaceutically acceptable salts of the components therein. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide) that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, tartaric, mandelic and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.Physiologically tolerable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline. Still further, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol and other solutes. Liquid compositions can also contain liquid phases in addition to and to the exclusion of w ater. Exemplary of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and w ater-oil emulsions. The amount of an active agent used in the methods described herein that will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences. A. Osol, a standard reference text in this field of art. For example, a parenteral composition suitable for administration by injection is prepared by dissolving 1.5% by weight of active ingredient in 0.9% sodium chloride solution.

[0142] The term "carrier" in the context of a pharmaceutical carrier refers to a diluent, adjuvant, excipient, or vehicle with w hich the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as w ater and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and gly cerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations, and the like. The composition can be formulated as a suppository', with traditional binders and carriers such as triglycerides. Oral fonnulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in28Attorney Docket No: 002806-000156WOPTRemington's Pharmaceutical Sciences, 18th Ed., Gennaro, cd. (Mack Publishing Co., 1990). The formulation should suit the mode of administration.

[0143] Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and / or dispersion media. The use of such carriers and diluents is well known in the art. Some non-limiting examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as coni starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil. olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar: (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin. HDL and LDL; (22) C2-C12alcohols, such as ethanol; and (23) other nontoxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation. The terms such as "excipient", "carrier", "pharmaceutically acceptable carrier" or the like are used interchangeably herein. In some embodiments, the carrier inhibits the degradation of the active compound. The term "pharmaceutically acceptable carrier" excludes tissue culture medium.

[0144] In some embodiments of any of the aspects, a composition as described herein or cell comprising a composition as described herein, can be formulated as an oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, cutaneous, injection, or intratumoral formulation.

[0145] In one aspect of any of the embodiments, a composition as described herein or cell comprising a composition as described herein for a method of administering or delivering at least biomolecule, e.g., for the treatment of a disease. In one aspect of any of the embodiments, described herein is a method of administering at least one biomolecule and / or active agent or cell as described herein, the method comprising administering a composition as described herein or cell comprising a composition as described herein. In one aspect of any of the embodiments, described herein is a method of treating a disease by administering at least one biomolecule and / or active agent or cell described herein, the method comprising administering a composition as described herein or cell comprising a composition as described herein. A biomolecule and / or active agent can be directly29Attorney Docket No: 002806-000156WOPTtherapeutic, or modulate the activity of the functionalized cell such that the cell exhibits a therapeutic effect. In some embodiments of any of the aspects, administering comprises intravenous injection of a composition described herein. In some embodiments of any of the aspects, administering comprises intravenous injection of a composition described herein, e.g., the composition adheres to at least one cell in vivo. In some embodiments of any of the aspects, administering comprises intravenous injection of a cell comprising a composition as described herein.

[0146] In one aspect of any of the embodiments, described herein is a method of administering a viral vector and / or reducing the immune clearance of viral vectors, the method comprising administering a viral particle or viral vector adhered to a cell (e.g., mammalian cell and / or a red blood cell). The adherence can be via or mediated by one or more compositions described herein. In some embodiments of any of the aspects, the viral particle or viral vector adhered to a cell can be a functionalized cell as described herein. In some embodiments of any of the aspects, the viral particle or viral vector adhered to a cell can be a nanocomplex as described herein. In some embodiments of any of the aspects, administering comprises intravenous injection of a composition described herein. In some embodiments of any of the aspects, administering comprises intravenous injection of a composition described herein, e.g., the composition adheres to at least one cell in vivo. In some embodiments of any of the aspects, administering comprises intravenous injection of a cell comprising a composition as described herein.

[0147] In one aspect of any of the embodiments, described herein is a method of gene therapy comprising administering a functionalized cell as described herein, wherein the biomolecule and / or active agent comprises a nucleic acid sequence, e.g., a nucleic acid sequence suitable for or configured for gene therapy. In some embodiments of any of the aspects, administering comprises intravenous injection of a composition described herein. In some embodiments of any of the aspects, administering comprises intravenous injection of a composition described herein, e.g., the composition adheres to at least one cell in vivo. In some embodiments of any of the aspects, administering comprises intravenous injection of a cell comprising a composition as described herein.

[0148] Nucleic acid sequences suitable or configured for gene therapy will vary in structure and sequence depending on the nature of the gene therapy desired. One of skill in the art is well aware of how to select and / or design such sequences. Merely by way of non-limiting example, such sequences can comprise one or more of: one or two homology arms to direct recombination, a sequence to be inserted into the genome, a sequence to direct repair of a mutation in the genome, a sequence comprising an expression cassette, and the like. In some embodiments of any of the aspects, the gene therapy target (e.g., the cells to be targeted, the cells which display the pathology or disease requiring gene therapy, or the like) is primarily in the lungs. In some embodiments of any of the aspects, the gene therapy target (e.g.. the cells to be targeted, the cells which display the pathology or disease requiring gene therapy, or the like) is primarily in the brain.30Attorney Docket No: 002806-000156WOPT

[0149] In some embodiments, the methods described herein relate to treating a subject having or diagnosed as having a condition with a composition as described herein, e.g., a composition as described herein or a cell comprising a composition described herein. In some embodiments of any of the aspects, the condition is cancer. Subjects having cancer can be identified by a physician using current methods of diagnosing cancer. Symptoms and / or complications of cancer which characterize these conditions and aid in diagnosis are well known in the art and include but are not limited to, fevers, weight loss, bumps or tumors. Tests that may aid in a diagnosis of, e.g. cancer include, but are not limited to, biopsy and imaging exams. A family history of cancer, or exposure to risk factors for cancer can also aid in determining if a subject is likely to have cancer or in making a diagnosis of cancer.

[0150] The compositions and methods described herein can be administered to a subject having or diagnosed as having a condition described herein. In some embodiments, the methods described herein comprise administering an effective amount of compositions described herein or a cell comprising a composition as described herein, to a subject in order to alleviate a symptom of a condition. As used herein, "alleviating a symptom " is ameliorating any marker or symptom associated with a condition. As compared with an equivalent untreated control, such reduction is by at least 5%, 10%. 20%, 40%, 50%, 60%. 80%, 90%, 95%, 99% or more as measured by any standard technique. A variety of means for administering the compositions described herein to subjects are known to those of skill in the art. Such methods can include, but are not limited to oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal. airway (aerosol), pulmonary, cutaneous, injection, or intratumoral administration. Administration can be local or systemic.

[0151] Oral administration can comprise providing tablets (including without limitation scored or coated tablets), pills, caplets, capsules, chewable tablets, powder packets, cachets, troches, wafers, aerosol sprays, or liquids, such as but not limited to, syrups, elixirs, solutions or suspensions in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion, or a water-in-oil emulsion. Oral formulations can comprise discrete dosage forms, such as, but not limited to, tablets (including without limitation scored or coated tablets), pills, caplets, capsules, chewable tablets, powder packets, cachets, troches, wafers, aerosol sprays, or liquids, such as but not limited to, syrups, elixirs, solutions or suspensions in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion, or a water-in-oil emulsion. Such compositions may be prepared by methods of pharmacy well known to those skilled in the art. See generally, Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams, and Wilkins, Philadelphia PA. (2005).

[0152] In some embodiments, parenteral administration comprises delivery to a tumor, e.g., a cancer tumor. In some embodiments of any of the aspects, a composition described herein can be a parenteral dose form. Since administration of parenteral dosage forms typically bypasses the patient's natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of31Attomey Docket No: 002806-000156WOPTbeing sterilized prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions. In addition, controlled-release parenteral dosage forms can be prepared for administration of a patient, including, but not limited to, DUROS®-type dosage forms and dose-dumping.

[0153] Suitable vehicles that can be used to provide parenteral dosage forms of a composition as described herein are well known to those skilled in the art. Examples include, without limitation: sterile water; water for injection USP; saline solution; glucose solution; aqueous vehicles such as but not limited to, sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate. Compounds that alter or modify the solubility of an ingredient in a composition as disclosed herein can also be incorporated into the parenteral dosage forms of the disclosure, including conventional and controlled-release parenteral dosage forms.

[0154] Conventional dosage forms generally provide rapid or immediate drug release from the formulation. Depending on the pharmacology and pharmacokinetics of the drug, use of conventional dosage forms can lead to wide fluctuations in the concentrations of the drug in a patient's blood and other tissues. These fluctuations can impact a number of parameters, such as dose frequency, onset of action, duration of efficacy, maintenance of therapeutic blood levels, toxicity, side effects, and the like. For example, controlled-release formulations can be used to control a drug's onset of action, duration of action, plasma levels within the therapeutic window, and peak blood levels. In particular, controlled- or extended-release dosage forms or formulations can be used to ensure that the maximum effectiveness of a drug is achieved while minimizing potential adverse effects and safety concerns, which can occur both from under-dosing a drug (i.e., going below the minimum therapeutic levels) as well as exceeding the toxicity level for the drug. In some embodiments, the composition comprising a functionalized mammalian cell can be administered in a sustained release formulation.

[0155] Controlled-release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non-controlled release counterparts. Ideally, the use of an optimally designed controlled-release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time. Advantages of controlled-release formulations include: 1) extended activity of the drug; 2) reduced dosage frequency; 3) increased patient compliance; 4) usage of less total drug; 5) reduction in local or systemic side effects; 6) minimization of drug accumulation; 7) reduction in blood level fluctuations; 8) improvement in efficacy of treatment; 9) reduction of potentiation or loss of drug activity; and 10)32Attorney Docket No: 002806-000156WOPTimprovement in speed of control of diseases or conditions. Kim, Cherng-ju, Controlled Release Dosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.: 2000).

[0156] Most controlled-release formulations are designed to initially release an amount of drug (active ingredient) that promptly produces the desired therapeutic effect, and gradually and continually release other amounts of drug to maintain this level of therapeutic or prophylactic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body. Controlled-release of an active ingredient can be stimulated by various conditions including, but not limited to, pH, ionic strength, osmotic pressure, temperature, enzymes, water, and other physiological conditions or compounds.

[0157] A variety of known controlled- or extended-release dosage forms, formulations, and devices can be adapted for use with the salts and compositions of the disclosure. Examples include, but are not limited to. those described in U. S. Pat. Nos.: 3,845,770; 3.916.899; 3.536,809; 3,598,123; 4.008.719; 5674,533; 5,059,595; 5,591 ,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185 Bl; each of which is incorporated herein by reference. These dosage forms can be used to provide slow or controlled-release of one or more active ingredients using, for example, hydroxypropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems (such as OROS® (Alza Corporation, Mountain View, Calif. USA)), or a combination thereof to provide the desired release profile in varying proportions.

[0158] The term “effective amount" as used herein refers to the amount of a composition needed to alleviate at least one or more symptom of the disease or disorder, and relates to a sufficient amount of pharmacological composition to provide the desired effect. The term "therapeutically effective amount" therefore refers to an amount of a composition that is sufficient to provide a particular effect when administered to a typical subject. An effective amount as used herein, in various contexts, would also include an amount sufficient to delay the development of a symptom of the disease, alter the course of a symptom disease (for example but not limited to, slowing the progression of a symptom of the disease), or reverse a symptom of the disease. Thus, it is not generally practicable to specify an exact “effective amount". However, for any given case, an appropriate “effective amount" can be determined by one of ordinary skill in the art using only routine experimentation.

[0159] Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g, for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dosage can vary depending upon the dosage fonn employed and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50 / ED50. Compositions and methods that exhibit large therapeutic indices are preferred. A therapeutically effective dose can be estimated initially from cell33Attorney Docket No: 002806-000156WOPTculture assays. Also, a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the active compound, which achieves a half-maximal inhibition of symptoms) as determined in cell culture, or in an appropriate animal model. Levels in plasma can be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by a suitable bioassay, e.g., assay for tumor growth, or inflammation, among others. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.

[0160] In some embodiments of any of the aspects, the composition as described herein, e.g., a composition comprising a composition described herein or a cell comprising a composition as described herein, is administered as a monotherapy, e.g.. another treatment for the condition is not administered to the subject.

[0161] In some embodiments of any of the aspects, the methods described herein can further comprise administering a second agent and / or treatment to the subject, e.g. as part of a combinatorial therapy, either in a composition described herein, or as a separate formulation. For example, nonlimiting examples of a second agent and / or treatment for treatment of cancer can include radiation therapy, surgery, gemcitabine, cisplastin, paclitaxel, carboplatin, bortezomib, AMG479, vorinostat, rituximab, temozolomide, rapamycin, ABT-737. PI- 103; alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinonc); a camptothecin (including the synthetic analogue topotecan); bryostatin: callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine. cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)): dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis. dactinomycin, daunorubicin. detorubicin. 6-diazo-5-oxo-L-norleucine,ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin. 2- 34Attorney Docket No: 002806-000156WOPTpyrrolino-doxorubicin and deoxy doxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products. Eugene. Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (" Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N. J.), ABRAXANE® Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chlorambucil; GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE. RTM. vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including the treatment regimen of irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including the oxaliplatin treatment regimen (FOLFOX); lapatinib (Tykerb. RTM.); inhibitors of PKC -alpha. Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva®)) and VEGF-A that reduce cell proliferation and pharmaceutically acceptable salts, acids or derivatives of any of the above. In addition, the methods of treatment can further include the use of radiation or radiation therapy. Further, the methods of treatment can further include the use of surgical treatments.

[0162] By way of non-limiting example, if a subject is to be treated for pain or inflammation according to the methods described herein, the subject can also be administered a second agent and / or treatment known to be beneficial for subjects suffering from pain or inflammation. Examples of such35Attorney Docket No: 002806-000156WOPTagents and / or treatments include, but are not limited to, non-steroidal anti-inflammatory drugs (NSAIDs - such as aspirin, ibuprofen, or naproxen); corticosteroids, including glucocorticoids (e g. cortisol, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, and beclometasone): methotrexate; sulfasalazine; leflunomide; anti-TNF medications; cyclophosphamide; pro-resolving drugs; mycophenolate; or opiates (e.g. endorphins, enkephalins, and dynorphin), steroids, analgesics, barbiturates, oxycodone, morphine, lidocaine, and the like.

[0163] In certain embodiments, an effective dose of a composition described herein, e.g., a composition as described herein or a cell comprising a composition as described herein, can be administered to a patient once. In certain embodiments, an effective dose a composition described herein, e.g., a composition as described herein or a cell comprising a composition as described herein can be administered to a patient repeatedly. For systemic administration, subjects can be administered a therapeutic amount of a composition described herein, e.g., a composition as described herein or a cell comprising a composition as described herein such as. e.g. 0.1 mg / kg, 0.5 mg / kg, 1.0 mg / kg. 2.0 mg / kg, 2.5 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, or more. In some embodiments of any of the aspects, the at least one biomolecule and / or active agent is present in the composition at a dose of from about 1.0-20.0 mg / kg. In some embodiments of any of the aspects, the at least one biomolecule and / or active agent is present in the composition at a dose of from 1.0-20.0 mg / kg.

[0164] Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the minimal effective dose and / or maximal tolerated dose. The dosage can vary depending upon the dosage form employed and the route of administration utilized. A therapeutically effective dose can be estimated initially from cell culture assays. Also, a dose can be formulated in animal models to achieve a dosage range between the minimal effective dose and the maximal tolerated dose. The effects of any particular dosage can be monitored by a suitable bioassay, e.g., assay for tumor growth and / or size among others. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.

[0165] In some embodiments, after an initial treatment regimen, the treatments can be administered on a less frequent basis. For example, after treatment biweekly for three months, treatment can be repeated once per month, for six months or a year or longer. Treatment according to the methods described herein can reduce levels of a marker or symptom of a condition, by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80 % or at least 90% or more.

[0166] The dosage of a composition as described herein can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment. With respect to duration and frequency of treatment, it is typical for skilled clinicians to monitor subjects in order to determine36Attorney Docket No: 002806-000156WOPTwhen the treatment is providing therapeutic benefit, and to determine whether to increase or decrease dosage, increase or decrease administration frequency, discontinue treatment, resume treatment, or make other alterations to the treatment regimen. The dosing schedule can vary from once a week to daily depending on a number of clinical factors, such as the subject's sensitivity to the composition. The desired dose or amount of activation can be administered at one time or divided into subdoses, e.g., 2-4 subdoses and administered over a period of time, e.g., at appropriate intervals through the day or other appropriate schedule. In some embodiments, administration can be chronic, e.g., one or more doses and / or treatments daily over a period of weeks or months. Examples of dosing and / or treatment schedules are administration daily, twice daily, three times daily or four or more times daily over a period of 1 week, 2 weeks, 3 weeks, 4 weeks. 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months, or more. A composition described herein, e.g., a composition comprising a functionalized mammalian cell, can be administered over a period of time, such as over a 5 minute. 10 minute. 15 minute, 20 minute, or 25 minute period.

[0167] The dosage ranges for the administration of the compositions described herein, according to the methods described herein depend upon, for example, the form of the active compound, its potency, and the extent to which symptoms, markers, or indicators of a condition described herein are desired to be reduced, for example the percentage reduction desired for symptoms or markers. The dosage should not be so large as to cause adverse side effects. Generally, the dosage will vary with the age, condition, and sex of the patient and can be determined by one of skill in the art. The dosage can also be adjusted by the individual physician in the event of any complication.

[0168] The efficacy of a composition described in, e.g. the treatment of a condition described herein, or to induce a response as described herein can be determined by the skilled clinician.However, a treatment is considered “effective treatment," as the term is used herein, if one or more of the signs or symptoms of a condition described herein are altered in a beneficial manner, other clinically accepted symptoms are improved, or even ameliorated, or a desired response is induced e.g., by at least 10% following treatment according to the methods described herein. Efficacy can be assessed, for example, by measuring a marker, indicator, symptom, and / or the incidence of a condition treated according to the methods described herein or any other measurable parameter appropriate. Efficacy can also be measured by a failure of an individual to worsen as assessed by hospitalization, or need for medical interventions (i.e., progression of the disease is halted). Methods of measuring these indicators are known to those of skill in the art and / or are described herein.Treatment includes any treatment of a disease in an individual or an animal (some non-limiting examples include a human or an animal) and includes: (1) inhibiting the disease, e.g., preventing a worsening of symptoms (e.g. tumor growth or inflammation); or (2) relieving the severity of the disease, e.g., causing regression of symptoms. An effective amount for the treatment of a disease means that amount which, when administered to a subject in need thereof, is sufficient to result in37Attorney Docket No: 002806-000156WOPTeffective treatment as that term is defined herein, for that disease. Efficacy of an agent can be determined by assessing physical indicators of a condition or desired response. It is well within the ability of one skilled in the art to monitor efficacy of administration and / or treatment by measuring any one of such parameters, or any combination of parameters. Efficacy can be assessed in animal models of a condition described herein, for example treatment of cancer or autoimmune conditions. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant change in a marker is observed.

[0169] In vitro and animal model assays are provided herein which allow the assessment of a given dose of a composition described herein, e.g., a composition as described herein or a cell comprising a composition as described herein. By way of non-limiting example, the effects of a dose of a composition comprising a functionalized mammalian cell can be assessed by using the models described in the Examples herein.

[0170] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.

[0171] For convenience, certain terms employed herein, in the specification, examples and appended claims are collected here.

[0172] The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments, “reduce,” “reduction" or “decrease" or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g. the absence of a given treatment or agent) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more. As used herein, “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.38Attorney Docket No: 002806-000156WOPT

[0173] The terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount. In some embodiments, die terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5 -fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker or symptom, a “increase” is a statistically significant increase in such level.

[0174] As used herein, a "subject" means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice. rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g.. dog. fox, wolf, avian species, e.g.. chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. In some embodiments, the subject is a mammal, e.g.. a primate, e.g., a human. The terms, “individual,” “patient” and “subject” are used interchangeably herein.

[0175] Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of a disease or condition. A subject can be male or female.

[0176] A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g. cancer) or one or more complications related to such a condition, and optionally, have already undergone treatment for the condition or the one or more complications related to the condition. Alternatively, a subject can also be one who has not been previously diagnosed as having the condition or one or more complications related to the condition. For example, a subject can be one who exhibits one or more risk factors for the condition or one or more complications related to the condition or a subject who does not exhibit risk factors.

[0177] A “subject in need” of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.

[0178] As used herein, the term “cancer” relates generally to a class of diseases or conditions in which abnormal cells divide without control and can invade nearby tissues. Cancer cells can also spread to other parts of the body through the blood and lymph systems. There are several main ty pes of cancer. Carcinoma is a cancer that begins in the skin or in tissues that line or cover internal organs. Sarcoma is a cancer that begins in bone, cartilage, fat, muscle, blood vessels, or other connective or39Attorney Docket No: 002806-000156WOPTsupportive tissue. Leukemia is a cancer that starts in blood-forming tissue such as the bone marrow, and causes large numbers of abnormal blood cells to be produced and enter the blood. Lymphoma and multiple myeloma are cancers that begin in the cells of the immune system. Central nervous system cancers are cancers that begin in the tissues of the brain and spinal cord.

[0179] In some embodiments of any of the aspects, the cancer is a primary cancer. In some embodiments of any of the aspects, the cancer is a malignant cancer. As used herein, the term “malignant” refers to a cancer in which a group of tumor cells display one or more of uncontrolled growth (i.e.. division beyond normal limits), invasion (i.e., intrusion on and destruction of adjacent tissues), and metastasis (i.e., spread to other locations in the body via lymph or blood). As used herein, the term “metastasize” refers to the spread of cancer from one part of the body to another. A tumor formed by cells that have spread is called a “metastatic tumor” or a “metastasis.” The metastatic tumor contains cells that are like those in the original (primary) tumor. As used herein, the term “benign” or “non-malignant” refers to tumors that may grow larger but do not spread to other parts of the body. Benign tumors are self-limited and typically do not invade or metastasize.

[0180] A “cancer cell” or “tumor cell” refers to an individual cell of a cancerous growth or tissue. A tumor refers generally to a swelling or lesion formed by an abnormal growth of cells, which may be benign, pre-malignant, or malignant. Most cancer cells form tumors, but some, e.g., leukemia, do not necessarily form tumors. For those cancer cells that form tumors, the terms cancer (cell) and tumor (cell) are used interchangeably.

[0181] As used herein the term "neoplasm" refers to any new and abnormal growth of tissue, e.g.. an abnormal mass of tissue, the growth of which exceeds and is uncoordinated with that of the normal tissues. Thus, a neoplasm can be a benign neoplasm, premalignant neoplasm, or a malignant neoplasm.

[0182] A subject that has a cancer or a tumor is a subject having objectively measurable cancer cells present in the subject's body. Included in this definition are malignant, actively proliferative cancers, as well as potentially dormant tumors or micrometastatses. Cancers which migrate from their original location and seed other vital organs can eventually lead to the death of the subject through the functional deterioration of the affected organs.

[0183] Examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, leukemia, basal cell carcinoma, biliary tract cancer: bladder cancer; bone cancer; brain and CNS cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma (GBM); hepatic carcinoma; hepatoma; intra-epithelial neoplasm.; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g.. small-cell lung cancer, nonsmall cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); lymphoma40Attorney Docket No: 002806-000156WOPTincluding Hodgkin’s and non-Hodgkin’s lymphoma; melanoma; myeloma; neuroblastoma; oral cavity cancer (e.g.. lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; as well as other carcinomas and sarcomas; as well as B-cell lymphoma (including low grade / follicular non-Hodgkin‘s lymphoma (NHL): small lymphocytic (SL) NHL; intermediate grade / follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom’s Macroglobulinemia); chronic lymphocytic leukemia (CLL): acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs’ syndrome

[0184] A “cancer cell” is a cancerous, pre-cancerous, or transformed cell, either in vivo, ex vivo, or in tissue culture, that has spontaneous or induced phenotypic changes that do not necessarily involve the uptake of new genetic material. Although transformation can arise from infection with a transforming virus and incorporation of new genomic nucleic acid, or uptake of exogenous nucleic acid, it can also arise spontaneously or following exposure to a carcinogen, thereby mutating an endogenous gene. Transformation / cancer is associated with, e.g.. morphological changes, immortalization of cells, aberrant growth control, foci formation, anchorage independence, malignancy, loss of contact inhibition and density limitation of growth, growth factor or serum independence, tumor specific markers, invasiveness or metastasis, and tumor growth in suitable animal hosts such as nude mice.

[0185] As used herein, “inhibitor” refers to an agent which can decrease the expression and / or activity of a target, e.g. by at least 10% or more, e.g. by 10% or more, 50% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 98 % or more. The efficacy of an inhibitor of one or more targets, e.g. its ability to decrease the level and / or activity of the target can be determined, e.g. by measuring the level of an expression product of the target and / or the activity of the target. In some embodiments of any of the aspects, the inhibitor can be an inhibitory nucleic acid; an aptamer; an antibody reagent; an antibody; or a small molecule. An inhibitor of a target described herein can inhibit the activity, expression, or accumulation of the target polypeptide. Inhibitors can include inhibitors that act directly on the target itself (e.g., that bind to the protein or transcript, e.g., direct inhibitors). In some embodiments of any of the aspects, an inhibitor of a specified target is an antibody, antibody reagent, or antigen-binding fragment thereof, that specifically binds to the target.

[0186] As used herein, the terms “protein" and “polypeptide" are used interchangeably herein to designate a series of amino acid residues, connected to each other by peptide bonds between the41Attorney Docket No: 002806-000156WOPTalpha-amino and carboxy groups of adjacent residues. The terms "protein", and "polypeptide" refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. " Protein" and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term "peptide" is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms "protein" and "polypeptide" are used interchangeably herein when referring to a gene product and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing. The terms also refer to fragments or variants of the polypeptide that maintain at least 50% of the activity or effect, of the full length polypeptide.Conservative substitution variants that maintain the activity of wildtype proteins will include a conservative substitution as defined herein. The identification of amino acids most likely to be tolerant of conservative substitution while maintaining at least 50% of the activity of the wildtype is guided by, for example, sequence alignment with homologs or paralogs from other species. Amino acids that are identical between homologs are less likely to tolerate change, while those showing conservative differences are obviously much more likely to tolerate conservative change in the context of an artificial variant. Similarly, positions with non-conservative differences are less likely to be critical to function and more likely to tolerate conservative substitution in an artificial variant. Variants, fragments, and / or fusion proteins can be tested for activity, for example, by administering the variant to an appropriate animal model of a disease as described herein.

[0187] As used herein, the term “nucleic acid” or “nucleic acid sequence” refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analog thereof. The nucleic acid can be either single-stranded or double-stranded. A single-stranded nucleic acid can be one nucleic acid strand of a denatured double- stranded DNA. Alternatively, it can be a single-stranded nucleic acid not derived from any double-stranded DNA. In one aspect, the nucleic acid can be DNA. In another aspect, the nucleic acid can be RNA. Suitable DNA can include, e.g., genomic DNA or cDNA. Suitable RNA can include, e.g., mRNA.

[0188] In some embodiments of any of the aspects, the nucleic acid is an inhibitory nucleic acid. In some embodiments of any of tire aspects, inhibitors of the expression of a given gene can be an inhibitory nucleic acid. As used herein, “inhibitory nucleic acid” refers to a nucleic acid molecule which can inhibit the expression of a target, e.g., double -stranded RNAs (dsRNAs), inhibitory RNAs (iRNAs), and the like. In some embodiments of any of the aspects, the inhibitory nucleic acid can be a silencing RNA (siRNA), microRNA (miRNA), or short hairpin RNA (shRNA).

[0189] Double-stranded RNA molecules (dsRNA) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi). The inhibitory nucleic acids described herein can include an RNA strand (the antisense strand) having a region which42Attorney Docket No: 002806-000156WOPTis 30 nucleotides or less in length, i.e., 15-30 nucleotides in length, generally 19-24 nucleotides in length, which region is substantially complementary to at least part the targeted mRNA transcript. The use of these iRNAs enables the targeted degradation of mRNA transcripts, resulting in decreased expression and / or activity of the target.

[0190] As used herein, the term "iRNA'' refers to an agent that contains RNA (or modified nucleic acids as described below herein) and which mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway. In some embodiments of any of the aspects, an iRNA as described herein effects inhibition of the expression and / or activity of a target. In some embodiments of any of the aspects, contacting a cell with the inhibitor (e.g. an iRNA) results in a decrease in the target mRNA level in a cell by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%. about 60%, about 70%, about 80%. about 90%, about 95%, about 99%, up to and including 100% of the target mRNA level found in the cell without the presence of the iRNA. In some embodiments of any of the aspects, administering an inhibitor (e.g. an iRNA) to a subject results in a decrease in the target mRNA level in the subject by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, up to and including 100% of the target mRNA level found in the subject without the presence of the iRNA.

[0191] In some embodiments of any of the aspects, the iRNA can be a dsRNA. A dsRNA includes two RNA strands that are sufficiently complementary to hybridize to form a duplex structure under conditions in which the dsRNA will be used. One strand of a dsRNA (the antisense strand) includes a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence. The target sequence can be derived from the sequence of an mRNA formed during the expression of the target, e.g., it can span one or more intron boundaries. The other strand (the sense strand) includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. Generally, the duplex structure is between 15 and 30 base pairs in length inclusive, more generally between 18 and 25 base pairs in length inclusive, yet more generally between 19 and 24 base pairs in length inclusive, and most generally between 19 and 21 base pairs in length, inclusive. Similarly, the region of complementarity to the target sequence is between 15 and 30 base pairs in length inclusive, more generally between 18 and 25 base pairs in length inclusive, yet more generally between 19 and 24 base pairs in length inclusive, and most generally between 19 and 21 base pairs in length nucleotides in length, inclusive. In some embodiments of any of the aspects, the dsRNA is between 15 and 20 nucleotides in length, inclusive, and in other embodiments, the dsRNA is between 25 and 30 nucleotides in length, inclusive. As the ordinarily skilled person will recognize, the targeted region of an RNA targeted for cleavage will most often be part of a larger RNA molecule, often an mRNA molecule. Where relevant, a ‘‘part” of an mRNA target is a contiguous sequence of43Attorney Docket No: 002806-000156WOPTan mRNA target of sufficient length to be a substrate for RNAi-directed cleavage (i.e., cleavage through a RISC pathway). dsRNAs having duplexes as short as 9 base pairs can, under some circumstances, mediate RNAi-directed RNA cleavage. Most often a target will be at least 15 nucleotides in length, preferably 15-30 nucleotides in length.

[0192] Exemplary embodiments of types of inhibitory nucleic acids can include, e.g., siRNA, shRNA, miRNA, and / or amiRNA, which are well known in the art. One skilled in the art would be able to design further siRNA, shRNA, or miRNA to target a particular nucleic acid sequence e g., using publically available design tools. siRNA, shRNA, or miRNA is commonly made using companies such as Dharmacon (Layfayette, CO) or Sigma Aldrich (St. Louis. MO).

[0193] In some embodiments of any of the aspects, the RNA of an iRNA, e.g., a dsRNA, is chemically modified to enhance stability or other beneficial characteristics. The nucleic acids described herein may be synthesized and / or modified by methods well established in the art. such as those described in " Current protocols in nucleic acid chemistry,” Beaucage. S. L. et al. (Edrs.). John Wiley & Sons, Inc., New York, NY, USA, which is hereby incorporated herein by reference.Modifications include, for example, (a) end modifications, e.g., 5’ end modifications (phosphorylation, conjugation, inverted linkages, etc.) 3’ end modifications (conjugation. DNA nucleotides, inverted linkages, etc ), (b) base modifications, e g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases, (c) sugar modifications (e.g., at the 2’ position or 4’ position) or replacement of the sugar, as well as (d) backbone modifications, including modification or replacement of the phosphodiester linkages. Specific examples of RNA compounds useful in the embodiments described herein include, but are not limited to RNAs containing modified backbones or no natural intemucleoside linkages. RNAs having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their intemucleoside backbone can also be considered to be oligonucleosides. In some embodiments of any of the aspects, the modified RNA will have a phosphorus atom in its intemucleoside backbone.

[0194] Modified RNA backbones can include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms are also included. Modified RNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl44Attorney Docket No: 002806-000156WOPTinternucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic intemucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones: methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; others having mixed N, O, S and CH2 component parts, and oligonucleosides with heteroatom backbones, and in particular -CH2-NH-CH2-. -CH2-N(CH3)— O-CH2-[known as a methylene (methylimino) or MMI backbone], -CH2-O-N(CH3)-CH2-, -CH2-N(CH3)-N(CH3)--CH2- and -N(CH3)--CH2--CH2-[wherein the native phosphodiester backbone is represented as - O-P-O-CH2-],

[0195] In other RNA mimetics suitable or contemplated for use in iRNAs, both the sugar and the intemucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an RNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an RNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.

[0196] The RNA of an iRNA can also be modified to include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2' and 4' carbons. This structure effectively "locks" the ribose in the 3'-endo structural conformation. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, and to reduce off -target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(l):439-447; Mook, OR. et al., (2007) Mol Cane Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).

[0197] Modified RNAs can also contain one or more substituted sugar moieties. The iRNAs, e.g., dsRNAs, described herein can include one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-. S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Cl to CIO alkyl or C2 to CIO alkenyl and alkynyl.Exemplary suitable modifications include O[(CH2)nO] mCH3. O(CH2).nOCH3, O(CH2)nNH2, O(CH2) nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. In some embodiments of any of the aspects, dsRNAs include one of the following at the 2' position: Cl to CIO lower alkyl, substituted lower alky l, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br. CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a45Attorney Docket No: 002806-000156WOPTreporter group, an intcrcalator, a group for improving the pharmacokinetic properties of an iRNA, or a group for improving the pharmacodynamic properties of an iRNA, and other substituents having similar properties. In some embodiments of any of the aspects, the modification includes a 2' methoxyethoxy (2'-O— CH2CH2OCH3, also known as 2'-O-(2-methoxy ethyl) or 2'-M0E) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy -alkoxy group. Another exemplary modification is 2'-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described in examples herein below, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O— CH2--O— CH2— N(CH2)2, also described in examples herein below.

[0198] Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2) and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of an iRNA, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked dsRNAs and the 5' position of 5' terminal nucleotide. iRNAs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.

[0199] An inhibitory nucleic acid can also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine. 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine. 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine. 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3 -deazaguanine and 3 -de azaadenine. Certain of these nucleobases are particularly useful for increasing the binding affinity of the inhibitory nucleic acids featured in the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5 -methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B.. Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications.

[0200] The preparation of the modified nucleic acids, backbones, and nucleobases described above are well known in the art.46Attorney Docket No: 002806-000156WOPT

[0201] Another modification of an inhibitory nucleic acid featured in the invention involves chemically linking to the inhibitory nucleic acid to one or more ligands, moieties or conjugates that enhance the activity, cellular distribution, pharmacokinetic properties, or cellular uptake of the iRNA. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan etal., Ann. N. Y. Acad. Sci., 1992, 660:306-309: Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.. EMBO J, 1991, 10: 1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium l,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al.. Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res.. 1990.18:3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al.. Tetrahedron Lett., 1995, 36:3651-3654), apahnityl moiety’ (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0202] The term "expression" refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification and processing. Expression can refer to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from a nucleic acid fragment or fragments of the invention and / or to the translation of mRNA into a polypeptide.

[0203] " Expression products" include RNA transcribed from a gene, and polypeptides obtained by translation of mRNA transcribed from a gene. The term "gene" means the nucleic acid sequence which is transcribed (DNA) to RNA in vitro or in vivo when operably linked to appropriate regulatory’ sequences. The gene may or may not include regions preceding and following tire coding region, e.g.5’ untranslated (5‘UTR) or "leader" sequences and 3’ UTR or "trailer" sequences, as well as intervening sequences (introns) between individual coding segments (exons).

[0204] “Operably lurked’’ refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, control elements operably linked to a coding sequence are capable of effecting the expression of the coding sequence. The control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered "operably linked" to the coding sequence.47Attorney Docket No: 002806-000156WOPT

[0205] In some embodiments of any of the aspects, a polypeptide, nucleic acid, or cell as described herein can be engineered. As used herein, “engineered" refers to the aspect of having been manipulated by the hand of man. For example, a polypeptide is considered to be “engineered" when at least one aspect of the polypeptide, e.g.. its sequence, has been manipulated by the hand of man to differ from the aspect as it exists in nature. As is common practice and is understood by those in the art, progeny of an engineered cell are typically still referred to as “engineered" even though the actual manipulation was performed on a prior entity.

[0206] In some embodiments of any of the aspects, the biomolecule and / or active agent described herein is exogenous. In some embodiments of any of the aspects, the biomolecule and / or active agent described herein is ectopic. In some embodiments of any of the aspects, the biomolecule and / or active agent described herein is not endogenous.

[0207] The term "exogenous" refers to a substance present in a cell other than its native source. The term "exogenous" when used herein can refer to a nucleic acid (e.g. a nucleic acid encoding a polypeptide) or a polypeptide that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is not normally found and one wishes to introduce the nucleic acid or polypeptide into such a cell or organism. Alternatively, “exogenous” can refer to a nucleic acid or a polypeptide that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is found in relatively low amounts and one wishes to increase the amount of the nucleic acid or polypeptide in the cell or organism, e.g., to create ectopic expression or levels. In contrast, the term "endogenous" refers to a substance that is native to the biological system or cell. As used herein, “ectopic” refers to a substance that is found in an unusual location and / or amount. An ectopic substance can be one that is normally found in a given cell, but at a much lower amount and / or at a different time. Ectopic also includes substance, such as a polypeptide or nucleic acid that is not naturally found or expressed in a given cell in its natural environment.

[0208] In some embodiments, a nucleic acid as described herein is comprised by a vector. In some of the aspects described herein, a nucleic acid sequence as described herein, or any module thereof, is operably linked to a vector. The term "vector", as used herein, refers to a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, a vector can be viral or non-viral. The term “vector” encompasses any genetic element that is capable of replication when associated with the proper control elements and that can transfer gene sequences to cells. A vector can include, but is not limited to, a cloning vector, an expression vector, a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc.

[0209] In some embodiments of any of the aspects, the vector is recombinant, e.g., it comprises sequences originating from at least tw o different sources. In some embodiments of any of the aspects, the vector comprises sequences originating from at least two different species. In some embodiments48Attorney Docket No: 002806-000156WOPTof any of the aspects, the vector comprises sequences originating from at least two different genes, e.g., it comprises a fusion protein or a nucleic acid encoding an expression product which is operably linked to at least one non-native (e.g., heterologous) genetic control element (e.g., a promoter, suppressor, activator, enhancer, response element, or the like).

[0210] In some embodiments of any of the aspects, the vector or nucleic acid described herein is codon-optimized, e.g., the native or wild-type sequence of the nucleic acid sequence has been altered or engineered to include alternative codons such that altered or engineered nucleic acid encodes the same polypeptide expression product as the native / wild-type sequence, but will be transcribed and / or translated at an improved efficiency in a desired expression system. In some embodiments of any of the aspects, the expression system is an organism other than the source of the native / wild-type sequence (or a cell obtained from such organism). In some embodiments of any of the aspects, the vector and / or nucleic acid sequence described herein is codon-optimized for expression in a mammal or mammalian cell, e.g., a mouse, a murine cell, or a human cell. In some embodiments of any of the aspects, the vector and / or nucleic acid sequence described herein is codon-optimized for expression in a human cell. In some embodiments of any of the aspects, the vector and / or nucleic acid sequence described herein is codon-optimized for expression in a yeast or yeast cell. In some embodiments of any of the aspects, the vector and / or nucleic acid sequence described herein is codon-optimized for expression in a bacterial cell. In some embodiments of any of the aspects, the vector and / or nucleic acid sequence described herein is codon-optimized for expression in an E. coli cell.

[0211] As used herein, the term "expression vector" refers to a vector that directs expression of an RNA or polypeptide from sequences linked to transcriptional regulatory sequences on the vector. The sequences expressed will often, but not necessarily, be heterologous to the cell. An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in human cells for expression and in a prokaryotic host for cloning and amplification.

[0212] As used herein, the term “viral vector" refers to a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral vector particle. The viral vector can contain the nucleic acid encoding a polypeptide as described herein in place of non-essential viral genes. The vector and / or particle may be utilized for the purpose of transferring any nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art.

[0213] Viral vector systems which can be utilized in the present invention include, but are not limited to. (a) adenovirus vectors; (b) retrovirus vectors, e.g.. lentivirus vectors, murine moloney leukemia virus, etc.; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (!) polyoma virus vectors; (g) papilloma virus vectors; (h) picomavirus vectors; (i) pox virus vectors such as an orthopox, e.g.. vaccinia virus vectors or avipox, e.g.. canary pox or fowl pox; and49Attorney Docket No: 002806-000156WOPT(j) a helper-dependent or gutless adenovirus. Replication-defective viruses can also be advantageous. In some embodiments, the vector is an adeno-associated virus vector.

[0214] In some embodiments, a viral vector such as an adeno-associated virus (AAV) vector is used. AAVs, which normally infect mammals, including humans, but are non-pathogenic, have been developed and employed as gene therapy vectors in clinical trials in the United States and Europe (Daya and Berns, Clinical Microbiology Reviews 2008, 21, 583-593). AAV vectors may be prepared using any one of a number of methods available to those of ordinary skill in the art. Exemplary AAV vectors are disclosed in Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U. S. Pat. No.5.436,146 which is incorporated herein by reference; Gao et al., Gene Therapy 2005, 5, 285-297: Vandenberghe et al.. Gene Therapy 2009. 16, 311-319; Gao et al., PNAS 2002, 99, 11854-11859; Gao et al., PNAS 2003. 100, 6081-6086; Gao et al., J. of Virology 2004, 78. 6381-6388.

[0215] In some embodiments, the vector is an adeno-associated virus (AAV) vector. In some embodiments, the AAV vector is an AAV1, AAV2, AAV4, AAV5, AAV6. AAV7, AAV8, AAV9, AAV9. HR, AAVrh.10, AAVMYO, or AAV2.5. In some embodiments, the AAV is AAV9. In some embodiments, the AAV is AAV9 or AAV6. In some embodiments, the AAV is AAV6.

[0216] It should be understood that the vectors described herein can, in some embodiments, be combined with other suitable compositions and therapies. In some embodiments, the vector is episomal. The use of a suitable episomal vector provides a means of maintaining the nucleotide of interest in the subject in high copy number extra chromosomal DNA thereby eliminating potential effects of chromosomal integration.

[0217] As non-limiting examples, in some embodiments, a plasmid expression vector can be used. Plasmid expression vectors include, but are not limited to, pcDNA3.1, pET vectors (Novagen®), pGEX vectors (GE Life Sciences), and pMAL vectors (New England labs. Inc.) for protein expression in E. coli host cell such as BL21, BL21(DE3) and AD494(DE3)pLysS, Rosetta (DE3), and Origami(DE3) (Novagen®); the strong CMV promoter-based pcDNA3.1 (Invitrogen™ Inc.) and pCIneo vectors (Promega) for expression in mammalian cell lines such as CHO, COS, HEK-293, Jurkat, and MCF-7; replication incompetent adenoviral vector vectors pAdeno X, pAd5F35, pLP-Adeno-X-CMV (Clontech®), pAd / CMV / V5-DEST, pAd-DEST vector (Invitrogen™ Inc.) for adenovirus-mediated gene transfer and expression in mammalian cells; pLNCX2, pLXSN, and pLAPSN retrovirus vectors for use with the Retro-X ™ system from Clontech for retro viral -mediated gene transfer and expression in mammalian cells; pLenti4 / V5-DEST™, pLenti6 / V5-DEST™, and pLenti6.2 / V5-GW / lacZ (INVITROGEN™ Inc.) for lentivirus-mediated gene transfer and expression in mammalian cells; adenovirus-associated virus expression vectors such as pAAV-MCS, pAAV-IRES-hrGFP, and pAAV-RC vector (Stratagene®) for adeno-associated virus-mediated gene transfer and expression in mammalian cells.50Attorney Docket No: 002806-000156WOPT

[0218] A retroviral vector can also be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA. In another embodiment, the vector is a pox virus such as a vaccinia virus, for example an attenuated vaccinia such as Modified Virus Ankara (MV A) or NYVAC, an avipox such as fowl pox or canary pox. In another embodiment, lentiviral vectors are used, such as the HIV based vectors described in U. S. Patent Nos. 6,143,520; 5,665,557; and 5,981,276, which are herein incorporated by reference. The vector may or may not be incorporated into the genome of a cell. The constructs may include viral sequences for transfection, if desired. Alternatively, the vector can be capable of episomal replication, e.g., EPV and EBV vectors.

[0219] As used herein, “viral particle” refers to a particle comprising at least one viral capsid polypeptide and a nucleic acid molecule, e.g., a viral genome and / or viral vector. Viral vectors are discussed elsewhere herein.

[0220] As used herein, “antiviral” refers to any chemical or biological agent with therapeutic usefulness in the inhibition of viral transmission, activity, or replication. Categories of antivirals can include, but are not limited to entry inhibitors, uncoating inhibitors, viral synthesis inhbitiors, assembly inhibitors, and release inhibitors. Exemplary, non-limiting antivirals include enfuvirtide, amantadine, rimantadine, pleconaril, acyclovir, zidovudine, lamivudine. fomivirsen, rifampicin, zanamivir. oseltamivir. peramivir. abacavir, acyclovir, adefovir, amprenavir, baloxavir marboxil, boceprevir, cobicistat, combivir, daclatasvir. doravirine, etravirine, ganciclovir, ibalizumab, letermovir, rilpivirine, simeprevir, telbivudine. and valciclovir. One of skill in the art can readily identify an antiviral agent of use e.g. see Antiviral Drugs, Wieslaw M. Kazmierski (ed.) Wiley and Sons (2011); Antiviral Drugs, John S. Driscoll. Wiley and Sons (2005); each of which is incorporated by reference herein in its entirety.

[0221] As used herein, “antibiotic” refers to any chemical or biological agent with therapeutic usefulness in the inhibition of bacterial cell growth or in killing bacteria, e.g, those that are bactericidal or bacteriostatic. Categories of antibiotics can include, but are not limited to those that target the bacterial cell wall (e.g., penicillins, cephalosporins), those that target the bacterial cell membrane (e.g., polymyxins), those that target bacterial enzymes (e.g., rifamycins, lipiarmycins, quinolones, sulfonamides), protein synthesis inhibitors (e.g., macrolides, lincosamides, and tetracyclines), aminoglycosides, cyclic lipopeptides, glycyclines, oxazolidinones, beta-lactams, and lipiarmycins. Exemplary, non-limiting antibiotics include penicillin, methicilling. nafcillin, oxacillin, cloxacillin, dicloxacillin, flucioxacillin, ampicillin, amoxicillin, pivampicillin, hetacillin, bacampicillin, metampicillin. talamipicillin. epicillin, cabenicillin, ticaricillin, temocillin, mezlocillin, piperacillin, azolocillin, clavulanic acid, sulbactam, tazobactam, cafadroxil, cephalexin, cefalotin, cefapirin, cefazolin, cefradine, cefaclor, cefonicid. cefprozil, cefuroxime, loracarbef, cefmetazole, cefotetan, cefoxitin, cefotiam, cefdinir, cefixime, cefotaxime, cefovecin, cefpodoxime, ceftibuten,51Attorney Docket No: 002806-000156WOPTceftiofur, ceftizoxime, ceftriaxone, cefoperazone, ceftazimdime, latamoxef, cefepime, cefiderocol, cefpriome, rifampicin, rifabutin, rifapentine, rifamixin, fidaxomicin, ciproflaxicin, moxifloxacin, levofloxacin, sulfafurzole, azithromycin, clarithromycin, erythromycin, fidaxomicin, spiramycin, telihtromycin, lincomycin, clindamycin, pirlimycin, tetracycline, eravacycline, sarecycline, omadacy cline, doxycycline, kanamycin, tobramycin, gentamicin, neomycin, streptomycin, vancomycin, tigecycline. linezolid, posizolid, tedizolid, radezolid, cycloserine, contezolid, and daptomycin. One of skill in the art can readily identify an antibiotic agent of use e.g. see Antibiotics in Laboratory Medicine, Victor Lorian (ed.) Wolters Kluwer; and Antibotics Manual, David Schlossberg and Rafik Samuel. John Wiley and Sons (2017); each of which is incorporated by reference herein in its entirety'.

[0222] As used herein, the term “antibody reagent" refers to a polypeptide that includes at least one immunoglobulin variable domain or immunoglobulin variable domain sequence and which specifically binds a given antigen. An antibody reagent can comprise an antibody or a polypeptide comprising an antigen-binding domain of an antibody. In some embodiments of any of the aspects, an antibody reagent can comprise a monoclonal antibody or a polypeptide comprising an antigenbinding domain of a monoclonal antibody. For example, an antibody can include a heavy (H) chain variable region (abbreviated herein as VH), and a light (L) chain variable region (abbreviated herein as VL). In another example, an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions. The term "antibody reagent" encompasses antigen-binding fragments of antibodies (e.g., single chain antibodies. Fab and sFab fragments, F(ab')2, Fd fragments, Fv fragments. scFv, and domain antibodies (dAb) fragments as well as complete antibodies.

[0223] As used herein, the term “antibody” refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. The term also refers to antibodies comprised of two immunoglobulin heavy chains and two immunoglobulin light chains as well as a variety of forms including full length antibodies and antigen-binding portions thereof; including, for example, an immunoglobulin molecule, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a humanized antibody, a Fab, a Fab', a F(ab')2, a Fv, a disulfide linked Fv, a scFv, a single domain antibody (dAb), a diabody, a multispecific antibody, a dual specific antibody, an anti-idiotypic antibody, a bispecific antibody, a functionally active epitope -binding portion thereof, and / or bifunctional hybrid antibodies. Each heavy chain is composed of a variable region of said heavy chain (abbreviated here as HCVR or VH) and a constant region of said heavy chain. The heavy chain constant region consists of three domains CHI, CH2 and CH3. Each light chain is composed of a variable region of said light chain (abbreviated here as LCVR or VL) and a constant region of said light chain. The light chain constant region consists of a CL domain. The VH and VL regions may be further divided into hypervariable regions referred to as complementarity-determining regions (CDRs)52Attorney Docket No: 002806-000156WOPTand interspersed with conserved regions referred to as framework regions (FR). Each VH and VL region thus consists of three CDRs and four FRs which are arranged from the N terminus to the C terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. This structure is well known to those skilled in the art.

[0224] Antibodies and / or antibody reagents can include an immunoglobulin molecule, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a humanized antibody, a fully human antibody, a Fab, a Fab', a F(ab')2, a Fv, a disulfide linked Fv, a scFv, a single domain antibody, a diabody, a multispecific antibody, a dual specific antibody, an anti-idiotypic antibody, a bispecific antibody, and a functionally active epitope-binding portion thereof.

[0225] As used herein, the term “nanobody” or single domain antibody (sdAb) refers to an antibody comprising the small single variable domain (VHH) of antibodies obtained from camelids and dromedaries. Antibody proteins obtained from members of the camel and dromedary (Camelus bactrianus and Calelus dromaderius) family including new world members such as llama species (Lama paccos, Lama glama and Lama vicugna) have been characterized with respect to size, structural complexity and antigenicity for human subjects. Certain IgG antibodies from this family of mammals as found in nature lack light chains, and are thus structurally distinct from the typical four chain quaternary structure having two heavy and two light chains, for antibodies from other animals. See PCT / EP93 / 02214 (WO 94 / 04678 published 3 Mar. 1994; which is incorporated by reference herein in its entirety).

[0226] A region of the camelid antibody which is the small single variable domain identified as VHH can be obtained by genetic engineering to yield a small protein having high afiinity for a target, resulting in a low molecular weight antibody -derived protein known as a “camelid nanobody”. See U. S. Pat. No. 5,759,808 issued Jun. 2, 1998; see also Stijlemans, B. et al., 2004 J Biol Chem 279: 1256-1261; Dumoulin, M. et al., 2003 Nature 424: 783-788; Pleschberger, M. et al. 2003 Bioconjugate Chem 14: 440-448; Cortez-Retamozo, V. et al. 2002 Int J Cancer 89: 456-62; and Lauwereys, M. et al. 1998 EMBO J. 17: 3512-3520; each of which is incorporated by reference herein in its entirety. Engineered libraries of camelid antibodies and antibody fragments are commercially available, for example, from Ablynx, Ghent, Belgium. As with other antibodies of non-human origin, an amino acid sequence of a camelid antibody can be altered recombinantly to obtain a sequence that more closely resembles a human sequence, i.e., the nanobody can be “humanized”. Thus the natural low antigenicity of camelid antibodies to humans can be further reduced.

[0227] The camelid nanobody has a molecular weight approximately one-tenth that of a human IgG molecule and the protein has a physical diameter of only a few nanometers. One consequence of the small size is the ability of camelid nanobodies to bind to antigenic sites that are functionally invisible to larger antibody proteins, i.e., camelid nanobodies are useful as reagents detect antigens that are otherwise cryptic using classical immunological techniques, and as possible therapeutic53Attorney Docket No: 002806-000156WOPTagents. Thus yet another consequence of small size is that a camelid nanobody can inhibit as a result of binding to a specific site in a groove or narrow cleft of a target protein, and hence can serve in a capacity that more closely resembles the function of a classical low molecular weight drug than that of a classical antibody. The low molecular weight and compact size further resultin camelid nanobodies being extremely thermostable, stable to extreme pH and to proteolytic digestion, and poorly antigenic. See U. S. patent application 20040161738 published Aug. 19, 2004; which is incorporated by reference herein in its entirety. These features combined with the low antigenicity to humans indicate great therapeutic potential.

[0228] Immune checkpoint inhibitors inhibit one or more immune checkpoint proteins. The immune system has multiple inhibitory pathways that are critical for maintaining self-tolerance and modulating immune responses. For example, in T-cells, the amplitude and quality of response is initiated through antigen recognition by the T-cell receptor and is regulated by immune checkpoint proteins that balance co-stimulatory and inhibitory signals. In some embodiments of any of the aspects, a subject or patient is treated with at least one inhibitor of an immune checkpoint protein. As used herein, "‘immune checkpoint protein” refers to a protein which, when active, exhibits an inhibitory effect on immune activity, e.g., T cell activity. Exemplary immune checkpoint proteins can include PD-1 (e.g.. NCBI Gene ID: 5133); PD-L1 (e.g., NCBI Gene ID: 29126); PD-L2 (e.g., NCBI Gene ID: 80380); TIM-3 (e.g., NCBI Gene ID: 84868); CTLA4 (e.g., NCBI Gene ID: 1493); TIGIT (e.g., NCBI Gene ID: 201633); KIR (e.g., NCBI Gene ID: 3811); LAG3 (e.g., NCBI Gene ID: 3902); DD1-α (e.g., NCBI Gene ID: 64115); A2AR (e.g., NCBI Gene ID: 135); B7-H3 (e.g., NCBI Gene ID: 80381); B7-H4 (e.g., NCBI Gene ID: 79679); BTLA (e.g., NCBI Gene ID: 151888); IDO (e.g., NCBI Gene ID: 3620); TDO (e.g., NCBI Gene ID: 6999); HVEM (e.g., NCBI Gene ID: 8764); GAL9 (e.g., NCBI Gene ID: 3965); 2B4 (belongs to the CD2 family of molecules and is expressed on all NK, y5, and memoryCD8+ (a|3) T cells) (e.g., NCBI Gene ID: 51744); CD160 (also referred to as BY55) (e.g., NCBI Gene ID: 11126); and various B-7 family ligands. B7 family ligands include, but are not limited to, B7- 1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6 and B7-H7.

[0229] Non-limiting examples of immune checkpoint inhibitors (with checkpoint targets and manufacturers noted in parantheses) can include:MGA271 (B7-H3: MacroGenics); ipilimumab (CTLA-4; Bristol Meyers Squibb); pembrolizumab (PD-1; Merck); nivolumab (PD-1; Bristol Meyers Squibb); atezolizumab (PD-L1; Genentech); galiximab (B7.1; Biogen); IMP321 (LAG3: Immuntep); BMS-986016 (LAG3; Bristol Meyers Squibb); SMB-663513 (CD137; Bristol-Meyers Squibb); PF-05082566 (CD137; Pfizer); IPH2101 (KIR; Innate Pharma); KW-0761 (CCR4; Kyowa Kirin); CDX-1127 (CD27; CellDex); MEDI-6769 (0x40: Medimmune); CP-870,893 (CD40; Genentech); tremelimumab (CTLA-4; Medimmune); pidilizumab (PD-1; Medivation); MPDL3280A (PD-L1; Roche); MEDI4736 (PD-L1; AstraZeneca); MSB0010718C (PD-L1; EMD Serono); AUNP12 (PD-1; Aurigene); avelumab (PD-L1; Merck); durvalumab (PD-L1; Medimmune); IMP321, a soluble Ig54Attorney Docket No: 002806-000156WOPTfusion protein (Brignone et al., 2007, J. Immunol. 179:4202-4211); the anti-B7-H3 antibody MGA271 (Loo et al., 2012, Clin. Cancer Res. July 15 (18) 3834); TIM3 (T-cell immunoglobulin domain and mucin domain 3) inhibitors (Fourcade et al., 2010, J. Exp. Med. 207:2175-86 and Sakuishi et al., 2010, J. Exp. Med. 207:2187-94); anti-CTLA-4 antibodies described in US Patent Nos: 5,811,097; 5.811,097; 5,855,887; 6,051,227; 6,207,157; 6,682,736; 6,984,720; and 7,605,238; tremelimumab, (ticilimumab, CP-675,206); ipilimumab (also known as 10D1, MDX-D010); PD-1 and PD-L1 blockers described in US Patent Nos. 7,488,802; 7,943,743; 8,008,449; 8,168,757; 8,217,149, and PCT Published Patent Application Nos: W003042402, WO2008156712, W02010089411, W02010036959, WO2011066342, WO2011159877, WO2011082400, and WO2011161699; nivolumab (MDX 1106, BMS 936558, ONO 4538); lambrolizumab (MK-3475 or SCH 900475): CT-011; AMP-224; and BMS-936559 (MDX- 1105-01). The foregoing references are incorporated by reference herein in their entireties.

[0230] In some embodiments of any of the aspects, the antibody or antibody reagent is selected from the group consisting of: MGA271; ipilimumab; pembrolizumab; nivolumab; atezolizumab; galiximab; IMP321; BMS-986016; SMB-663513; PF-05082566; IPH2101; KW-0761; CDX-1127; MEDI-6769; CP-870,893; tremelimumab; pidilizumab; MPDL3280A; MEDI4736; MSB0010718C; AUNP12; avelumab; durvalumab; IMP321; MGA271; tremelimumab; ipilimumab; nivolumab; lambrolizumab; CT-011; AMP-224; BMS-936559; cemiplimab; dostarlimab; retifanlimab; toripalimab; tislelizumab; vopratelimab; spartalizumab; camrelizumab; sintilimab; MGA012; AMP-224; AMP -514; acrixolimba; sansalimab;

[0231] In some embodiments of any of the aspects, the antibody or antibody reagent is an anti-PD-1 antibody or antibody reagent selected from the group consisting of: pembrolizumab; nivolumab; cemiplimab; dostarlimab; retifanlimab; toripalimab; tislelizumab; vopratelimab; spartalizumab; camrelizumab; sintilimab; MGA012; AMP -224; AMP-514; acrixolimba; sansalimab; and pidilizumab.

[0232] In some embodiments of any of the aspects, the antibody or antibody reagent is an anti-PD-L1 antibody or antibody reagent selected from the group consisting of: atezolizumab;MPDL3280A; MEDI4736; MSB0010718C; avelumab; durvalumab; cosibelimab; and KN035.

[0233] In some embodiments of any of the aspects, the antibody or antibody reagent is an anti-VEGF antibody or antibody reagent selected from the group consisting of: bevacizumab; ranibizumab; ramucirumab; and brolucizumab.

[0234] In some embodiments of any of the aspects, the active agent and / or biomolecule is a PD-1 inhibitor selected from: pembrolizumab; nivolumab: cemiplimab; dostarlimab; retifanlimab; toripalimab; tislelizumab; vopratelimab; spartalizumab; camrelizumab; sintilimab; MGA012; AMP-224; AMP -514; acrixolimba; sansalimab; and pidilizumab.55Attorney Docket No: 002806-000156WOPT

[0235] In some embodiments of any of the aspects, the active agent and / or biomolecule is a PD-L1 inhibitor selected from: atezolizumab; MPDL3280A; MEDI4736; MSB0010718C; avelumab; durvalumab; cosibelimab; KN035; AUNP12; CA-170; and BMS-986189.

[0236] In some embodiments of any of the aspects, the active agent and / or biomolecule is a VEGF inhibitor selected from: bevacizumab; ranibizumab: ramucirumab; brolucizumab; sunitinib; sorafenib; axitinib; pazopanib; vandetanib; regorafenib; ponatinib; Lenvatinib; lapatinib; and cabozantinib.

[0237] In some embodiments of any of the aspects, the one or more biomolecules and one or more active agents comprise: i) an immune checkpoint inhibitor; and 2) a VEGF inhibitor. In some embodiments of any of the aspects, the immune checkpoint inhibitor is an antibody or antibody reagent that specifically binds to an immune checkpoint protein (e.g.. an anti-PD-1 or anti-PDLl antibody or antibody reagent). In some embodiments of any of the aspects, the VEGF inhibitor is an antibody or antibody reagent that specifically binds to VEGF (i.e., an anti-VEGF antibody or antibody reagent). In some embodiments of any of the aspects, the immune checkpoint protein is PD-1. In some embodiments of any of the aspects, the immune checkpoint protein is PD-L1. In some embodiments of any of the aspects, the i) an immune checkpoint inhibitor; and 2) a VEGF inhibitor are provided as a bispecific antibody or bispecific antibody reagent.

[0238] In some embodiments of any of the aspects, the one or more biomolecules and one or more active agents comprise: i) an IL-4 inhibitor and ii) IL- 13 inhibitor. In some embodiments of any of the aspects, the one or more biomolecules and one or more active agents comprise: i) an IL-4 inhibitor and / or ii) IL-13 inhibitor. In some embodiments of any of the aspects, the one or more biomolecules and one or more active agents comprise: i) an IL-4R inhibitor and ii) IL-13R inhibitor. In some embodiments of any of the aspects, the one or more biomolecules and one or more active agents comprise: i) an IL-4R inhibitor and / or ii) IL-13R inhibitor. In some embodiments of any of the aspects, the inhibitor is an antibody or antibody reagent that specifically binds to the target (e.g., an anti-IL-4R or anti-IL-13R antibody or antibody reagent). In some embodiments of any of the aspects, the antibody or antibody reagent that specifically binds to IL-4R and / or IL-13R is duplimab.

[0239] In some embodiments of any of the aspects, the antibody or antibody reagent is an antibody or antibody reagent for the treatment of pulmonary disease(s). In some embodiments of any of the aspects, the antibody or antibody reagent binds specifically to IL17A, IL17, IL-5, IL-13, EGFR, CD3, and / or CD19. In some embodiments of any of the aspects, the antibody or antibody reagent is obiltoxaximab, ixekizumab, reslizumab, benralizumab, lebrikizumab. tralokinumab, palivizumab, panobacumab. necitumumab. cetuximab, panitumumab, SAR156597, blinatumomab, emicizumab, or omalizumab. See also, Desoubeaux et al. MAbs. 2016 Jun 6;8(6):999-1009. doi:10.1080 / 19420862.2016.1196521; which is incorporated by reference herein in its entirety.56Attorney Docket No: 002806-000156WOPT

[0240] In some embodiments of any of the aspects, the biomolecule and / or active agent can be a therapeutic compound or drug, e.g., an agent or compound which is therapeutically effective for the treatment of at least one condition in a subject. Therapeutic compounds are known in the art for a variety of conditions, see, e.g., the database available on the world wide web at drugs.com or the catalog of FDA-approved compounds available on the world wide web at catalog.data.gov / dataset / drugsfda-database; each of which is incorporated by reference herein in its entirety.

[0241] As used herein the term “chemotherapeutic agent" refers to any chemical or biological agent with therapeutic usefulness in the treatment of diseases characterized by abnormal cell growth. Such diseases include tumors, neoplasms and cancer as well as diseases characterized by hyperplastic growth. These agents can function to inhibit a cellular activity upon which the cancer cell depends for continued proliferation. In some aspect of all the embodiments, a chemotherapeutic agent is a cell cycle inhibitor or a cell division inhibitor. Categories of chemotherapeutic agents that are useful in the methods of the invention include alkylating / alkaloid agents, antimetabolites, hormones or hormone analogs, and miscellaneous antineoplastic drugs. Most of these agents are directly or indirectly toxic to cancer cells. In one embodiment, a chemotherapeutic agent is a radioactive molecule. One of skill in the art can readily identify a chemotherapeutic agent of use (e.g. see Physicians' Cancer Chemotherapy Drug Manual 2014, Edward Chu, Vincent T. DeVita Jr.. Jones & Bartlett Learning; Principles of Cancer Therapy, Chapter 85 in Harrison's Principles of Internal Medicine, 18th edition; Therapeutic Targeting of Cancer Cells: Era of Molecularly Targeted Agents and Cancer Pharmacology, Chs. 28-29 in AbelofT s Clinical Oncology, 2013 Elsevier; and Fischer D S (ed): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 2003).

[0242] Exemplary chemotherapeutics include an anthracycline (e.g., doxorubicin (e.g., liposomal doxorubicin)), a vinca alkaloid (e.g., vinblastine, vincristine, vindesine, vinorelbine), an alkylating agent (e.g., cyclophosphamide, decarbazine, melphalan, ifosfamide, temozolomide), an antibody (e.g., alemtuzamab, bevacizumab (Avastin®), gemtuzumab, nivolumab (Opdivo®), pembrolizumab (Keytruda®), rituximab (Rituxan®), traztuzumab (Herceptin®) tositumomab), an antimetabolite (including, e.g., folic acid antagonists, pyrimidine analogs, purine analogs and adenosine deaminase inhibitors (e.g., fludarabine)), an mTOR inhibitor, a TNFR glucocorticoid induced TNFR related protein (GITR) agonist, a proteasome inhibitor (e.g., aclacinomycin A, gliotoxin or bortezomib), an immunomodulator such as thalidomide or a thalidomide derivative (e.g., lenalidomide (Revlimid®)), a kinase inhibitor (e.g.. palbociclib (Ibrance®), or a hormone therapy (e.g., abiraterone acetate (Zytiga®)). General chemotherapeutic agents include anastrozole (Arimidex®). bicalutamide (Casodex®), bleomycin sulfate (Blenoxane®), busulfan (Myleran®). busulfan injection (Busulfex®), capecitabine (Xeloda®), N4-pentoxycarbonyl-5- deoxy-5-fluorocytidine, carboplatin (Paraplatin®), carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Platinol®), cladribine (Leustatin®),57Attorney Docket No: 002806-000156WOPTcyclophosphamide (Cytoxan® or Neosar®), cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposome injection (DepoCyt®), dacarbazine (DTIC-Dome®), dactinomycin (Actinomycin D, Cosmegan), daunorubicin hydrochloride (Cerubidine®), daunorubicin citrate liposome injection (DaunoXome®), dexamethasone, docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin®, Rubex®), etoposide (Vepesid®, Etopophos®, Toposar®), fludarabine phosphate (Fludara®), 5-fluorouracil (Adrucil®, Efudex®), flutamide (Eulexin®), tezacitibine, gemcitabine (difluorodeoxycitidine), hydroxyurea (Hydrea®), ibrutinib (Imbruvica®). Idarubicin (Idamycin®), ifosfamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (ELSPAR®), leucovorin calcium, melphalan (Alkeran®), 6-mercaptopurine (Purinethol®). methotrexate (Folex®), mitoxantrone (Novantrone®), mylotarg, paclitaxel (Taxol®), phoenix (Yttrium90 / MX-DTPA), pentostatin, polifeprosan 20 with carmustine implant (Gliadel®), tamoxifen citrate (Nolvadex®), teniposide (Vumon®), 6-thioguanine. thiotepa, tirapazamine (Tirazone®), topotecan hydrochloride for injection (Hy camptin®), vinblastine (Velban®), vincristine (Oncovin®), and vinorelbine (Navelbine®). Exemplary alkylating agents include, without limitation, nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas and triazenes): uracil mustard (Aminouracil Mustard®, Chlorethaminacil®, Demethyldopan®, Desmethyldopan®, Haemanthamine®. Nordopan®, Uracil nitrogen mustard®. Uracillost®, Uracilmostaza®, Uramustin®. Uramustine®), chlormethine (Mustargen®). cyclophosphamide (Cytoxan®. Neosar®, Clafen®, Endoxan®, Procytox®.Revimmune™), ifosfamide (Mitoxana®), melphalan (Alkeran®). Chlorambucil (Leukeran®), pipobroman (Amedel®, Vercyte®), triethylenemelamine (Kernel®. Hexalen®, Hexastat®), triethylenethiophosphoramine, Temozolomide (Temodar®), thiotepa (Thioplex®, Tepadina®). busulfan (Busilvex®, Myleran®). improsulfan, piposulfan, carmustine (BiCNU®), lomustine (CeeNU®), streptozocin (Zanosar®), and Dacarbazine (DTIC-Dome®). Additional exemplary alkylating agents include, without limitation, Oxaliplatin (Eloxatin®); Temozolomide (Temodar® and Temodal®); Dactinomycin (also known as actinomycin-D, Cosmegen®); Melphalan (also known as L-PAM, L-sarcolysin, and phenylalanine mustard, Alkeran®); Altretamine (also known as hexamethylmelamine (HMM), Hexalen®); Carmustine (BiCNU®); Bendamustine (Treanda®); Busulfan (Busulfex® and Myleran®); carboplatin (Paraplatin®); Lomustine (also known as CCNU, CeeNU®); Cisplatin (also known as CDDP. Platinol® and Platinol®-AQ); Chlorambucil (Leukeran®); Cyclophosphamide (Cytoxan® and Neosar®); Dacarbazine (also known as DTIC, DIC and imidazole carboxamide, DTIC-Dome®); Altretamine (also known as hexamethylmelamine (HMM), Hexalen®): Ifosfamide (Ifex®); Prednumustine; Procarbazine (Matulane®);Mechlorethamine (also known as nitrogen mustard, mustine and mechloroethamine hydrochloride, Mustargen®); Streptozocin (Zanosar®); Thiotepa (also known as thiophosphoamide, TESPA and TSP A, Thioplex®); Cyclophosphamide (Endoxan®. Cytoxan®, Neosar®, Procytox®, Revimmune®); and Bendamustine HC1 (Treanda®). Exemplary mTOR inhibitors include, e.g.,58Attorney Docket No: 002806-000156WOPTtemsirolimus; ridaforolimus (formally known as deferolimus, (1R,2R,4S)-4-[(2R)-2 [(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28Z,30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35- hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.04,9] hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyl dimethylphosphinate, also known as AP23573 and MK8669, and described in PCT Publication No. WO 03 / 064383); everolimus (Afinitor® or RAD001); rapamycin (AY22989, Sirolimus®); simapimod (CAS 164301-51-3); emsirolimus. (5-{2,4-Bis[(3S,)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl}-2-methoxyphenyl)methanol (AZD8055); 2-Amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one (PF04691502, CAS 1013101-36-4); and N2-[1,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)morpholinium-4-yl]methoxy]butyl]-L-arginylglycyl-L-α-aspartylL-serine- (SEQ ID NO: 62), inner salt (SF1126, CAS 936487-67-1), and XL765. Exemplary immunomodulators include, e.g., afutuzumab (available from Roche®); pegfilgrastim (Neulasta®); lenalidomide (CC-5013.Revlimid®); thalidomide (Thalomid®), actimid (CC4047); and IRX-2 (mixture of human cytokines including interleukin 1, interleukin 2, and interferon y, CAS 951209-71-5, available from IRX Therapeutics). Exemplary anthracyclines include, e.g., doxorubicin (Adriamycin® and Rubex®); bleomycin (lenoxane®); daunorubicin (dauorubicin hydrochloride, daunomycin. and rubidomycin hydrochloride, Cerubidine®); daunorubicin liposomal (daunorubicin citrate liposome, DaunoXome®); mitoxantrone (DHAD, Novantrone®); epirubicin (Ellence™); idarubicin (Idamycin®. Idamycin PFS®); mitomycin C (Mutamycin®); geldanamycin; herbimycin; ravidomycin; and desacetylravidomycin. Exemplary’ vinca alkaloids include, e.g., vinorelbine tartrate (Navelbine®), Vincristine (Oncovin®), and Vindesine (Eldisine®)); vinblastine (also known as vinblastine sulfate, vincaleukoblastine and VLB, Alkaban-AQ® and Velban®); and vinorelbine (Navelbine®). Exemplary’ proteosome inhibitors include bortezomib (Velcade®); carfilzomib (PX-171-007, (S)-4-Methyl-N-((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S,)-2-(2-morpholinoacetamido)-4-phenylbutanamido)-pentanamide); marizomib (NPT0052); ixazomib citrate (MLN-9708); delanzomib (CEP-18770); and O-Methyl-N-[(2-methyl-5-thiazolyl)carbonyl]-L-seryl-O- methyl-N-[(1lS')-2-[(2R)-2-methyl-2-oxiranyl]-2-oxo-1-(phenylmethyl)ethyl]-L-serinamide (ONX-0912). Additional exemplary anti-cancer agents also include AMG479, vorinostat, ABT-737, PI-103; aziridines such as benzodopa, carboquone, meturedopa. and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine: acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-59Attorney Docket No: 002806-000156WOPTTM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phene sterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g, calicheamicin, especially calicheamicin gammall and calicheamicin omegall (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin. 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholinodoxorubicin. cyanomorpholino-doxorubicin. 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin. esorubicin, idarubicin. marcellomycin, mitomycins such as mitomycin C. mycophenolic acid, nogalamycin. olivomycins, peplomycin, potfiromycin, puromycin. quelamycin. rodorubicin, streptonigrin, streptozocin, tubercidin. ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues such as denopterin. methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, cannofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone. dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane. trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamidc glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spiro germanium; tenuazonic acid; triaziquone; 2.2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (" Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL® paclitaxel (Bristol-Myers Squibb Oncology. Princeton, N. J.), ABRAXANE® Cremophor-free. albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chlorambucil; GEMZAR® gemcitabine; 6-thioguanine; mercaptopmine; methotrexate; platinum analogs such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE. RTM. vinorelbine; novantrone; teniposide;60Attorney Docket No: 002806-000156WOPTedatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including the treatment regimen of irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including the oxaliplatin treatment regimen (FOLFOX); lapatinib (Tykerb. RTM.); inhibitors of PKC-alpha, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva®)) and VEGF-A that reduce cell proliferation.

[0243] As used herein, the terms "treat,” "treatment," "treating,” or "amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder. The term "treating" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder. Treatment is generally “effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is "effective" if the progression of a disease is reduced or halted. That is, "treatment" includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and / or decreased mortality, whether detectable or undetectable. The term "treatment" of a disease also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).

[0244] In some embodiments of any of the aspects, described herein is a prophylactic method of treatment. As used herein "prophylactic” refers to the timing and intent of a treatment relative to a disease or symptom, that is, the treatment is administered prior to clinical detection or diagnosis of that particular disease or symptom in order to protect the patient from the disease or symptom.Prophylactic treatment can encompass a reduction in the severity or speed of onset of the disease or symptom, or contribute to faster recovery from the disease or symptom. In some embodiments of any of the aspects, prophylactic treatment is not prevention of all symptoms or signs of a disease.

[0245] As used herein, the term "pharmaceutical composition” refers to the active agent in combination with a pharmaceutically acceptable carrier e.g. a carrier commonly used in the pharmaceutical industry. The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be a carrier other than water. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be a cream, emulsion, gel. liposome, nanoparticle, and / or61Attorney Docket No: 002806-000156WOPTointment. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be an artificial or engineered carrier, e.g., a carrier that the active ingredient would not be found to occur in in nature.

[0246] As used herein, the term "administering." refers to the placement of a compound as disclosed herein into a subject by a method or route which results in at least partial delivery of the agent at a desired site. Pharmaceutical compositions comprising the compounds disclosed herein can be administered by any appropriate route which results in an effective treatment in the subject. In some embodiments, administration comprises physical human activity, e.g.. an injection, act of ingestion, an act of application, and / or manipulation of a delivery device or machine. Such activity' can be performed, e.g.. by a medical professional and / or the subject being treated.

[0247] As used herein, "contacting" refers to any suitable means for delivering, or exposing, an agent to at least one cell. Exemplary delivery methods include, but are not limited to, direct delivery to cell culture medium, perfusion, injection, or other delivery' method well known to one skilled in the art. In some embodiments, contacting comprises physical human activity, e.g., an injection; an act of dispensing, mixing, and / or decanting; and / or manipulation of a delivery’ device or machine.

[0248] The term “statistically significant" or “significantly" refers to statistical significance and generally means a two standard deviation (2SD) or greater difference.

[0249] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used in connection with percentages can mean ±1%.

[0250] As used herein, the term “comprising” means that other elements can also be present in addition to the defined elements presented. The use of “comprising” indicates inclusion rather than limitation.

[0251] The term "consisting of refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[0252] As used herein the term "consisting essentially of refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[0253] As used herein, the term “specific binding” refers to a chemical interaction between two molecules, compounds, cells and / or particles wherein the first entity binds to the second, target entity with greater specificity and affinity than it binds to a third entity which is a non-target. In some embodiments, specific binding can refer to an affinity of the first entity for the second target entity which is at least 10 times, at least 50 times, at least 100 times, at least 500 times, at least 1000 times or greater than the affinity for the third nontarget entity. A reagent specific for a given target is one62Attorney Docket No: 002806-000156WOPTthat exhibits specific binding for that target under the conditions of the assay being utilized.

[0254] The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example."

[0255] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in. or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0256] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 20th Edition, published by Merck Sharp & Dohme Corp.. 2018 (ISBN 0911910190, 978-0911910421); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); I munology by Werner Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), W. W. Norton & Company, 2016 (ISBN 0815345054, 978-0815345053); Lewin's Genes XL published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual. 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc.. New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X. 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons. Inc., 2005; and63Attorney Docket No: 002806-000156WOPTCurrent Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are all incorporated by reference herein in their entireties.

[0257] Other terms are defined herein within the description of the various aspects of the invention.

[0258] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[0259] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of. and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0260] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.

[0261] In some embodiments, the present technology may be defined in any of the following numbered paragraphs:1. A composition comprising:64Attorney Docket No: 002806-000156WOPTa) one or more polyphenol molecules;b) one or more biomolecules or active agents; andc) at least one of:i) at least one multivalent ion; andii) at least one endothelium permeabilization agent.The composition of any one of the preceding paragraphs, comprising:a) one or more polyphenol molecules;b) one or more biomolecules or active agents; andc) at least one multivalent ion.The composition of any one of the preceding paragraphs, comprising:a) one or more polyphenol molecules;b) one or more biomolecules or active agents; andc) at least one endothelium permeabilization agent.The composition of any one of the preceding paragraphs, comprising:a) one or more polyphenol molecules;b) one or more biomolecules or active agents;c) at least one multivalent ion; andd) at least one endothelium permeabilization agent.The composition of any one of the preceding paragraphs, wherein the at least one multivalent ion is selected from the group consisting of: Al, Gd(III), Au(III), Fe(III), Mn (II), Ni(II), Sr, Ti(IV), and Zn.The composition of any one of the preceding paragraphs, wherein the at least one multivalent ion comprises Fe(III).The composition of any one of the preceding paragraphs, wherein the at least one multivalent ion is at a concentration of no more than 1 mM.The composition of any one of the preceding paragraphs, wherein the at least one multivalent ion is at a concentration of at least 2 mM.The composition of any one of the preceding paragraphs, wherein the at least one multivalent ion is at a concentration of 2 mM to 37 mM.The composition of any one of the preceding paragraphs, wherein the multivalent ion is at a concentration of 5 mM to 20 mM.The composition of any one of the preceding paragraphs, wherein the polyphenol is at a concentration of at least 5 μM.The composition of any one of the preceding paragraphs, wherein the polyphenol is at a concentration of at least 10 pM.65Attorney Docket No: 002806-000156WOPTThe composition of any one of the preceding paragraphs, wherein the polyphenol is at a concentration of at least 10 μM and the at least one multivalent ion is at a concentration of no more than 1 mM.The composition of any one of the preceding paragraphs, wherein the one or more polyphenols collectively comprise at least one galloyl moiety and / or at least one catechol moiety.The composition of any one of the preceding paragraphs, wherein the one or more polyphenols collectively comprise at least one galloyl moiety and at least one catechol moiety.The composition of any one of the preceding paragraphs, wherein the one or more polyphenols each comprise at least one galloyl moiety and at least one catechol moiety. The composition of any one of the preceding paragraphs, wherein the polyphenol is tannic acid.The composition of any one of the preceding paragraphs, wherein the stoichiometric ratio of polyphenol molecules to biomolecules is 570 or less relative polyphenol.The composition of any one of the preceding paragraphs, wherein the stoichiometric ratio of tannic acid molecules to biomolecules is 190 to 570.The composition of any one of the preceding paragraphs, wherein the stoichiometric ratio of tannic acid molecules to biomolecules is 190.The composition of any one of the preceding paragraphs, wherein the biomolecule and / or active agent is a nucleic acid, protein, a viral particle, a viral vector, a lipid nanoparticle, a polymer, alkaloid, polysaccharide, anthocyanin, lipid, antiviral drug, antibiotic, chemotherapeutic, or combination thereof.The composition of paragraph 21, wherein the biomolecule and / or active agent comprises or is a protein.The composition of paragraph 22, wherein the biomolecule and / or active agent is ovalbumin, serum albumin, interleukin-4, an antibody or antibody reagent, cholera toxin subunit B, biotin, cytokine, or lectin.The composition of paragraph 23, wherein the antibody or antibody reagent is specific for an immune checkpoint protein.The composition of paragraph 23, wherein the antibody or antibody reagent is specific for PD1 or PD-L1.The composition of paragraph 21, wherein the biomolecule and / or active agent is a viral particle to viral vector.The composition of paragraph 26, wherein the viral particle or viral vector is an adeno-associated virus vector.66Attorney Docket No: 002806-000156WOPTThe composition of paragraph 27. wherein the adcno-associatcd virus vector is AAV9 or AAV6.The composition of any one of the preceding paragraphs, wherein the at least one endothelium permeabilization agent is selected from the group consisting of:a Vascular Endothelial Growth Factor (VEGF) polypeptide; histamine; bradykinin; and serotonin.The composition of any one of the preceding paragraphs, wherein the at least one endothelium permeabilization agent is a Vascular Endothelial Growth Factor (VEGF) polypeptide.A functionalized mammalian cell comprising at least one composition of any one of paragraphs 1-30 adhered to the surface of the cell.The cell of paragraph 31. wherein the cell is a hematopoietic cell.The cell of paragraph 31. wherein the cell is an erythrocyte. B cell, T cell, monocyte, macrophage, neutrophil or natural killer cell.The cell of any one of paragraphs 31-33, wherein the biomolecule and / or active agent is an antibody or antibody reagent specific for an immune checkpoint protein and the cell is a macrophage.The cell of any one of paragraphs 31-33. wherein the biomolecule and / or active agent is an antibody or antibody reagent, cytokine, antiviral drug, antibiotic, viral particle, viral vector, or siRNA and the cell is an erythrocyte.The cell of any one of paragraphs 31-33, wherein the biomolecule and / or active agent is an antibody or antibody reagent, siRNA, or chemotherapeutic and the cell is a natural killer cell. The cell of any one of paragraphs 31-33, wherein the biomolecule and / or active agent is cytokine and the cell is a T cell.The cell of any one of paragraphs 31-33, wherein the biomolecule and / or active agent is an anti-inflammatory drug and the cell is a neutrophil.The cell of any one of paragraphs 31-33, wherein compositions of paragraphs 1-30 collectively comprising 10 to 1 trillion biomolecules are adhered to the surface of die cell. A method of functionalizing a mammalian cell, the method comprising: contacting a mammalian cell with the composition of any one of paragraphs 1-30;whereby the combination adheres to the surface of the cell.A method of administering a biomolecule and / or active agent to a patient in need of treatment with the biomolecule and / or active agent, the method comprising administering the composition of any one of paragraphs 1-30 or the cell of any of paragraphs 31-39 to the patient.The method of paragraph 41, wherein the cell is autologous to the patient.67Attorney Docket No: 002806-000156WOPTThe method of any one of paragraphs 41-42. wherein the administration is via intravenous injection and a plurality of the biomolecule administered to the patient is delivered to the lungs.The method of any one of paragraphs 41-42, wherein the administration is via intravenous injection in a vein in a limb and a plurality of the biomolecule administered to the patient is delivered to the lungs.The method of any one of paragraphs 41-42, wherein the administration is via injection to the carotid artery and a plurality of the biomolecule administered to the patient is delivered to the brain.The method of any one of paragraphs 41-42, wherein the cell is an erythrocyte and a plurality of the biomolecule and / or active agent administered to the patient is delivered to the lungs. The method of any one of paragraphs 41-42, wherein the cell is a macrophage and a plurality of the biomolecule and / or active agent administered to the patient is delivered to the brain, a tumor, or a site of inflammation or autoimmune inflammation.The method of any one of paragraphs 41-42, wherein the cell is a natural killer cell and a plurality’ of the biomolecule and / or active agent administered to the patient is delivered to a tumor.The method of any one of paragraphs 41-42, wherein the cell is a T cell and a plurality' of the biomolecule and / or active agent administered to the patient is delivered to a tumor.The method of any one of paragraphs 41-42, wherein the cell is a neutrophil and a plurality of the biomolecule and / or active agent administered to the patient is delivered to the lungs or a site of inflammation.A cell of any one of paragraphs 31-39, for use in a method of administering a biomolecule and / or active agent to a patient in need of treatment with the biomolecule, the method comprising administering the functionalized cell to the patient.The cell of paragraph 51, wherein the cell is autologous to the patient.The cell of any one of paragraphs 51-52, wherein the cell is an erythrocyte and a plurality’ of the biomolecule and / or active agent administered to the patient is delivered to the lungs. The cell of any one of paragraphs 51-52, wherein the cell is a macrophage and a plurality of the biomolecule and / or active agent administered to the patient is delivered to the brain, a tumor, or a site of inflammation or autoimmune inflammation.The cell of any one of paragraphs 51-52, wherein the cell is a natural killer cell and a plurality of the biomolecule and / or active agent administered to the patient is delivered to a tumor. The cell of any one of paragraphs 51-52, wherein the cell is a T cell and a plurality of the biomolecule and / or active agent administered to the patient is delivered to a tumor.68Attorney Docket No: 002806-000156WOPT57. The cell of any one of paragraphs 51-52, wherein the cell is a neutrophil and a plurality of the biomolecule and / or active agent administered to the patient is delivered to the lungs or a site of inflammation.58. A method of administering a viral vector and / or reducing the immune clearance of viral vectors, the method comprising administering the composition of any one of paragraphs 1-30 or the cell of any of paragraphs 31-39, wherein the biomolecule and / or active agent is a viral vector or viral particle.59. The method of paragraph 58, wherein the cell is a red blood cell.60. The method of any one of paragraphs 58-59, wherein the viral vector or viral particle is an AAV viral vector or AAV viral particle.61. The method of paragraph 60, wherein the AAV is AAV9 or AAV6.62. The method of paragraph 60. wherein the AAV is AAV9.63. The method of paragraph 60. wherein the AAV is AAV6.64. A method of gene therapy comprising administering the composition of any one of paragraphs 1-30 or the cell of any of paragraphs 31-39 to the patient, wherein the biomolecule and / or active agent comprises a nucleic acid sequence, e.g.. a nucleic acid sequence suitable for or configured for gene therapy.65. The method of paragraph 64, wherein the cell is a red blood cell.66. The method of paragraph 64 or 65, wherein the gene therapy target is primarily in the lungs.67. The method of paragraph 64 or 65, wherein the gene therapy target is primarily in the brain.68. The method of any one of paragraphs 64-67, wherein the biomolecule and / or active agent is a viral vector or viral particle.69. The method of any one of paragraphs 64-68, wherein the biomolecule and / or active agent is an AAV viral vector or AAV viral particle.70. The method of paragraph 69, wherein the AAV is AAV9 or AAV6.71. The method of paragraph 69, wherein the AAV is AAV9.72. The method of paragraph 69, wherein the AAV is AAV6.

[0262] In some embodiments, the present technology may be defined in any of the following numbered paragraphs:1. A composition comprising:a) one or more polyphenol molecules;b) one or more biomolecules or active agents; andc) at least one of:i) at least one multivalent ion: andii) at least one endothelium permeabilization agent.2. The composition of any one of the preceding paragraphs, comprising:69Attorney Docket No: 002806-000156WOPTa) one or more polyphenol molecules;b) one or more biomolecules or active agents; andc) at least one multivalent ion.The composition of any one of the preceding paragraphs, comprising:a) one or more polyphenol molecules;b) one or more biomolecules or active agents; andc) at least one endothelium permeabilization agent.The composition of any one of the preceding paragraphs, comprising:a) one or more polyphenol molecules;b) one or more biomolecules or active agents;c) at least one multivalent ion; andd) at least one endothelium permeabilization agent.The composition of any one of the preceding paragraphs, wherein the at least one multivalent ion is selected from the group consisting of: Al, Gd(III), Au(III), Fe(III), Mn (II), Ni(II), Sr, Ti(IV), and Zn.The composition of any one of the preceding paragraphs, wherein the at least one multivalent ion comprises Fe(III).The composition of any one of the preceding paragraphs, wherein the at least one multivalent ion is at a concentration of no more than 1 mM.The composition of any one of the preceding paragraphs, wherein the at least one multivalent ion is at a concentration of at least 2 mM.The composition of any one of the preceding paragraphs, wherein the at least one multivalent ion is at a concentration of 2 mM to 37 mM.The composition of any one of the preceding paragraphs, wherein the multivalent ion is at a concentration of 5 mM to 20 mM.The composition of any one of the preceding paragraphs, wherein the multivalent ion is at a concentration of 0.01 mM to 10 mM.The composition of any one of the preceding paragraphs, wherein the multivalent ion is at a concentration of 0.05 mM to 10 mM.The composition of any one of the preceding paragraphs, wherein the multivalent ion is at a concentration of 0.01 mM to 0.5 mM.The composition of any one of the preceding paragraphs, wherein the multivalent ion is at a concentration of 0.05 mM to 0.5 mM.The composition of any one of the preceding paragraphs, wherein the polyphenol is at a concentration of at least 0.1 μM.70Attorney Docket No: 002806-000156WOPTThe composition of any one of the preceding paragraphs, wherein the polyphenol is at a concentration of at least 0.5 μM.The composition of any one of the preceding paragraphs, wherein the polyphenol is at a concentration of 0.5 μM to 1.5 μM.The composition of any one of the preceding paragraphs, wherein the polyphenol is at a concentration of at least 5 μM.The composition of any one of the preceding paragraphs, wherein the polyphenol is at a concentration of at least 10 pM.The composition of any one of the preceding paragraphs, wherein the polyphenol is at a concentration of at least 10 μM and the at least one multivalent ion is at a concentration of no more than 1 mM.The composition of any one of the preceding paragraphs, wherein the polyphenol is at a concentration of at least 0.1 μM and the at least one multivalent ion is at a concentration of 0.01 mM to 10 mM.The composition of any one of the preceding paragraphs, wherein the one or more polyphenols collectively comprise at least one galloyl moiety and / or at least one catechol moiety.The composition of any one of the preceding paragraphs, wherein the one or more polyphenols collectively comprise at least one galloyl moiety’ and at least one catechol moiety.The composition of any one of the preceding paragraphs, wherein the one or more polyphenols each comprise at least one galloyl moiety and at least one catechol moiety. The composition of any one of the preceding paragraphs, wherein the polyphenol is tannic acid.The composition of any one of the preceding paragraphs, wherein the stoichiometric ratio of polyphenol molecules to biomolecules is 570 or less relative polyphenol.The composition of any one of the preceding paragraphs, wherein the stoichiometric ratio of tannic acid molecules to biomolecules is 190 to 570.The composition of any one of the preceding paragraphs, wherein the stoichiometric ratio of tannic acid molecules to biomolecules is 190.The composition of any one of the preceding paragraphs, wherein the one or more biomolecules and / or active agents are selected from: a nucleic acid, protein, a viral particle, a viral vector, a lipid nanoparticle, a polymer, alkaloid, polysaccharide, anthocyanin, lipid, antiviral drug, antibiotic, chemotherapeutic, or combination thereof.The composition of paragraph 29, wherein the one or more biomolecules and / or active agents comprises or is a protein.71Attorney Docket No: 002806-000156WOPTThe composition of paragraph 30, wherein the protein is selected from: ovalbumin, serum albumin, interleukin-4, an antibody or antibody reagent, cholera toxin subunit B, biotin, cytokine, or lectin.The composition of paragraph 31, wherein the antibody or antibody reagent is specific for an immune checkpoint protein.The composition of paragraph 31, wherein the antibody or antibody reagent is specific for PD1 or PD-L1.The composition of paragraph 31, wherein the antibody or antibody reagent is specific for an immune checkpoint protein and VEGF.The composition of paragraph 31, wherein the antibody or antibody reagent is specific for i) PD1 or PD-L1 and ii) VEGF.The composition of paragraph 29, wherein the one or more biomolecules and / or active agents comprise i) an immune checkpoint protein inhibitor and ii) a VEGF inhibitor.The composition of paragraph 29, wherein the one or more biomolecules and / or active agents comprise i) an PD-1 inhibitor and / or a PD-L1 inhibitor and ii) a VEGF inhibitor.The composition of paragraph 29, wherein the biomolecule and / or active agent is a viral particle or viral vector.The composition of paragraph 38, wherein the viral particle or viral vector is an adeno-associated virus vector.The composition of paragraph 39, wherein the adeno-associated virus vector is AAV9 or AAV6.The composition of any one of the preceding paragraphs, wherein the at least one endothelium permeabilization agent is selected from the group consisting of:a Vascular Endothelial Growth Factor (VEGF) polypeptide; histamine; bradykinin; and serotonin.The composition of any one of the preceding paragraphs, wherein the at least one endothelium permeabilization agent is a Vascular Endothelial Growth Factor (VEGF) polypeptide.The composition of any one of the preceding paragraphs, not comprising a cell.The composition of any one of the preceding paragraphs, wherein the cell is a hematopoietic cell.The composition of any one of the preceding paragraphs, wherein the hematopoietic cells is a red blood cell.A functionalized mammalian cell comprising at least one composition of any one of paragraphs 1-45 adhered to the surface of the cell.The cell of paragraph 46. wherein the cell is a hematopoietic cell.72Attorney Docket No: 002806-000156WOPTThe cell of paragraph 46, wherein the cell is an erythrocyte. B cell, T cell, monocyte, macrophage, neutrophil or natural killer cell.The cell of any one of paragraphs 46-48, wherein the biomolecule and / or active agent is an antibody or antibody reagent specific for an immune checkpoint protein and the cell is a macrophage.The cell of any one of paragraphs 46-48, wherein the biomolecule and / or active agent is an antibody or antibody reagent, cytokine, antiviral drug, antibiotic, viral particle, viral vector, or siRNA and the cell is an erythrocyte.The cell of any one of paragraphs 46-48, wherein the biomolecule and / or active agent is an antibody or antibody reagent, siRNA, or chemotherapeutic and the cell is a natural killer cell. The cell of any one of paragraphs 46-48, wherein the biomolecule and / or active agent is cytokine and the cell is a T cell.The cell of any one of paragraphs 46-48, wherein the biomolecule and / or active agent is an anti-inflammatory drug and the cell is a neutrophil.The cell of any one of paragraphs 46-53, wherein compositions of paragraphs 1-45 collectively comprising 10 to 1 trillion biomolecules are adhered to the surface of the cell. A method of functionalizing a mammalian cell, the method comprising: contacting a mammalian cell with the composition of any one of paragraphs 1-45;whereby the combination adheres to the surface of the cell.A method of administering a biomolecule and / or active agent to a patient in need of treatment with the biomolecule and / or active agent, the method comprising administering the composition of any one of paragraphs 1-45 or the cell of any of paragraphs 46-54 to the patient.The method of paragraph 56, wherein the cell is autologous to the patient.The method of any one of paragraphs 56-57, wherein the administration is via intravenous injection and a plurality' of the biomolecule administered to the patient is delivered to the lungs.The method of any one of paragraphs 56-57, wherein the administration is via intravenous injection in a vein in a limb and a plurality of the biomolecule administered to the patient is delivered to the lungs.The method of any one of paragraphs 56-57, wherein the administration is via injection to the carotid artery and a plurality of the biomolecule administered to the patient is delivered to the brain.The method of any one of paragraphs 56-57, wherein the cell is an erythrocyte and a plurality of the biomolecule and / or active agent administered to the patient is delivered to the lungs.73Attorney Docket No: 002806-000156WOPTThe method of any one of paragraphs 56-57. wherein the cell is a macrophage and a plurality of the biomolecule and / or active agent administered to the patient is delivered to the brain, a tumor, or a site of inflammation or autoimmune inflammation.The method of any one of paragraphs 56-57, wherein the cell is a natural killer cell and a plurality of the biomolecule and / or active agent administered to the patient is delivered to a tumor.The method of any one of paragraphs 56-57, wherein the cell is a T cell and a plurality of the biomolecule and / or active agent administered to the patient is delivered to a tumor.The method of any one of paragraphs 56-57, wherein the cell is a neutrophil and a plurality of the biomolecule and / or active agent administered to the patient is delivered to the lungs or a site of inflammation.The method of any one of paragraphs 56-65, wherein the tumor, inflammation, or autoimmune inflammation is in the lung.The method of any one of paragraphs 56-65, wherein the tumor is in the lung.The method of any one of paragraphs 56-65, wherein the patient is in need of treatment for lung cancer.The method of any one of paragraphs 56-65, wherein the patient is in need of treatment of a disease of or in the lung.A cell of any one of paragraphs 46-54, for use in a method of administering a biomolecule and / or active agent to a patient in need of treatment with the biomolecule and / or active agent, the method comprising administering the functionalized cell to the patient.The cell of paragraph 70, wherein the cell is autologous to the patient.The cell of any one of paragraphs 70-71, wherein the cell is an erythrocyte and a plurality of the biomolecule and / or active agent administered to the patient is delivered to the lungs. The cell of any one of paragraphs 70-71, wherein the cell is a macrophage and a plurality of the biomolecule and / or active agent administered to the patient is delivered to the brain, a tumor, or a site of inflammation or autoimmune inflammation.The cell of any one of paragraphs 70-71, wherein the cell is a natural killer cell and a plurality of the biomolecule and / or active agent administered to the patient is delivered to a tumor. The cell of any one of paragraphs 70-71, wherein the cell is a T cell and a plurality of the biomolecule and / or active agent administered to the patient is delivered to a tumor.The cell of any one of paragraphs 70-71, wherein the cell is a neutrophil and a plurality of the biomolecule and / or active agent administered to the patient is delivered to the lungs or a site of inflammation.74Attorney Docket No: 002806-000156WOPT77. A composition of any one of paragraphs 1-45, for use in a method of administering a biomolecule and / or active agent to a patient in need of treatment with the biomolecule and / or active agent.78. The method of paragraph 77, wherein patient is in need of treatment of a tumor, inflammation, or autoimmune inflammation in the lung.79. The method of paragraph 77 or 78, wherein the tumor is in the lung.80. The method of any one of paragraphs 77-79, wherein the patient is in need of treatment for lung cancer.81. The method of any one of paragraphs 77-80, wherein the patient is in need of treatment of a disease of or in the lung.82. A method of administering a viral vector and / or reducing the immune clearance of viral vectors, the method comprising administering the composition of any one of paragraphs 1-45 or the cell of any of paragraphs 46-54, wherein the biomolecule and / or active agent is a viral vector or viral particle.83. The method of paragraph 82, wherein the cell is a red blood cell.84. The method of any one of paragraphs 82-83, wherein the viral vector or viral particle is an AAV viral vector or AAV viral particle.85. The method of paragraph 84, wherein the AAV is AAV9 or AAV6.86. The method of paragraph 84, wherein the AAV is AAV9.87. The method of paragraph 84, wherein the AAV is AAV6.88. A method of gene therapy comprising administering the composition of any one of paragraphs 1-45 or the cell of any of paragraphs 46-54 to the patient, wherein the biomolecule and / or active agent comprises a nucleic acid sequence, e.g„ a nucleic acid sequence suitable for or configured for gene therapy.89. The method of paragraph 88, wherein the cell is a red blood cell.90. The method of paragraph 88 or 89, wherein the gene therapy target is primarily in the lungs.91. The method of paragraph 88 or 89, wherein the gene therapy target is primarily in the brain.92. The method of any one of paragraphs 88-91, wherein the biomolecule and / or active agent is a viral vector or viral particle.93. The method of any one of paragraphs 88-92, wherein the biomolecule and / or active agent is an AAV viral vector or AAV viral particle.94. The method of paragraph 93, wherein the AAV is AAV9 or AAV6.95. The method of paragraph 93, wherein the AAV is AAV9.96. The method of paragraph 93, wherein the AAV is AAV6.

[0263] The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting.75Attorney Docket No: 002806-000156WOPTEXAMPLESExample 1 - Multiscale Red Blood Cell Hitchhiking for Targeted Deep Tissue Gene Delivery in Lungs

[0264] The applications of gene therapies based on mRNA lipid nanoparticles or adeno-associated viruses (AAVs) are often hindered by the difficulty of achieving specific and efficient delivery to target tissues beyond the liver, particularly following intravenous administration. Current strategies have focused on molecular targeting strategies, for example, the engineering of AAV capsids to improve tissue tropism or surface modification of lipid nanoparticles. However, these strategies have had limited success due to their inability to overcome the hierarchical barriers from the injection site to deep tissue transduction. To address these limitations, the inventors developed a Multiscale Approach using RBC-mediated hitchhiking and Vascular Endothelium Leakage (MARVEL). This new paradigm combines RBC-mediated hitchhiking with VEGF-induced vascular permeabilization to enhance targeted delivery and tissue penetration of cargoes. Demonstrated herein is the principle and capabilities of MARVEL using AAVs as an example. In vitro and in vivo studies demonstrated that MARVEL significantly increases AAV accumulation in the lungs, improves transcytosis across the endothelial barrier, and enhances gene expression in deep tissue layers while maintaining a favorable safety profile. Furthermore, it is demonstrated herein that MARVEL can be adopted into an in situ hitchhiking approach thereby bypassing the necessity of ex vivo formulation, thus simplifying the application process. These results highlight MARVEL'S potential to overcome the delivery challenges that have long restricted the broader applicability of gene therapy, offering a versatile and scalable solution for more effective gene delivery.

[0265] Introduction

[0266] Adeno-associated viruses (AAVs) have become a leading gene delivery platform for multiple indications owing to their high in vivo transduction efficiency and safety profile in animals and humans (7, 2). Attempts have been made to improve the efficacy of AAV-based therapeutics by engineering tissue tropism, increasing transduction efficiency, and decreasing immunogenicity (3-5). These improvements have led to promising preclinical and clinical results in recent years, leading to the FDA approval of AAV-based therapeutics for Duchenne muscular dystrophy and Leber congenital amaurosis, among others (6-9).

[0267] Despite their clinical success, the broad applicability of AAV-based gene therapy is limited by the challenges associated with targeting specific cells and tissues outside the liver upon intravenous administration, which poses limitations on efficacy as well as safety. The challenge lies in the fact that AAVs need to overcome multiple hierarchical hurdles after intravenous administration before exhibiting tissue and ccll-spccific deliver}’, including (i) avoiding liver clearance and toxicity (70) by accumulating on the endothelium of tire target tissue, (ii) crossing tire endothelium to infiltrate the target tissue and (iii) transducing the target cells within the tissue.76Attorney Docket No: 002806-000156WOPT

[0268] Past efforts to improve AAV-mediated gene therapy have focused primarily on virology, aiming at serotype selection and modification of the transgene cassette and capsid components (77-16). However, without explicitly addressing the delivery challenges, the applications of AAVs remain restricted to cases where delivery hurdles are less relevant, for example, directly injecting AAVs into the tissue, such as the retina, brain, and muscle, or exploring applications in the liver, the natural target site of AAVs after intravenous administration (77-20). Some efforts have aimed at engineering the AAV to improve tissue tropism. However, simultaneous improvement of tissue accumulation and transduction in deeper tissue regions has proved challenging.

[0269] The challenge in achieving high tissue tropism can be appreciated from the master targeting equation (1). which describes the flux. J, of the capsid, or any other gene carrier, into tissues after injection into a blood vessel.J —T(1)where Cois the initial carrier concentration in blood upon injection, is the enhancement of localtissue concentration of the carrier achieved by the targeting strategy,is the vascular endothelialpermeability in the target tissue, and r is the time constant of blood clearance of the carrier. Classical targeting dogma uses polyethylene glycol (PEG) to increase r, and relies either on targeting ligands to increase or enhanced permeation and retention (EPR) to achieve highfor tumor targeting.

[0270] The efficacy of such approaches is limited by the inherent chemical conflict between PEG-mediated enhanced circulation and ligand-mediated improved vascular binding. The efficacy of ligand binding is further limited by inadequate margination of cargoes to the vascular endothelium. This lowers the likelihood that the targeting ligands actually engage with their receptors on vascular endothelium. The variability of EPR also makes it a challenge to rely entirely on it for targeting purposes.

[0271] Drug loading on red blood cells (RBC). called RBC hitchhiking, increases the accumulation of nanoparticles in various organs, including the lungs, brain, and kidneys (21-27). This approach has also been shown to deliver AAVs to the lungs and reduce their off-target gene expression (28). While RBC hitchhiking increases AAV concentration in tissue endothelium, the hitchhiking strategy is not inherently designed to overcome the endothelial barrier.

[0272] Here, that challenge is addressed by incorporating the vascular endothelial growth factor (VEGF) into the carrier design. Leveraging the ability of VEGF to improve endothelial permeability,77Attorney Docket No: 002806-000156WOPTdescribed herein is a new Multiscale Approach using RBC-mediated hitchhiking and Vascular Endothelium Leakage (MARVEL), which leverages (i) RBC-mediated improved circulation (τ), (ii) RBC-mediated enhanced tissue accumulation (λtis), and (iii) VEGF-mediated vascularpermeabilization (μvas). MARVEL uses concurrent adsorption of AAVs and VEGF on the RBCsurface using polyphenol-mediated complexation. RBC-adsorbed AAVs and VEGF are dislodged in the vascular endothelium upon intravenous administration due to contact-mediated transfer. This increases local AAV concentration and VEGF-mediated vascular permeabilization, leading to deep tissue penetration of AAVs (Fig. 1). Furthermore, demonstrated herein is that hitchhiking can be achieved in situ, where AAV-VEGF complexes, upon intravenous injection, bind to native RBCs and achieve organ-targeted, deep-tissue transduction. This simplified process demonstrates MARVEL’s potential for translation and applicability to various indications, thus offering a new tool for effective gene therapy.

[0273] Results

[0274] RBCs were loaded with AAV alone (RBC / AAV) or AAV+VEGF (MARVEL) by complexation with tannic acid (TA) and iron chloride (III) (FeCl3) to induce metal-phenolic network formation (Fig. 2A). TA and FeCl3concentrations were first optimized with AAV alone to minimize RBC hemolysis and aggregation during formulation (Fig. 6). The AAV, TA, VEGF, and FcCL complexes were visible on the surface of RBCs after formulation, in contrast to the smooth surfaces of bare RBCs (Fig. 2B).

[0275] AAV loading can be controlled by adjusting the concentration of AAV in the incubation solution (Fig 3A). AAVs are stably associated with RBCs and released in a shear-dependent manner. When RBC / AAV was placed at room temperature with mild shaking (250 rpm), a cumulative % AAV release of 40% was observed within 48 hours (Fig 3B). In contrast, when the RBC / AAV was exposed to shear stresses by placing under flow conditions, more than 65% of the AAV was released within 10 minutes, reaching nearly 95% release at 0.1 Pa in 2 minutes (Fig 3C), demonstrating shear-induced release of AAVs from the RBC surface.

[0276] The abi lity of AAVs loaded on RBC to transfer to endothelial cells was confirmed ex vivo in a microfluidic chip laden with endothelial cells (EA.hy926) on the channel surface (Fig. 7).Significantly higher cell-associated AAVs were observed in the RBC / AAV group than in the free AAV group (Fig 3D, red signal indicates Alexa Fluor 647-labeled AAVs). Quantitatively, RBC association led to a 3 -fold increase in AAV deposition onto the endothelial cells (Fig. 3E). Even under static conditions, RBC -associated AAVs were able to induce gene expression, but the expression was significantly improved under the flow, supporting the role of the shear-induced detachment of AAVs from RBCs (Fig 3F). The degree of transduction under the flow condition was78Attorney Docket No: 002806-000156WOPTsignificantly higher for RBC / AAV than for free AAVs (Fig. 8), indicating the significance of the combination of physical contact provided by RBC hitchhiking and shear-induced release of AAVs to maximize transduction.

[0277] RBC hitchhiking led to a significantly higher accumulation of AAVs in the lungs in vivo after tail vein injection than free AAVs (Fig 3G). While free AAVs accumulated primarily in the liver, the RBC / AAVs accumulated mainly in the lungs, resulting in a 90-fold higher lung-to-liver ratio for the RBC / AAV than the free AAVs (Fig 3H and 31). When used with GFP-encoded AAVs, RBC / AAVs successfully transduced cells throughout the lungs as assessed 3 weeks post-injection (Fig. 9A). The difference in expression between free AAVs and RBC / AAVs was also evident 12 weeks post-injection (Fig. 9B). The target tissue for expression can be altered by changing the injection location, thus changing the location for contact-mediated detachment of AAVs from RBCs. For example, when RBC / AAV was injected via the carotid artery, transgene expression in the brain significantly increased compared to free AAVs (Fig. 10).

[0278] VEGF was loaded onto RBCs by co-incubating AAVs and VEGF with TA, FeCl3, and RBC through one-pot synthesis to accomplish vascular endothelial permeabilization and deep tissue expression. The VEGF loading on RBCs can be controlled by adjusting the feed amount (Fig. 4A).VEGF did not adversely impact AAV loading on RBCs (Fig. 11). We also confirmed the loading of a model protein. FITC-BSA. which exhibits a negative charge at neutral pH, in contrast to VEGF, which has a positive charge, demonstrating MARVEL’s flexibility in loading cargoes of different therapeutic interests (Fig. 12). VEGF maintained its bioactivity after complexation with TA and Fe (VEGF / TA / Fe) as indicated by its ability' to impact vascular endothelial cadherin (VE-cadherin) signals in endothelial cells (Fig. 4B). Specifically, upon incubation with endothelial cells, VEGF released from the VEGF / TA / Fe complex disrupted the junctional proteins, decreasing the VE-cadherin signal comparable to the positive control (Fig. 4B). The cell surface area, perimeter, and Feret's diameter were significantly increased in VEGF / TA / Fe-treated group, supporting junctional disruption (Fig. 13). In a transwell setting, free AAVs or RBC / AAVs with or without VEGF were applied to a layer of EA.hy926 cells (Fig. 14). Only upon loading VEGF on RBCs (MARVEL) was a statistically significant increase in AAV translocation compared to other groups, likely due to the increased AAV and VEGF concentrations near the endothelial cells.

[0279] VEGF, either in the free form or loaded on MARVEL, was administered by a tail vein injection in vivo (Fig. 4C). While free VEGF. which was co-delivered with free AAV, did not lead to a detectable increase in lung VEGF concentration. MARVEL led to a significant lung accumulation of VEGF. MARVEL also led to increased lung transgene expression compared to RBC / AAV. Four weeks after the injection, MARVEL showed a trend of higher endothelial transduction than RBC / AAV (Fig. 4D) and a significant increase in epithelial transduction (Fig. 4E). Notably, RBC / AAV without VEGF did not effectively transduce the epithelial cells. However, MARVEL79Attorney Docket No: 002806-000156WOPTsignificantly increased the transduction, attesting to the significance of VEGF in inducing deep tissue transfection.

[0280] MARVEL was well tolerated in all animals. Four hours after injection, neither AAV + VEGF combination nor MARVEL showed an increase in IL-6 and TNF-a levels in the lungs, while the systemic levels of IL-6 and TNF-a returned to baseline within an hour after injection (Figs 15A, 15B). AAV or RBC / AAV, with or without VEGF, did not cause mouse bodyweight change, indicating the tolerance of the formulation at the tested doses (Fig. 15C).

[0281] It was next assessed whether MARVEL allows AAVs to bind to and hitchhike on RBCs in situ spontaneously since bypassing ex vivo attachment could substantially simplify the administration process.

[0282] Given that the association of MARVEL-AAVs ex vivo with RBCs upon incubation is rapid and that RBCs are the predominant cell type in blood, it was hypothesized that MARVEL-AAVs may also associate with RBCs upon direct intravenous injection. This was first validated using a model drug carrier, fluorescence-labeled polystyrene (PS) beads of 200 nm in diameter. PS beads were added to whole blood with or without TA and Fe. The binding between the PS beads and RBC was significantly greater when combined with TA and Fe (Fig. 5A). RBC association of PS beads in the presence of TA and Fe also occurred under flow conditions (Figs 16A, 16B). PS beads successfully adhered to RBCs within a short tune (< 2 sec) in the presence of TA and Fe. A similar study was performed with lipid nanoparticles (LNPs) containing a model mRNA encoding luciferase. The addition of TA and Fe significantly improved the binding of LNPs to RBC in whole blood, demonstrating the potential generalizability for drug loading onto RBCs within the blood in situ (Fig.16C)

[0283] AAV formed complexes in the presence of TA and Fe, and these complexes bound to RBCs in whole blood (Fig. 5B and Fig. 17). Upon direct intravenous injection, MARVEL-AAV led to the accumulation of AAVs in the lungs within an hour, giving a trend of improved lung-to-liver ratio (based on absolute titers) compared to AAV injection alone (Figs 5C and 5D). Notably, the AAV accumulation per mg of organ increased by 6.2-fold in the lungs and decreased by 2.3-fold and 2.7-fold in the liver and spleen, respectively, with MARVEL-AAV. Thirty days after injection, the mice injected with MARVEL-AAV exhibited significantly greater transgene expression in the endothelial (Fig. 5E) and epithelial (Fig. 5F) cells compared to AAV alone. MARVEL-AAV led to a 4-fold improvement of endothelial gene expression and a 2.6-fold improvement of epithelial gene expression compared to free AAV, which were 388- and 52-fold compared to the respective notreatment baseline signals. These results confirm that MARVEL improves gene expression through a combination of RBC hitchhiking-mediated lung accumulation of AAVs on the endothelium and subsequent deeper tissue transduction into the epithelial tissue achieved by VEGF.

[0284] Discussion80Attorney Docket No: 002806-000156WOPT

[0285] In this study, MARVEL is introduced and validated as a platform strategy for die targeted delivery of AAVs and enabling their deep-tissue transduction. Traditional drug delivery strategies, which often focus on molecular-level targeting modifications, such as PEGylation or targeting ligand conjugation, can pose inherent constraints since they apply competing chemistries at the same molecular scale, potentially limiting their combined effectiveness. Further, the efficacy of molecular-scale targeting ligands is limited by the low likelihood of these ligands making molecular-scale contact with their corresponding receptors. MARVEL employs a multi-scale approach to propose a new paradigm in drug delivery: achieving organ-targeted drug accumulation at the microscale and enhancing drug infiltration into deeper tissue regions at the sub-microscale. By combining the RBC hitchhiking approach with VEGF-mediated vascular permeabilization, we achieved a significantly improved accumulation of AAVs in lung tissues through a concurrent improvement in 2^.and T, surpassing the efficacy of traditional methods based on free AAV. This dual-action approach increased the concentration of AAVs at the endothelial interface and facilitated their transcytosis into deeper tissue layers, leading to enhanced gene expression in target cells. The enhanced lung transduction with MARVEL is especially significant, given that AAV6 used in these lung studies has already been optimized for lung delivery by engineering tropism (30). This indicates that MARVEL can be broadly applied to enhance the targeted transduction of recombinant AAVs (rAAV) orthogonal to existing engineering approaches for specific tropisms. The loading of other model therapeutic cargoes, including proteins, polymers, and LNPs, onto the RBC surface in whole blood further underscores MARVEL’S versatility, potentially broadening its application to other therapeutic agents and disease targets. It was found that the LNP loading results particularly promising, as they indicate an approach to enhance genetic transfection via non-viral vectors - a rapidly advancing area in the field with growing applications for LNP technologies (31, 32).

[0286] Overall, MARVEL represents a substantial advancement in gene therapy, offering a robust and scalable method for overcoming the long-standing challenges of targeted delivery and tissue-specific transduction. The multi-scale delivery approach is a fundamentally different approach and a paradigm shift from traditional targeting methods. It offers an orthogonal strategy to achieve spatially and quantitatively improved drug delivery to target tissues in vivo. Upon further studies focused on optimization and safety. MARVEL offers a new tool for designing gene therapies for treating a range of genetic disorders.

[0287] Materials and methods

[0288] RBC preparation. Blood was collected from mice via submandibular bleeding using 4 mm lancets and placed in EDTA-coated blood collection tubes. The blood was centrifuged at 1000 g for 10 min at 4 °C. The serum and the buffy coat were removed by pipetting. The RBC pellet at the bottom was transferred into a 15 mL tube filled with ~14 mL of ice-cold PBS. The sample was centrifuged at 650 g for 15 min at 4 °C. The supernatant was removed, followed by resuspension of81Attorney Docket No: 002806-000156WOPTthe pellet with 15 mL of cold PBS. The washing step was repeated twice. After removing die supernatant following the final centrifugation, the packed RBC at the bottom of the tube was transferred into a new tube using a pipette, and 9x the volume of cold PBS was added to achieve a 10% hematocrit RBC solution.

[0289] Ex vivo MAR VEL preparation. The stock AAV (AAV6-CMV-GFP and AAV6-CMV-Luc (SignaGen Laboratories)) solution containing 5e+11 AAV and 10 pg of recombinant murine VEGF (#450-32; Peprotech) was mixed with PBS to a 390 pL intermediate volume. 10 pL of 10 mg / mL TA (Sigma- Aldrich, 16201) (dissolved in deionized water) was added, followed by a brief vortex to mix. The sample was incubated at room temperature for 2 min. Next, 100 pL of RBC (10% hematocrit) in PBS was added. The solution was mixed by manually inverting the tube 3-4 times. 3 pL of 10 mg / mL Iron (III) chloride hexahydrate (FeCl₃ Sigma- Aldrich, 236489) (dissolved in deionized water) was added, followed by an addition of 500 pL of PBS. The tube was manually inverted several times for mixing, then centrifuged at 100 g for 4 min. The supernatant was removed, and the pellet was resuspended with 1 mL of PBS. This washing step was repeated twice. After the final centrifugation, the pellet was resuspended with 90 pL of PBS to a final volume of 100 pL of RBC / AAV formulation.

[0290] For in situ MARVEL, le+11 vg AAV. 450 ng VEGF, 20 pg TA, and 6 pg Fe were mixed in water and adjusted to IX PBS by mixing 10X PBS to a final volume of 200 pL.

[0291] Microfluidic shear stress-induced AAV release from RBC. Microfluidics chips (Ibidi uChips 0.2 Luer) were treated with 100 pL of poly-L-lysine for 20 min at RT, then washed with 100 pL PBS twice. 100 pL of RBC / AAV (1.25e+9 / mL) was treated on each chip and incubated for 20 min on ice. The chips were washed three times with cold PBS to remove any unbound RBC / AAV. Next, the flow was turned on for 2 minutes using a peristaltic pump in a closed-loop system with cold PBS in the tubing. The entire chip’s channels were imaged using a bright field microscope to count the RBC / AAV before and after the flow.

[0292] Transduction study in microfluidic chips. To prepare microfluidics chips coated with a model endothelial cell line, EA.hy926, cells were seeded in microfluidics chips (Ibidi uChips 0.2 Luer) in 100 pL of 3e+6 cells / mL concentration in complete DMEM media (supplemented with 10% FBS and 1% Pen-Strep), and incubated at 37 °C for 3 hours to allow the cells to adhere. The chips were washed with 120 pL of fresh media tw ice to remove any unbound cells and were further incubated for two days until AAV treatment, with a daily media change. Depending on the experimental design, RBC / AAV or free AAV were treated either by a syringe, pipette, or tubing. After treatment, chips were thoroughly washed with complete DMEM and further incubated for 10-14 days until analysis.

[0293] In Vitro functional study using endothelial cells. Primary human brain endothelial cells were obtained from CellSystems and cultured according to the manufacturer's instructions. For the functional assay, cells were seeded into an 8-well confocal chamber at a density of 3e+5 cells per well82Atorney Docket No: 002806-000156WOPTand maintained in an EGM-2 medium (Lonza). After two days, once a monolayer had formed, cells were cultured with cAMP, hydrocortisone, and other supplements to enhance junctional function. On the day of the experiment, cells were treated with 20 ng / mL of free VEGF or 20 ng / mL of VEGF / TA / Fe. After a 30-minute incubation, the medium was replaced with fresh medium, and the cells were cultured for an additional 6 hours. For the positive control, cells were treated with 100 ng / mL of VEGF and incubated for 24 hours. Following the designated incubation times, cells were fixed, blocked, and immunostained with an anti-VE-cadherin antibody (Santa Cruz, sc-9989). Samples were washed three times before incubation with a secondary antibody (Invitrogen, A11001) and counterstained with DAPI. Confocal images were captured using a Zeiss LSM980 with AIRYSCAN™. Fluorescent intensity of the junctional marker and cellular morphology were quantified using ImageJ.

[0294] Fluorophore labeling on AAV. The NHS-amine reaction chemistry was used to label AAVs with Alexa Fluor 647. 1 mg of lyophilized Alexa Fluor™ 647 NHS Ester (Succinimidyl Ester) (Invitrogen; cat. no. A20006) was dissolved into 135 pL of 20 mM phosphate buffer containing le+12 vg AAV. The solution was incubated at room temperature for an hour under constant shaking. The labeled AAVs were diluted by adding 715 pL of PBS and then purified by running through 7k Zeba desalting columns (Thermo Scientific; cat. no. 89891) twice. The resulting solution was concentrated using 3k Amicon Ultra-0.5. The resulting Alexa Fluor 647-labeled AAV was quantified using PCR following the protocol described later in the Methods section.

[0295] Transmission electron microscopy (TEM) imaging. Samples were prepared at a high concentration (> 1 mg / mL). Hexagonal carbon TEM grids were placed on a glass cover slide wrapped in parafilm, followed by glow discharge cleaning (Pelco EASY GLOW™) on the grids. Next, on the surface of a stretched piece of parafilm taut across a glass bench surface. 15 pL drops of each sample were placed. Inverted carbon grids were placed on top of the drops (carbon side down) and waited 1 minute. The grids were retrieved with fine-tipped forceps, handling only the grid border. The carbon surface was washed with 5 drops of 3% uranyl acetate in deionized water passed through a 0.22 pm syringe filter. Then, the grid edges were blotted to remove excess moisture, followed by air drying overnight. The resulting samples were imaged on Hitachi 7800 TEM.

[0296] Scanning electron microscopy (SEM) imaging. Samples were fixed with a solution containing 2% glutaraldehyde overnight at 4°C and adsorbed onto poly-L-lysine coated 12 mm² coverslips. Samples were dehydrated by incubating in increasing concentrations (50%, 75%, 90%, 95%, 100%. 100%, 100%) of ethanol for 10 min, then dried using a critical point dryer. Samples were mounted and then sputter coated with 5 nm Pt / Pd. Samples were then imaged on the Zeiss GEMINI™ 360 SEM using the SE2 detector and accelerating voltage of 1.5 keV.

[0297] PCR. The forward and reverse primers were prepared at 0.5 pM and probe oligo at 0.15 pM, respectively. Then, the PCR master mix (Prime Time Gene Expression Master Mix (2X),83Attorney Docket No: 002806-000156WOPTIDTDNA) was added to a IX final concentration for PCR. The thermal cycle shown in Table 1 was used for running PCR (BioRad CFX 96). TE (pH 8.0) buffer was used for dilutions when necessary. The nucleic acid sequences of primers and probes are listed in Table 2.

[0298] Table 1. Thermal cycleStep 1 95 °C 5 minStep 2 95 °C 15 secStep 3 55 °C 1 minRepeat steps 2-3 39 timesStep 4 4 °C Done

[0299] Table 2. List of primers and probesCMV primers and probeCMV FP Sequence TCA TAT GCC AAG TAC GCC CC (SEQ ID NO: 63)CMV RP Sequence CCC GTG AGT CAA ACC GCT AT (SEQ ID NO: 64) CMV_probe Sequence / 56-FAM / TG GGA CTT T / ZEN / C CTA CTT GGC AGT AC (SEQ ID NO: 65)EGFP primers and probeEGFP FP Sequence AGC AAA GAC CCC AAC GAG AA (SEQ ID NO: 66) EGFP RP Sequence GGC GGC GGT CAC GAA (SEQ ID NO: 67) EGFP_FAM_probe Sequence / 56-FAM / CG CGA TCA C / ZEN / A TGG TCC TGC TGG (SEQ ID NO: 68)

[0300] In vivo transduction study. All experiments were performed according to the approved protocols by the Institutional Animal Care and Use Committee (IACUC) of the Faculty of Arts and Sciences (FAS), Harvard University (protocol number 18-02-320-1). Female C57BL / 6 mice (aged 5- 6 weeks) were purchased from the Jackson Laboratory (Bar Harbor. ME). 100 pL of the RBC / AAV formulation containing le+11 vg of AAV6-CMV-GFP was administered via the tail vein using a syringe with a 29G needle. 450 ng of VEGF was included in all VEGF-containing formulations.

[0301] For carotid artery injections, carotid artery -catheterized C57BL / 6 mice (Surgery code: CARART-CD; catheter inserted into the left carotid artery and advanced toward the aortic arch), aged 10-11 weeks and weighing 20-25 g, were purchased from Charles River Laboratories (Wilmington, MA). 1.51e+10 vg of AAV9-CMV-dsRED was prepared in 100 pL of PBS and was injected via the catheter as free AAVs or loaded in RBC / AAV. Following the manufacturer's instruction, each dose was injected into the catheters using the Vascular Access Button (Instech) connected to 1 mL syringes with an adaptor, PINPORTS™ (Instech; PNP3M), over 2 minutes.84Atorney Docket No: 002806-000156WOPT

[0302] In vivo biodistribution study, 1e+11 vg of AAV6-CMV-Luc was IV injected via the tail vein. For the in situ drug loading condition, 20 gg TA and 6 pg FeCL were co-injected. 1 hour after administration, the mice were sacrificed by CO2 euthanasia. Immediately after the euthanasia, mice were perfused with 20 niL of cold PBS, and the organs, including the brain, lung, heart, spleen, kidney, and liver, were collected. These were homogenized in TE buffer using a homogenizer (IKA T10 Basic ULTRA-TURRAX™, NC). The viral genome was extracted from the homogenates using a DNA extraction kit (Thermo Scientific, K0721, GeneJet Genomic DNA Purification kit) and quantified using qPCR.

[0303] Flow cytometry. 3 weeks after RBC / AAV administration, mice were euthanized via CO2 euthanasia. AAVs encoding GFP were used. The lungs were harvested after perfusion and placed in PBS containing 1% FBS. The lungs were cut into small pieces (<1 mm), then digested and processed using a lung dissociation kit (Miltenyi, 130-095-927) into single cells. Briefly, digestion enzymes were added to the samples and then incubated at 37 °C for 30 minutes. The samples were placed on top of pre-wet 70 pm strainers, mashed with plungers, and rinsed with 10 mL PBS. The cell suspensions were collected in 50 mL tubes and centrifuged at 500 g for 5 min. The supernatant was removed, and the samples were washed twice with a cell staining buffer (BioLegend, 420201). If necessary, red blood cells were lysed with ACK buffer (Gibco, A1049201) before the washing step.

[0304] Following a typical flow cytometry sample preparation protocol, the resulting cells were stained with antibodies. Briefly, sample pellets were resuspended with 20 pL of 1 / 30 diluted anti- CD16 / 32 antibody (Invitrogen, 14-0161-85) for 10 min at 4 °C to block Fc receptors. Next, 20 pL of the antibody cocktail (list of antibodies shown in Table 3, for lung and brain samples, respectively) was added and incubated for 25 min at 4 °C. For the cocktail, each antibody was diluted by 1 / 40, which was then diluted to a final dilution of 1 / 80 once mixed with the sample solution containing CD16 / 32-coated cells. After the incubation, the samples were washed twice with PBS at 250 g for 5 min via centrifugation, followed by live / dead staining with LIVE / DEAD Fixable Blue (Invitrogen, L23105). Next, the samples were fixed and permeabilized using the BD perm / fix kit (BD, 554714) following the manufacturer’s protocol for intracellular staining. Anti-GFP antibody was diluted by 1 / 160 for staining. After the staining, samples were washed tw ice with the perm / wash buffer, then finally resuspended into 200 pL of the staining buffer and stored at 4 °C until analysis.

[0305] Table 3. List of antibodies for flow cytometric analysis of the lungsTarget Fluor Manufacturer Cat. No.CD326 AF594 Bioss BS-4889R-A594CD31 BV711 Invitrogen 407-0311-82CD45 BUV395 BD 564616GFP AF488 BioLegend 33800785Attorney Docket No: 002806-000156WOPT

[0306] Binding study in whole blood. Binding of polystyrene beads on RBC in whole blood in a static condition was done by mixing 5 pL of fluorescent polystyrene beads (FLUOROBRITE®, cat# 17151; 200 nm diameter. 5.68 x 1012particles / mL), 10 pg TA and 3 g Fe to a final volume of 10 pL of PBS (pH 7.4), followed by mixing with 10 pL of whole blood. The mixture was immediately diluted with 200 pL of PBS containing 5 w / v% BSA to quench further binding of the beads. The samples were washed twice with PBS. The fluorescence from the resulting samples was measured with a plate reader (BioTek SYNERGY™ Hl).

[0307] For the binding study in the presence of flow, 77 pL of fluorescent polystyrene beads (5.68 x 1012particles / mL) mixed with 100 pg TA and 30 pg Fe was injected into whole blood flowing in the tubing (0.02 inches inner diameter) at a 10 pL / sec flow rate by a syringe pump, connected via a 25 gauge needle. The samples from the outlet were collected into PBS containing 4 w / v% BSA and were run immediately on a flow cytometer. Red blood cells were gated based on size, followed by gating on the PS bead-positive population.

[0308] For studying the binding of AAV to RBC in whole blood, Alexa Fluor 647-labeled AAV6-CMV-GFP was used. 8e+9 vg of AAV was mixed with 16 pg TA and 4.8 pg Fe to a final volume of 8 pL, then mixed into 10 pL of whole blood. The samples were quenched with PBS with 5 w / v% BSA. The resulting samples were fixed with 4% PFA for 10 min at room temperature. Next, the RBC membrane was stained with a lipophilic dye, DiO, following the manufacturer’s protocol. The resulting samples were run on a flow cytometer.

[0309] Statistical analysis. Data are presented as mean ± SEM. Data were analyzed using Student’s t-test, one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test, or two-way ANOVA followed by Sidak’s multiple comparisons test to compare multiple groups using Prism 10 (GraphPad Software). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

[0310] References1. S. Maurya, P. Sarangi, G. R. Jayandharan. Safety’ of Adeno-associated virus-based vector- mediated gene therapy -impact of vector dose. Cancer Gene Ther 29, 1305-1306 (2022). 2. C. E. Dunbar et al., Gene therapy conies of age. Science 359, (2018).3. Y. Meng et al., Erratum: Cell-penetrating peptides enhance the transduction of adeno- associated virus serotype 9 in the central nervous system. Mol Ther Methods Clin Dev 26, 1-3 (2022).4. C. Barnes, O. Scheideler. D. Schaffer, Engineering the AAV capsid to evade immune responses. Curr Opin Biotechnol 60, 99-103 (2019).5. C. Li, R. J. Samulski, Engineering adeno-associated virus vectors for gene therapy. Nat Rev Genet 21, 255-272 (2020).6. E. Smalley, First AAV gene therapy poised for landmark approval. Nat Biotechnol 35, 998- 999 (2017).86Attorney Docket No: 002806-000156WOPTA. Mullard, FDA approves first gene therapy for Duchenne muscular dystrophy, despite internal objections. Nat Rev Drug Discov 22, 610 (2023).F. Arabi, V. Mansouri, N. Ahmadbeigi, Gene therapy clinical trials, where do we go? An overview. Biomed Pharmacother 153, 113324 (2022).H. K. E. Au, M. Isalan, M. Mielcarek, Gene Therapy Advances: A Meta-Analysis of AAV Usage in Clinical Settings. Front Med (Lausanne) 8, 809118 (2021).L. O. Whiteley, An Overview of Nonclinical and Clinical Liver Toxicity Associated With AAV Gene Therapy. Toxicol Pathol 51. 400-404 (2023).M. F. Naso, B. Tomkowicz, W. L. Perry. 3rd, W. R. Strohl. Adeno-Associated Virus (AAV) as a Vector for Gene Therapy. BioDrugs 31.317-334 (2017).G. E. Berry. A. Asokan, Cellular transduction mechanisms of adeno-associated viral vectors. Curr Opin Virol 21, 54-60 (2016).M. A. Kotterman. D. V. Schaffer, Engineering adeno-associated viruses for clinical gene therapy. Nat Rev Genet 15, 445-451 (2014).M. S. Ghauri, L. Ou, AAV Engineering for Improving Tropism to the Central Nervous System. Biology (Basel) 12. (2023).A. Srivastava, Rationale and strategies for the development of safe and effective optimized AAV vectors for human gene therapy. Mol Ther Nucleic Acids 32, 949-959 (2023).D. Goertsen. N. Goeden, N. C. Flytzanis. V. Gradinaru. Targeting the lung epithelium after intravenous delivery by directed evolution of underexplored sites on the AAV capsid. Mol Ther Methods Clin Dev 26, 331-342 (2022).S. Russell et al., Efficacy and safety of voretigene neparvovec (AAV2-hRPE65v2) in patients with RPE65-mediated inherited retinal dystrophy: a randomised, controlled, open-label, phase 3 trial. Lancet 390, 849-860 (2017).P. Batty, D. Lillicrap, Advances and challenges for hemophilia gene therapy. Hum Mol Genet 28, R95-R101 (2019).D. Duan, Systemic delivery of adeno-associated viral vectors. Curr Opin Virol 21, 16-25 (2016).T. R. Flotte et al., AAV gene therapy for Tay-Sachs disease. Nat Med 28, 251-259 (2022). A. C. Anselmo et al., Delivering nanoparticles to lungs while avoiding liver and spleen through adsorption on red blood cells. ACS Nano 1, 11129-11137 (2013).J. S. Brenner et al.. Red blood cell-hitchhiking boosts delivery of nanocarriers to chosen organs by orders of magnitude. Nat Commun 9, 2684 (2018).A. Ukidve et al., Erythrocyte-driven immunization via biomimicry of their natural antigen-presenting function. Proc Natl Acad Sci U S A 117, 17727-17736 (2020).87Attorney Docket No: 002806-000156WOPT24. J. S. Brenner. S. Mitragotri, V. R. Muzykantov, Red Blood Cell Hitchhiking: A Novel Approach for Vascular Delivery of Nanocarriers. Annu Rev Biomed Eng 23, 225-248 (2021).25. I. V. Zelepukin et al., Nanoparticle-based drug delivery via RBC-hitchhiking for the inhibition of lung metastases growth. Nanoscale 11, 1636-1646 (2019).26. Z. Zhao, A. Ukidve, Y. Gao, J. Kim, S. Mitragotri, Erythrocyte leveraged chemotherapy (ELeCt): Nanoparticle assembly on erythrocyte surface to combat lung metastasis. Sci Adv 5, eaax9250 (2019).27. V. Lenders et al., Modularity of RBC hitchhiking with polymeric nanoparticles: testing the limits of non-covalent adsorption. J Nanobiotechnology 20, 333 (2022).28. Z. Zhao et al., Red Blood Cell Anchoring Enables Targeted Transduction and ReAdministration of AAV-Mediated Gene Therapy. Adv Sci (Weinh) 9, e2201293 (2022). 29. R. S. Apte, D. S. Chen, N. Ferrara, VEGF in Signaling and Disease: Beyond Discovery and Development. Cell 176, 1248-1264 (2019).30. C. L. Halbert, J. M. Allen, A. D. Miller, Adeno-associated virus type 6 (AAV6) vectors mediate efficient transduction of airway epithelial cells in mouse lungs compared to that of AAV2 vectors. J Virol 75, 6615-6624 (2001).31. D. Pozzi. G. Caracciolo, Looking Back, Moving Forward: Lipid Nanoparticles as a Promising Frontier in Gene Delivery. ACS Pharmacol Transl Sci 6, 1561-1573 (2023).32. H. Mukai, K. Ogawa, N. Kato, S. Kawakami. Recent advances in lipid nanoparticles for delivery of nucleic acid. mRNA, and gene editing-based therapeutics. Drug Metab Pharmacokinet 44, 100450 (2022).Example 2

[0311] Preparation of LNP

[0312] LNP was prepared based on our previous report (7). Briefly, powdered lipids (DSPC, DMG-PEG, and cholesterol) were weighed and dissolved in pure ethanol to make 5 mg / mL stocks of each lipid. These stocks were then mixed with (6Z,9Z,28Z,31Z)-heptatriacont-6,9,28,31-tetraene-19-yl4-(dimethylamino)butanoate (DLin-MC3-DMA) to give a final DLin-MC3-DMA:cholesterol: DSPC: DSPE-Cy7: DMG-PEG molar ratio of 50:34.65:3.85:10:1.5. Lastly, the final total lipid concentration was adjusted to 6.25 mM by adding ethanol.

[0313] Luciferase mRNA (L-7202; TriLink BioTechnologies) was prepared at 55.8 pg / mL in 100 mM citrate buffer pH 4), mixed with the lipid stocks, and loaded into syringes. Precision Nanosystems NanoAssemblr Benchtopand / or Ignite was used to flow the mixtures through a microfluidic device for LNP synthesis. The resulting LNP had an average hydrodynamic diameter of 88 nm, PDK0.02, and a zeta potential average of -30 mV.88Attorney Docket No: 002806-000156WOPT

[0314] Cryosectioning & imaging of tissues. After CO2 euthanasia, mice were perfused with 10- 20 mL of PBS, and then the lungs were extracted and stored in PBS containing 1% FBS. The lungs were submerged in OCT compound (Sakura) inside mini cassettes and stored at -80 °C overnight. Samples were sectioned with a cryostat (Thermo Scientific) to a 10 pm thickness, mounted onto a slide-glass (Fisherbrand, FIS 1255015), and stored at -80 °C until further processing. For staining, samples were brought to room temperature for 30 min, fixed with acetone at -20 °C for 10 min, and then placed back at room temperature for 10 min to dry the acetone. Next, the samples were washed with PBS. blocked with a blocking buffer (10% goat serum and 1% BSA in PBS) for 1 hour at room temperature, and stained with an antibody cocktail prepared by mixing the antibodies listed in Table 4

[0315] Table 4. List of antibodies for tissue section stainingTarget Fluor Dilution Manufacturer Cat. No.Rabbit anti-mouse GFP 1:200 Fabgennix GFP-101APGoat anti-rabbit IgG AF488 4 pg / mL Invitrogen A-11008

[0316] The resulting samples were washed with PBS twice, then covered with a cover slip using DAPI-containing mounting media (Invitrogen. P36961). They were stored at room temperature overnight until imaged with a fluorescence microscope (Axioscan, Zeiss).

[0317] Flow cytometry gating strategy. Gating on endothelial (CD31) and epithelial (CD326) cells for flow cytometry was done following previously reported gating strategies for the two cell types (2, 3). A detailed gating scheme is shown in Fig. 18.

[0318] Overview of the master targeting equation. The drug flux, / , across a vascular membrane with a permeability, Pvas. due to a concentration difference of Ac, can be expressed from Fick’s law7as,i = J AUnder the following assumptions.(a) The concentration of the drug on the exterior of the vasculature (in the tissue of interest) remains approximately 0 (or is much smaller than the concentration in blood),(b) Drug distribution and clearance follows a single-compartment PK model, and(c) Drug concentration in blood changes solely due to clearance (i.e., clearance rates are much higher than tissue adsorption rates),The flux can coimect to vascular drug concentration evolution analytically, giving the master targeting equation as follows:89Attorney Docket No: 002806-000156WOPTJ = CAAfT(1) J, drug flux into tissues from blood circulation is a function of the initial drug concentration in blood upon injection (Cs), the enhancement of local tissue concentration of the drug achieved by the targeting strategythe vascular endothelial permeability in the target tissueand the time constant of blood clearance of the drug (T).

[0319] In the case of RBC hitchhiking,and r can be depicted asand τ where ARBCis the amplification in local concentration at the target tissue due to the drug being loaded on the surface of RBC relative to free drug, and τRBCis the half-life of drugs loaded on RBC, giving die equation below (2):This equation can be plotted into graphs with several example valuesfor and τ as shown in Fig. 19:

[0320] Theoretical improvement in drug flux into tissue based on specific values. The total accumulated drug mass in the tissue can be obtained by recognizing J = — — —, and integrating theabove equation with respect to time, yielding,Q=^saas^S& C- Here, Q is the amount of drug accumulated, Avasis the area of vasculature. The enhancement in drug accumulation for RBC hitching with VEGF as compared to the current state of the art can be obtained as,_T«BC Aits.®q ~ r R,is£C' The following geometrical and RBC-migration argument can be made to estimating λRBCRBC squeezing through a capillary of radius J? with a gap & R. traps a volume per unit length of ZxR& R (Fig. 20A). The mass of drugs released from RBCs is distributed in this smaller volume rather than a volume per unit length ^-srR^of a large blood vessel. Assuming perfect drug detachment from the surface, this implies, an enhancement in drug concentration can be expected of the order ofwhich is about 10 based on known RBC hemodynamics (4).

[0321] In larger blood vessels, RBCs migrate away from the vascular surface due to shear-induced lift forces (5). As a result, the gap size increases, and shear-induced detachment decreases. Consequently,is expected to have a low value (at or below 1).

[0322] Compared to the traditional case, where we assume P, A, and r as (1, 1, 1), 2 fold increase in vascular permeability achieved by VEGF 2 fold increase in the availability of AAV in circulation (τRBC), and 10-fold increase in local tissue accumulation of AAV lead to significantly90Attorney Docket No: 002806-000156WOPThigher and durable drug flux into tissue (Fig. 20B), and overall 40-fold increased accumulation of AAV at the target tissue (Fig. 20C), based on the master targeting equation (1).

[0323] Putting2 and 2 into equation (3),jsexpected to beapproximately equal to 40, matching the enhancement value from above.

[0324] References1. A. M. Curreri et al., Localization of Intramuscular mRNA Delivery Using Deep Eutectic-Lipid Nanocomposites. Adv Healthc Mater 13, e2400327 (2024).2. S. Ghosh et al., Enteric viruses replicate in salivary glands and infect through saliva. Nature 607, 345-350 (2022).3. F. Palumbo et al., Impact of different tissue dissociation protocols on endothelial cell recovery from developing mouse lungs. Cytometry A 105, 521-535 (2024).4. T. W. Secomb, R. Hsu, A. R. Pries, Motion of red blood cells in a capillary with an endothelial surface layer: effect of flow velocity. Am J Physiol Heart Circ Physiol 281, H629-636 (2001).5. Z. Zhao et al., Engineering of Living Cells with Polyphenol-Functionalized Biologically Active Nanocomplexes. Adv Mater 32, e2003492 (2020).Example 3

[0325] IgG (tagged with Alexa Fluor 647) was intravenously injected into mice either as free IgG or IgG (100 pg) + tannic acid (TA) (100 pg) + Iron (FeCl3) (30 pg). The biodistribution was examined after 1 hour (Fig. 30, 31). Delivery with TA and iron enhanced delivery' to the lungs (Fig.32). Delivery' with TA and iron reduced antibody signals in the serum. (Fig. 33).

[0326] 100 pg of AF647-labeled mouse IgG was intravenously injected into the tail vein. Lungs were harvested 1 hr after the injection, fixed in 4% PFA overnight, and transferred to 30% sucrose for freezing. Images are taken right before freezing. Lungs from mice treated with IgG+TA+Fe appear visibly darker compared to those treated with free IgG (Fig. 34). Cryo sectioning of the lungs and examination with a fluorescence microscope showed that IgG+TA+Fe was well distributed throughout the lung tissue (Fig. 35).

[0327] These results were obtained without having to attach the IgG+TA+Fe to red blood cells ex vivo. IgG accumulated in high quantities in the lungs, which is the therapeutic target.

[0328] This demonstrates that the compositions and methods described herein provide greater ease of use than prior technologies. Technologies which require ex vivo attachment to red blood cells are "cell products”, which are more logistically challenging to manufacture and deliver. There is greater risk of failure and contamination with such technologies. This currently described technology91Attorney Docket No: 002806-000156WOPTavoids these disadvantages as it is like a 'drug product', injected directly in vivo without cells being present in the formulation.

[0329] The current technology can be used, e.g., to treat lung cancer (primary or metastasis), in particular, using a combination of antibodies (PD-1 and VEGF). Contemplated herein is a mixture of these two antibodies in our IgG+TA+Fe system described herein or a bispecific PD-l / VEGF antibody, providing improved delivery characteristics compared to free combinations or bispecific PD-l / VEGF antibodies.Example 4

[0330] Red blood cells were contacted with different combinations of tannic acid (at 0.625, 1.25, 2.5, 5, 10, 20, or 80 pM) and a metal (strontium, iron, nickel, titanium, gold, aluminum, zinc, manganese, or gadolinium; at 0.039063, 0.078125. 0.15625, 0.3125. 0.625, 1.25, 2.5, 5, 10, 20, or 40 mM) in 60% PBS. The cells were then assessed for agglutination and hemolysis to determine the levels of TA and metal that were effective for each metal. Figs. 36-44.92

Claims

Attorney Docket No: 002806-000156WOPTWhat is claimed herein is:

1. A composition comprising:a) one or more polyphenol molecules;b) one or more biomolecules or active agents; andc) at least one of:i) at least one multivalent ion; andii) at least one endothelium permeabilization agent.

2. The composition of any one of the preceding claims, comprising:a) one or more polyphenol molecules;b) one or more biomolecules or active agents; andc) at least one multivalent ion.

3. The composition of any one of the preceding claims, comprising:a) one or more polyphenol molecules;b) one or more biomolecules or active agents; andc) at least one endothelium permeabilization agent.

4. The composition of any one of the preceding claims, comprising:a) one or more polyphenol molecules;b) one or more biomolecules or active agents;c) at least one multivalent ion; andd) at least one endothelium permeabilization agent.

5. The composition of any one of the preceding claims, wherein the at least one multivalent ion is selected from the group consisting of: Al, Gd(III), Au(III), Fe(III), Mn (II), Ni(II), Sr, Ti(IV). and Zn.

6. The composition of any one of the preceding claims, wherein the at least one multivalent ion comprises Fe(III).

7. The composition of any one of the preceding claims, wherein the at least one multivalent ion is at a concentration of no more than 1 mM.

8. The composition of any one of the preceding claims, wherein the at least one multivalent ion is at a concentration of at least 2 mM.

9. The composition of any one of the preceding claims, wherein the at least one multivalent ion is at a concentration of 2 mM to 37 mM.

10. The composition of any one of the preceding claims, wherein the multivalent ion is at a concentration of 5 mM to 20 mM.

11. The composition of any one of the preceding claims, wherein the multivalent ion is at a concentration of 0.01 mM to 10 mM.93Attorney Docket No: 002806-000156WOPT12. The composition of any one of the preceding claims, wherein the multivalent ion is at a concentration of 0.05 mM to 10 mM.

13. The composition of any one of the preceding claims, wherein the multivalent ion is at a concentration of 0.01 mM to 0.5 mM.

14. The composition of any one of the preceding claims, wherein the multivalent ion is at a concentration of 0.05 mM to 0.5 mM.

15. The composition of any one of the preceding claims, wherein the polyphenol is at a concentration of at least 0.1 μM.

16. The composition of any one of the preceding claims, wherein the polyphenol is at a concentration of at least 0.5 μM.

17. The composition of any one of the preceding claims, wherein the polyphenol is at a concentration of 0.5 pM to 1.5 pM.

18. The composition of any one of the preceding claims, wherein the polyphenol is at a concentration of at least 5 μM.

19. The composition of any one of the preceding claims, wherein the polyphenol is at a concentration of at least 10 pM.

20. The composition of any one of the preceding claims, wherein the polyphenol is at a concentration of at least 10 μM and the at least one multivalent ion is at a concentration of no more than 1 mM.

21. The composition of any one of the preceding claims, wherein die polyphenol is at a concentration of at least 0.1 μM and the at least one multivalent ion is at a concentration of 0.01 mM to 10 mM.

22. The composition of any one of the preceding claims, wherein the one or more polyphenols collectively comprise at least one galloyl moiety and / or at least one catechol moiety.

23. The composition of any one of the preceding claims, wherein the one or more polyphenols collectively comprise at least one galloyl moiety and at least one catechol moiety.

24. The composition of any one of the preceding claims, wherein the one or more polyphenols each comprise at least one galloyl moiety and at least one catechol moiety.

25. The composition of any one of the preceding claims, wherein the polyphenol is tannic acid.

26. The composition of any one of the preceding claims, wherein the stoichiometric ratio of polyphenol molecules to biomolecules is 570 or less relative polyphenol.

27. The composition of any one of the preceding claims, wherein the stoichiometric ratio of tannic acid molecules to biomolecules is 190 to 570.

28. The composition of any one of the preceding claims, wherein the stoichiometric ratio of tannic acid molecules to biomolecules is 190.94Attorney Docket No: 002806-000156WOPT29. The composition of any one of the preceding claims, wherein the one or more biomolecules and / or active agents are selected from: a nucleic acid, protein, a viral particle, a viral vector, a lipid nanoparticle, a polymer, alkaloid, polysaccharide, anthocyanin, lipid, antiviral drug, antibiotic, chemotherapeutic, or combination thereof.

30. The composition of claim 29, wherein the one or more biomolecules and / or active agents comprises or is a protein.

31. The composition of claim 30. wherein the protein is selected from: ovalbumin, serum albumin, interleukin-4, an antibody or antibody reagent, cholera toxin subunit B, biotin, cytokine, or lectin.

32. The composition of claim 31. wherein the antibody or antibody reagent is specific for an immune checkpoint protein.

33. The composition of claim 31. wherein the antibody or antibody reagent is specific for PD1 or PD-L1.

34. The composition of claim 31, wherein the antibody or antibody reagent is specific for an immune checkpoint protein and VEGF.

35. The composition of claim 31, wherein the antibody or antibody reagent is specific for i) PD1 or PD-L1 and ii) VEGF.

36. The composition of claim 29, wherein the one or more biomolecules and / or active agents comprise i) an immune checkpoint protein inhibitor and ii) a VEGF inhibitor.

37. The composition of claim 29, wherein the one or more biomolecules and / or active agents comprise i) an PD-1 inhibitor and / or a PD-L1 inhibitor and ii) a VEGF inhibitor.

38. The composition of claim 29, wherein the biomolecule and / or active agent is a viral particle or viral vector.

39. The composition of claim 38, wherein the viral particle or viral vector is an adeno-associated virus vector.

40. The composition of claim 39, wherein the adeno-associated virus vector is AAV9 or AAV6.

41. The composition of any one of the preceding claims, wherein the at least one endothelium permeabilization agent is selected from the group consisting of:a Vascular Endothelial Growth Factor (VEGF) polypeptide; histamine; bradykinin; and serotonin.

42. The composition of any one of the preceding claims, wherein the at least one endothelium permeabilization agent is a Vascular Endothelial Growth Factor (VEGF) polypeptide.

43. The composition of any one of the preceding claims, not comprising a cell.

44. The composition of any one of the preceding claims, wherein the cell is a hematopoietic cell.

45. The composition of any one of the preceding claims, wherein the hematopoietic cells is a red blood cell.95Attorney Docket No: 002806-000156WOPT46. A functionalized mammalian cell comprising at least one composition of any one of claims 1- 45 adhered to the surface of the cell.

47. The cell of claim 46, wherein the cell is a hematopoietic cell.

48. The cell of claim 46, wherein the cell is an erythrocyte, B cell, T cell, monocyte, macrophage, neutrophil or natural killer cell.

49. The cell of any one of claims 46-48, wherein the biomolecule and / or active agent is an antibody or antibody reagent specific for an immune checkpoint protein and the cell is a macrophage.

50. The cell of any one of claims 46-48, wherein the biomolecule and / or active agent is an antibody or antibody reagent, cytokine, antiviral drug, antibiotic, viral particle, viral vector, or siRNA and the cell is an erythrocyte.

51. The cell of any one of claims 46-48, wherein the biomolecule and / or active agent is an antibody or antibody reagent, siRNA, or chemotherapeutic and the cell is a natural killer cell.

52. The cell of any one of claims 46-48, wherein the biomolecule and / or active agent is cytokine and the cell is a T cell.

53. The cell of any one of claims 46-48. wherein the biomolecule and / or active agent is an antiinflammatory drug and the cell is a neutrophil.

54. The cell of any one of claims 46-53. wherein compositions of claims 1-45 collectively comprising 10 to 1 trillion biomolecules are adhered to the surface of the cell.

55. A method of functionalizing a mammalian cell, the method comprising: contacting a mammalian cell with the composition of any one of claims 1-45;whereby the combination adheres to the surface of the cell.

56. A method of administering a biomolecule and / or active agent to a patient in need of treatment with the biomolecule and / or active agent, the method comprising administering the composition of any one of claims 1-45 or the cell of any of claims 46-54 to the patient.

57. The method of claim 56, wherein the cell is autologous to the patient.

58. The method of any one of claims 56-57, wherein the administration is via intravenous injection and a plurality of the biomolecule administered to the patient is delivered to the lungs.

59. The method of any one of claims 56-57. wherein the administration is via intravenous injection in a vein in a limb and a plurality of the biomolecule administered to the patient is delivered to the lungs.96Attorney Docket No: 002806-000156WOPT60. The method of any one of claims 56-57, wherein the administration is via injection to the carotid artery and a plurality of the biomolecule administered to the patient is delivered to the brain.

61. The method of any one of claims 56-57, wherein the cell is an erythrocyte and a plurality of the biomolecule and / or active agent administered to the patient is delivered to the lungs.

62. The method of any one of claims 56-57. wherein the cell is a macrophage and a plurality of the biomolecule and / or active agent administered to the patient is delivered to the brain, a tumor, or a site of inflammation or autoimmune inflammation.

63. The method of any one of claims 56-57. wherein the cell is a natural killer cell and a plurality of the biomolecule and / or active agent administered to the patient is delivered to a tumor.

64. The method of any one of claims 56-57. wherein the cell is a T cell and a plurality of the biomolecule and / or active agent administered to the patient is delivered to a tumor.

65. The method of any one of claims 56-57. wherein the cell is a neutrophil and a plurality of the biomolecule and / or active agent administered to the patient is delivered to the lungs or a site of inflammation.

66. The method of any one of claims 56-65, wherein the tumor, inflammation, or autoimmune inflammation is in the lung.

67. The method of any one of claims 56-65, wherein the tumor is in the lung.

68. The method of any one of claims 56-65, wherein the patient is in need of treatment for lung cancer.

69. The method of any one of claims 56-65, wherein the patient is in need of treatment of a disease of or in the lung.

70. A cell of any one of claims 46-54, for use in a method of administering a biomolecule and / or active agent to a patient in need of treatment with the biomolecule and / or active agent, the method comprising administering the functionalized cell to the patient.

71. The cell of claim 70, wherein the cell is autologous to the patient.

72. The cell of any one of claims 70-71, wherein the cell is an erythrocyte and a plurality of the biomolecule and / or active agent administered to the patient is delivered to the lungs.

73. The cell of any one of claims 70-71, wherein the cell is a macrophage and a plurality of the biomolecule and / or active agent administered to the patient is delivered to the brain, a tumor, or a site of inflammation or autoimmune inflammation.

74. The cell of any one of claims 70-71, wherein the cell is a natural killer cell and a plurality of the biomolecule and / or active agent administered to the patient is delivered to a tumor.

75. The cell of any one of claims 70-71. wherein the cell is a T cell and a plurality' of the biomolecule and / or active agent administered to the patient is delivered to a tumor.97Attorney Docket No: 002806-000156WOPT76. The cell of any one of claims 70-71, wherein the cell is a neutrophil and a plurality of the biomolecule and / or active agent administered to the patient is delivered to the lungs or a site of inflammation.

77. A composition of any one of claims 1-45, for use in a method of administering a biomolecule and / or active agent to a patient in need of treatment with the biomolecule and / or active agent.

78. The method of claim 77, wherein patient is in need of treatment of a tumor, inflammation, or autoimmune inflammation in the lung.

79. The method of claim 77 or 78, wherein the tumor is in the lung.

80. The method of any one of claims 77-79. wherein the patient is in need of treatment for lung cancer.

81. The method of any one of claims 77-80, wherein the patient is in need of treatment of a disease of or in the lung.

82. A method of administering a viral vector and / or reducing the immune clearance of viral vectors, the method comprising administering the composition of any one of claims 1-45 or the cell of any of claims 46-54, wherein the biomolecule and / or active agent is a viral vector or viral particle.

83. The method of claim 82, wherein the cell is a red blood cell.

84. The method of any one of claims 82-83, wherein the viral vector or viral particle is an AAV viral vector or AAV viral particle.

85. The method of claim 84, wherein the AAV is AAV9 or AAV6.

86. The method of claim 84, wherein the AAV is AAV9.

87. The method of claim 84, wherein the AAV is AAV6.

88. A method of gene therapy comprising administering the composition of any one of claims 1- 45 or the cell of any of claims 46-54 to the patient, wherein the biomolecule and / or active agent comprises a nucleic acid sequence, e.g., a nucleic acid sequence suitable for or configured for gene therapy.

89. The method of claim 88, wherein the cell is a red blood cell.

90. The method of claim 88 or 89, wherein the gene therapy target is primarily in the lungs.

91. The method of claim 88 or 89, wherein the gene therapy target is primarily in the brain.

92. The method of any one of claims 88-91. wherein the biomolecule and / or active agent is a viral vector or viral particle.98Attorney Docket No: 002806-000156WOPT93. The method of any one of claims 88-92, wherein the biomolecule and / or active agent is an AAV viral vector or AAV viral particle.

94. The method of claim 93, wherein the AAV is AAV9 or AAV6.

95. The method of claim 93, wherein the AAV is AAV9.

96. The method of claim 93, wherein the AAV is AAV6.99

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