WO2025217538A2 - Délivrance ciblée d'agents thérapeutiques à base d'arnm au parenchyme pulmonaire à l'aide d'un système de délivrance à un composant pour acides nucléiques - Google Patents

Délivrance ciblée d'agents thérapeutiques à base d'arnm au parenchyme pulmonaire à l'aide d'un système de délivrance à un composant pour acides nucléiques

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Publication number
WO2025217538A2
WO2025217538A2 PCT/US2025/024306 US2025024306W WO2025217538A2 WO 2025217538 A2 WO2025217538 A2 WO 2025217538A2 US 2025024306 W US2025024306 W US 2025024306W WO 2025217538 A2 WO2025217538 A2 WO 2025217538A2
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Prior art keywords
mrna
seq
iajd34
tgf
composition
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WO2025217538A3 (fr
Inventor
Elena ATOCHINA-VASSERMAN
Drew Weissman
Andrew Gow
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Rutgers State University of New Jersey
University of Pennsylvania Penn
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Rutgers State University of New Jersey
University of Pennsylvania Penn
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Publication of WO2025217538A3 publication Critical patent/WO2025217538A3/fr
Pending legal-status Critical Current
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7115—Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine

Definitions

  • This application contains a Sequence Listing, which is submitted electronically via EFS-Web as an XML Document formatted sequence listing with a file name “046483- 6284-OOWO Sequence Listing. xml,” having a creation date of April 11, 2025, and having a size of 46,487 bytes.
  • the sequence listing submitted via EFS-Web is part of the specification and is herein incorporated by reference in its entirety.
  • Acute lung injury is a prevalent condition in the United States, with 200,000 new diagnoses each year, resulting in high morbidity and mortality (Johnson et al., 2010, J. Aerosol Med. Pulm. Drug Deliv. 23, 243-252; Dushianthan et al., 2011, Postgrad. Med. J. 87, 612-622; Stevenson et al., 2022, J. Pharmacol. Exp. Ther. 382, 356-365; Wilkinson et al., 2020, Toxicol. Appl. Pharmacol. 407, 115236).
  • LNP lipid nanoparticle
  • the invention relates to a composition for delivering an agent to the lung parenchyma of a subject in need thereof, wherein the method comprises administering at least one nanoparticle comprising an ionizable amphiphilic Janus dendrimer (IAJD), wherein the IAJD encapsulates a therapeutic agent, or a composition comprising the same to the subject.
  • IAJD comprises a formula of:
  • the therapeutic agent comprises an anti-inflammatory agent.
  • the therapeutic agent comprises TGF-P, Alpha-1 - antitrypsin (A1AT), soluble IL-1R, CFTR, IL-4, IL-13, Surfactant Protein D fragment (SP-D fragment), surfactant protein B (SP-B) or surfactant protein C (SP-C) or a fragment or variant thereof.
  • the therapeutic agent comprises a nucleic acid molecule comprising SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO: 11.
  • the invention relates to a method of delivering an agent to the lung parenchyma of a subject in need thereof, wherein the method comprises administering at least one nanoparticle comprising an ionizable amphiphilic Janus dendrimer (IAJD), wherein the IAJD encapsulates a therapeutic agent, or a composition comprising the same to the subject.
  • IAJD ionizable amphiphilic Janus dendrimer
  • the IAJD comprises a formula of:
  • the method treats or inflammation, a viral infection, bacterial infection, fungal infection, parasitic infection, cancer, disease or disorder associated with cancer, autoimmune disease or disorder, or any combination thereof.
  • the method treats or prevents asthma, chronic obstructive pulmonary disease (COPD), emphysema, acute respiratory distress syndrome (ARDS), lung cancer, sarcoidosis, asbestosis, chronic cough, pneumothorax, pulmonary embolism, pleural effusion, rheumatoid lung disease, pulmonary fibrosis, bronchiectasis, tuberculosis, bronchitis, or pneumonia.
  • the lung cancer is non-small cell lung cancer.
  • the method treats or prevents acute lung injury (ALI), emphysema, cystic fibrosis or idiopathic fibrosis.
  • ALI acute lung injury
  • emphysema emphysema
  • cystic fibrosis emphysema
  • idiopathic fibrosis emphysema
  • the therapeutic agent comprises at least one selected from the group consisting of cDNA, cRNA, CirRNA, mRNA, miRNA, siRNA, sgRNA, modified RNA, tRNA, antagomir, antisense molecule, targeted nucleic acid, and any combination thereof.
  • the therapeutic agent is an mRNA molecule.
  • the therapeutic agent is an mRNA molecule encoding TGF-P, Alpha-1- antitrypsin (A1AT), soluble IL-1R, CFTR, IL-4, IL-13, Surfactant Protein D fragment (SP-D fragment), surfactant protein B (SP-B) or surfactant protein C (SP-C) or a fragment or variant thereof.
  • the therapeutic agent is an mRNA molecule comprising SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:11.
  • the therapeutic agent is an mRNA molecule transcribed from SEQ ID NO:2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO:8, SEQ ID NO: 10 or SEQ ID NO: 12
  • the therapeutic agent is a nucleoside-modified RNA.
  • the nucleoside-modified RNA comprises pseudouridine.
  • the nucleoside-modified RNA comprises pseudouridine plus 5 -methyl -cytosine.
  • the nucleoside-modified RNA comprises 5-methyl-uridine.
  • the nucleoside-modified RNA comprises 1-methyl-pseudouridine.
  • Figure 1 depicts data demonstrating the characterization of mRNA-IAJD34 formulation.
  • IAJD34 formulated as empty-IAID34, Luc mRNA-IAJD34, and TGF-[3 -IAJD34 were evaluated using ( Figure 1 A) size (nm), poly dispersity index (PDI), zeta potential, encapsulation efficiency (% EE) (Data presented as mean + SE) and ( Figure IB) Cryo-TEM.
  • Figure 2 depicts data demonstrating the specificity of IAJD34 for targeted mRNA delivery to the lung.
  • IAJD34 formulated with luciferase mRNA (Luc mRNA-IAID34) was i.v. injected into BALB/c mice and analyzed for efficient pulmonary mRNA using IVIS.
  • Figure 2A depicts data demonstrating the chemical structure of one- component IAJD3440.
  • Figure 2B depicts data demonstrating the specificity of Luc mRNA- IAJD34 delivery to the lung.
  • IVIS images of whole-body and organs of mice at 4 h postinjection of 10 pg Luc mRNA-IAID34 show luminescent biodistribution of luciferase expression predominantly in the lungs. IVIS images were analyzed, and the photon radiance (s ' sr 1 m 2 ) of each image was quantified. The quantitative bioluminescence imaging (BLI) data are shown as total flux (p/s).
  • Figure 2C depicts data demonstrating a time course of luciferase expression in vivo.
  • Figure 2E depicts data demonstrating the dose-dependent pulmonary delivery of Luc mRNA-IAJD34. Representative IVIS images of whole-body and organs of mice injected with Luc mRNAIAJD34 at doses of 10, 20, or 30 pg, imaged at 24 h post-injection.
  • FIG. 2F depicts data demonstrating the delivery of Luc mRNA-IAJD34 to the lung parenchyma.
  • IHC immunohistochemistry
  • FIG. 3 depicts a table of the specificity of IAJD34 for targeted mRNA delivery to the lung over time.
  • IAJD34 formulated with luciferase mRNA (Luc mRNA-IAJD34) was i.v. injected into BALB/c mice and analyzed for efficient pulmonary delivery of mRNA using IVIS.
  • IVIS images were analyzed and the photon radiance (s 'sr 'm 2 ) of each image was quantified.
  • the quantitative bioluminescence imaging (BLI) data are shown as total flux photon/sec (p/s) at various time points following the injection of 10 pg Luc mRNA-IAJD34. Exact n is indicated in the table. Data presented as mean + SE
  • Figure 4 depicts data demonstrating the evaluation of stability of Luc and TGF-P mRNA-IAJD34 formulation.
  • Figure 4A depicts data demonstrating Luc mRNA-IAJD34 at dose 10 pg per mouse was injected into BALB/c mice 1 hour after formulating (fresh), or 5 days after storing formulations at 4C.
  • IVIS images of whole-body of mice at 4, 24, and 48 hrs post-injection of 10 pg Luc mRNA-IAJD34 were analyzed. IVIS images were analyzed and the photon radiance (s 'sr 'm 2 ) of each image was quantified. The quantitative BLI data are shown as total flux photon/sec (p/s).
  • FIG. 4B depicts data demonstrating TGF-P mRNA-IAJD34 formulation at dose 10 pg was evaluated for size (nm) and PDI at 1,2, 3, 4, and 5 weeks after storing formulations at 4°C. Data presented as mean + SE. Exact n is indicated in the table, depicts a table demonstrating an inflammatory response to TGF-P mRNA delivery with IAJD34 only at high doses.
  • TGF mRNA formulated with IAID34 IAJD34 [TGF-P] 10-30 pg/mouse
  • BAL bronchoalveolar lavage
  • NP neutrophil
  • LP Lymphocyte
  • FIG. 5 depicts a table of the specificity of IAJD34 for targeted mRNA delivery to the lung at multiple doses.
  • IAJD34 formulated with luciferase mRNA (Luc mRNA-IAJD34) was i.v. injected into BALB/c mice and analyzed for efficient pulmonary delivery of mRNA using IVIS in whole body and organs 24 hrs post injection.
  • IVIS images were analyzed and the photon radiance (s 'sr 'm 2 ) of each image was quantified.
  • the quantitative BLI data are shown as total flux photon/sec (p/s). Exact n is indicated in the table. Data presented as mean + SE.
  • FIG. 6 depicts data demonstrating the specificity of IAJD34 for targeted mRNA delivery to the lung at multiple doses.
  • IAJD34 formulated with luciferase mRNA (Luc mRNA-IAJD34) was i.v. injected into BALB/c mice and analyzed for efficient pulmonary delivery of mRNA using IVIS.
  • Data are individual presentation of whole-body measurements 24 hrs post injection from Figure 3.
  • IVIS images were analyzed and the photon radiance (s -1 sr _1 m" 2 ) of each image was quantified.
  • the quantitative BLI data are shown as total flux photon/sec (p/s). Exact n are indicated in Figure 5.
  • Figure 7 depicts data demonstrating the confirmation of TGF-0 mRNA expression and protein production.
  • Figure 7A depicts data demonstrating that TGF-P mRNA was produced from plasmids, purified using cellulose, and imaged using gel electrophoresis to confirm mRNA presence and quality.
  • Figure 7B depicts data demonstrating that human embryonic kidney (HEK) 293 cells were seeded into a 24 well plate at 150,000 cells/well and transfected with 500 ng mRNA/well or no mRNA for negative control (NC) according to Methods and Materials.
  • HEK human embryonic kidney
  • Figure 8 depicts data demonstrating the evaluation of dose-dependent toxicity of TGFp mRNA delivery with IAJD34.
  • Various doses of TGFp mRNA-IAJD34 (10, 20, and 30 pg per mouse) were delivered to BALB/c mice and compared to naive animals.
  • BAL fluid and cells, whole lung tissue, and serum were collected.
  • Figure 8A depicts data demonstrating that BAL cells were fixed and stained using a Hema 3 Stat kit. Total BAL protein, differential BAL cell count, and BAL phospholipid levels were quantified as markers of lung injury: MP, Macrophage, NP, neutrophil, LP, Lymphocyte.
  • Figure 8B depicts representative cell differentials.
  • Figure 8C depicts data demonstrating that formaldehyde-fixed lung tissue was processed, stained with H&E, and scored by a board-certified pathologist. Representative H&E images are shown.
  • FIG. 9 depicts data demonstrating the evaluation of dose-dependent toxicity of TGF-P mRNA delivery with IAJD34.
  • BALB/c mice were injected with various doses of TGF-P mRNAIAJD34 (10, 20, and 30 pg per mouse).
  • Figure 10 depicts the gating strategy for alveolar and Interstitial macrophage characterization.
  • Cells were analyzed using a Gallios 10-color flow cytometer (Beckman Coulter, Brea, CA). Using Kaluza software (Beckman Coulter, Brea, CA) cells were gated upon size and complexity. Cells were confirmed to be CD45+ and viable.
  • Figure 10A depicts data demonstrating that cells that were positively stained for both Siglec F and F4/80 were determined to be alveolar macrophages (AMs).
  • AMs alveolar macrophages
  • Resident macrophages can be differentiated from recruited (CD11c- /CD1 lb+) or migratory macrophages (CD1 lc+/CDl lb+).
  • Figure 10B depicts data demonstrating that cells from digested lung tissue were immunomagnetically separated based upon CD45 expression. CD45+ cells were isolated, immunostained, and analyzed. Cells that expressed F4/80 and CD1 lb in the absence of Siglec F were categorized as interstitial macrophages (IMs).
  • IMs interstitial macrophages
  • Figure 11 depicts data demonstrating the absence of inflammatory activation in response to increasing doses of TGF-P mRNA- IAJD34 in alveolar macrophages and interstitial macrophages.
  • Various doses of TGFP mRNA- IAJD34 (10, 20, and 30 pg per mouse) were delivered to BALB/c mice and compared to naive animals at 24 h post-injection.
  • Figure 11 A depicts data demonstrating that cells from the BAL were isolated, immunostained, and analyzed by flow cytometry.
  • FIG 12 depicts data demonstrating the evaluation of alveolar macrophages containing Ly6c and CD206.
  • BALB/c mice were injected with various doses of TGF-[3 mRNA- IAFD34 (10, 20, and 30 pg per mouse).
  • BAL bronchoalveolar lavage fluid
  • Cells that were positively stained for both Siglec F and F4/80 were determined to be AMs and were evaluated for Ly6c and CD206 populations.
  • Figure 13 comprising Figure 13 A and Figure 13B depicts data demonstrating the evaluation of interstitial macrophages containing Ly6c and CD206.
  • BALB/c mice were injected with various doses of TGF-P mRNA-IAJD34 (10, 20, and 30 pg per mouse).
  • Figure 13A depicts data demonstrating that, at 24 hrs post-injection, cells from digested lung tissue were immunomagnetically separated based upon CD45 expression. CD45+ cells were isolated, immunostained, and analyzed. Cells that expressed F4/80 and CD1 lb in the absence of Siglec F were categorized as IMs and were analyzed for Ly6c and CD206 expression.
  • Figure 14 depicts data demonstrating Dose-dependent expression of TGF-P mRNA-IAJD34 in lung tissue.
  • Various doses of TGF-P mRNA-IAJD34 (10, 20, and 30 pg per mouse) were delivered to BALB/c mice, and lung tissue was collected 24 h post-injection and analyzed for TGF-P protein expression.
  • Figure 14A depicts western blots for TGF-P were performed on homogenized whole lung tissue at indicated time points. Representative images are shown.
  • Figure 14B depicts a table demonstrating that western blots results were quantified using densitometry.
  • FIG. 15 depicts data demonstrating that TGF-P mRNA-IAJD34-induced alterations in BAL cytokine production.
  • BALB/c mice were injected with various doses of TGF-P mRNA-IAJD34 (10, 20, and 30 pg per mouse).
  • IAJD34 [Empty] 10 pg TGF-P mRNA formulated with IAJD34
  • IAJD34 [TGF-P] 10 pg TGF-P mRNA formulated with IAJD34
  • Figure 16 depicts data demonstrating TGF-P mRNA-IAJD34-induced alterations in BAL cytokine production and cellular metabolic function.
  • Various doses of TGF-P mRNA-IAJD34 (10, 20, and 30 pg per mouse) were delivered to BALB/c mice, and BAL fluid was collected 24 h post-injection.
  • a D Cell- free BAL was evaluated for cytokines using a Milliplex Max Mouse Cytokine/Chemokine Magnetic Bead Panel - Premixed 32 Plex - Immunology Multiplex Assay.
  • CD45 + cells were isolated and analyzed metabolically using an Agilent Seahorse according to manufacturer’s guidelines;
  • Figure 16E ECAR was measured following injections of glucose, oligomycin (Oligo), and 2-DG, and glycolysis was determined following injection of glucose;
  • Figure 17 depicts data demonstrating the effects of TGF-P mRNA-IAJD34 delivery on BAL content over time.
  • BALB/c mice were injected with 10 pg of TGF-P mRNA-IAJD34 or empty IAJD34, BAL fluid and large aggregate surfactant fractions were collected at 4, 24, and 48 hrs post-injection.
  • Figure 17A depicts data demonstrating that cell-free BAL fluid was evaluated for total protein content using a BCA assay.
  • Figure 17B depicts data demonstrating that total phospholipids were determined from the large aggregate surfactant fraction and normalized to naive phospholipid content.
  • n/group 5/Naive, 3/4hr empty-IAJD34, 3/4hr TGFP mRNA-IAJD34, 4/24hr empty-IAJD34, 4/24hr TGF0 mRNA-IAJD34, 3/48hr empty- 1AJD34, 5/48hr TGF mRNAIAJD34.
  • FIG. 18 depicts data demonstrating the cell specific expression of TGF-P mRNA-IAJD34 in the lung over time.
  • Empty IAJD34 or 10 pg TGF-P mRNA-IAJD34 were delivered to BALB/c mice, and lung tissues were evaluated for TGF-P protein expression at 4, 24, and 48 h postinjection.
  • Figure 19 depicts data demonstrating that TGF-P protein expression is transiently induced after mRNA delivery to lung via IAJD34.
  • Empty IAJD34 or 10 ug TGF-P mRNA-IAJD34 were delivered to BALB/c mice, and lung tissue homogenates were evaluated for TGF-P protein expression at 4, 24, and 48 h postinjection.
  • Figure 19A depicts western blots for TGF-P were performed on homogenized whole lung tissue. Representative images are shown.
  • FIG. 20 depicts data demonstrating the absence of time-dependent Inflammatory activation in response to TGF-P mRNA-IAJD34 in alveolar macrophages.
  • BALB/c mice were injected with 10 pg of TGF-P mRNA-IAJD34 or empty IAJD34.
  • BAL cells were isolated, immunostained, and analyzed by flow cytometry. Cells that were positively stained for both Siglec F and F4/80 were determined to be AMs.
  • Mature macrophages (CD1 lc+/CDl lb-), can be differentiated from recruited or migratory macrophages (CD1 lb+).
  • Figure 21 depicts data demonstrating the Absence of time-dependent Inflammatory activation in response to TGF-P mRNA-IAJD34 in interstitial macrophages.
  • BALB/c mice were injected with 10 pg of TGF-P mRNA-IAJD34 or empty IAJD34.
  • cells from digested lung tissue were immunomagnetically separated based upon CD45 expression.
  • CD45+ cells were isolated, immunostained, and analyzed by flow cytometry.
  • Cells that expressed F4/80 and CD1 lb in the absence of Siglec F were categorized as interstitial macrophages and were analyzed for Ly6c and CD206 expression.
  • n/group 5/Naive, 3/4hr empty-IAJD34, 3/4hr TGFp mRNA-IAJD34, 4/24hr empty-IAJD34, 4/24hr TGFp mRNA-IAJD34, 3/48hr empty-IAJD34, 5/48hr TGFp mRNA-IAJD34.
  • Figure 22 depicts data demonstrating the effects of TGF-P mRNA-IAJD34 on bleomycin-induced changes in body weight.
  • BALB/c mice were exposed to PBS or bleomycin (ITB) and subsequently received either an empty IAJD34 or 10 pg TGF-P mRNA-IAJD34. Baseline body weights were measured, and then daily body weights were recorded. Percent change in body weight was determined based on baseline weights.
  • FIG. 23 depicts data demonstrating the effects of TGF-P mRNA-IAJD34 on bleomycin-induced histological alterations.
  • BALB/c mice were exposed to PBS or bleomycin (ITB) and subsequently received either an empty IAJD34 or 10 pg TGF-P mRNA-IAJD34.
  • Lung tissue was collected 3 days post-exposure and injection. Fixed lung tissue was processed and stained with H&E. Representative H&E images are shown. The slides are shown at 400x magnification, with scale bars indicating 50 pm.
  • Figure 24 depicts data demonstrating the effects of TGF-P mRNA-IAJD34 bleomycin-induced injury.
  • BALB/c mice were exposed to PBS or bleomycin (ITB) and subsequently received either an empty IAJD34 or 10 pg TGF-P mRNA-IAJD34.
  • IB bleomycin
  • Cell-free BAL fluid, BAL cells, and large aggregate surfactant fractions were collected 3 days post-exposure and injection.
  • Figure 24D and Figure 24E depict data demonstrating that BAL cells were immunostained for flow cytometric analysis. Cells that were positively stained for both Siglec F and F4/80 were determined to be alveolar macrophages (AMs).
  • AMs alveolar macrophages
  • Resident AMs exposed to PBS and ITB were compared using a 2- way ANOVA.
  • recruited AMs were compared using a two-tailed Wilcoxon Signed Rank test.
  • Figure 24F depicts data demonstrating that cells from digested lung tissue were immunomagnetically separated based upon CD45 expression.
  • CD45+ cells were isolated, immunostained, and analyzed.
  • Figure 25 depicts data demonstrating the effects of TGF-P mRNA-IAID34 bleomycin-induced cytokine production.
  • BALB/c mice were exposed to PBS or bleomycin (ITB) and subsequently received either an empty IAJD34 or 10 pg TGF-P mRNA-IAJD34.
  • Cell-free BAL fluid was collected 3 days postexposure and injection.
  • A-E Cell-free BAL fluid was evaluated for cytokines using a Milliplex Max Mouse Cytokine/Chemokine Magnetic Bead Panel - Premixed 32 Plex - Immunology Multiplex Assay.
  • FIG. 26 depicts data demonstrating the effect of TGF-P mRNA-IAJD34 on bleomycin induced alterations in BAL cytokine production.
  • BALB/c mice were exposed to PBS or bleomycin (ITB) and subsequently received either an empty IAJD34 or 10 pg TGF-P mRNA-IAJD34. 3 days post exposure and injection, BAL fluid was collected. Cell-free BAL fluid was evaluated for cytokines using a Milliplex Max Mouse Cytokine/Chemokine Magnetic Bead Panel (Premixed 32 Plex) Immunology Multiplex Assay and are presented as pg/mL.
  • Figure 27 depicts data demonstrating that Alpha- 1 -Antitrypsin mRNA treatment is useful for the treatment of genetically induced chronic obstructive pulmonary disease in a genetic knockout model.
  • 10 pg of luciferase mRNA was co-assemble with IAFD34 or IAJD34 formulated with 1.5% PEG, followed by dialysis in PBS, and then administered intravenously into C57BL/6 mice.
  • Representative IVIS images of whole-body mice and organs were taken 4 hours post-injection and show a strong luciferase signal in the lung of C57B1/6 mice.
  • Figure 28 depicts data demonstrating that 10 pg of luciferase mRNA was coassemble with IAJD33 or IAID33 formulated with 1.5% PEG, followed by dialysis in PBS, and then administered intravenously into C57BL/6 mice.
  • Representative IVIS images of wholebody mice and organs were taken 4 hours post-injection and show a strong luciferase signal in the lung of C57B1/6 mice.
  • Figure 29 depicts data demonstrating that 10 pg of luciferase mRNA was coassemble with IAJD33 (Luc mRNA-IAID33) and injected by intravenous administration into BALB/c mice. Representative IVIS images of whole-body mice and organs were taken 4 hours post-injection and show a strong luciferase signal in the lung of BALB/c mice
  • Figure 29 depicts data demonstrating that 5 pg of luciferase mRNA was coassemble with IAJD34 and then administered intranasally into BALB/c mice. Representative IVIS images of whole-body mice and organs were taken 4 hours post-injection and show a strong luciferase signal in the lung of BALB/c mice.
  • the present invention is based, in part, on the unexpected results that nanoparticles comprising at least one ionizable amphiphilic Janus dendrimer having the structure of Formula (I) or Formula (II) effectively and efficiently delivered a therapeutic agent to the lung parenchyma.
  • the present invention relates to methods of use of an ionizable amphiphilic Janus dendrimer having the structure of Formula (I) or Formula (II) for delivery of therapeutic mRNA to the lung parenchyma.
  • the nanoparticle further comprises at least one therapeutic mRNA that is encapsulated by an ionizable amphiphilic Janus dendrimer of the present invention.
  • the present invention relates to a composition comprising at least one ionizable amphiphilic Janus dendrimer of the present invention or a nanoparticle thereof.
  • the present invention relates to methods of delivering an agent to a target of interest using at least one ionizable amphiphilic Janus dendrimer of the present invention or a nanoparticle or a composition thereof.
  • the present invention relates to methods of preventing or treating a disease or disorder in a subject using at least one ionizable amphiphilic Janus dendrimer of the present invention or a nanoparticle or a composition thereof.
  • the present invention relates to methods of treating or preventing a lung disease or disorder in a subject using at least one ionizable amphiphilic Janus dendrimer of the present invention or a nanoparticle or a composition thereof.
  • the present invention relates to methods of reducing lung inflammation in a subject using at least one ionizable amphiphilic Janus dendrimer of the present invention or a nanoparticle or a composition thereof.
  • “about 40 [units]” may mean within ⁇ 25% of 40 (e.g., from 30 to 50), within ⁇ 20%, ⁇ 15%, ⁇ 10%, ⁇ 9%, ⁇ 8%, ⁇ 7%, ⁇ 6%, ⁇ 5%, ⁇ 4%, ⁇ 3%, ⁇ 2%, ⁇ 1%, less than ⁇ 1%, or any other value or range of values therein or therebelow.
  • the phrases “less than about [a value]” or “greater than about [a value]” should be understood in view of the definition of the term “about” provided herein.
  • compound refers to any specific chemical compound disclosed herein. In one embodiment, the term also refers to stereoisomers and/or optical isomers (including racemic mixtures) or enantiomerically enriched mixtures of disclosed compounds.
  • an analog is meant to refer to a chemical compound or molecule made from a parent compound or molecule by one or more chemical reactions.
  • an analog can be a structure having a structure similar to that of the small molecule therapeutic agents described herein or can be based on a scaffold of a small molecule therapeutic agents described herein, but differing from it in respect to certain components or structural makeup, which may have a similar or opposite action metabolically.
  • An analog or derivative can also be a small molecule that differs in structure from the reference molecule, but retains the essential properties of the reference molecule.
  • An analog or derivative may change its interaction with certain other molecules relative to the reference molecule.
  • An analog or derivative molecule may also include a salt, an adduct, tautomer, isomer, prodrug, or other variant of the reference molecule.
  • prodrug refers to an agent that is converted into the parent drug in vivo.
  • prodrug refers to a derivative of a known direct acting drug, which derivative has enhanced delivery characteristics and therapeutic value as compared to the drug, and is transformed into the active drug by an enzymatic or chemical process.
  • prodrug refers to an inactive or relatively less active form of an active agent that becomes active by undergoing a chemical conversion through one or more metabolic processes.
  • a prodrug upon in vivo administration, is chemically converted to the biologically, pharmaceutically, or therapeutically active form of the compound.
  • a prodrug is enzymatically metabolized by one or more steps or processes to the biologically, pharmaceutically, or therapeutically active form of the compound.
  • the present compounds can be administered to a subject as a prodrug that includes an initiator bound to an active agent, and, by virtue of being degraded by a metabolic process, the active agent is released in its active form.
  • tautomers are constitutional isomers of organic compounds that readily interconvert by a chemical process (tautomerization).
  • isomers or “stereoisomers” refers to compounds, which have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups in space.
  • nanoparticle refers to a particle having at least one dimension on the order of nanometers (e.g., 1-1,000 nm), which includes one or more amphiphilic Janus dendrimer of Formula (I) or Formula (II).
  • nanoparticles are included in a formulation comprising a nucleoside-modified RNA as described herein.
  • such nanoparticles an ionizable hydrophilic group and a lipophilic (hydrophobic) group.
  • the nanoparticles further comprise one or more excipient selected from neutral lipids, charged lipids, steroids and polymer conjugated lipids.
  • the nanoparticles do not comprise additional excipients. In one embodiment, the nanoparticles do not comprise any of additional lipids, additional cationic polymers, steroids, neutral lipids, charged lipids, or polymer conjugated lipids, besides the at least one compound of Formula (I) or Formula (II).
  • the nucleoside-modified RNA is encapsulated in the lipid portion of the nanoparticle or an aqueous space enveloped by some or all of the lipid portion of the nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells, e.g., an adverse immune response.
  • Encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA, circularRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
  • Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the noncoding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
  • a “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell.
  • vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.
  • the term “vector” includes an autonomously replicating plasmid or a virus.
  • the term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like.
  • viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.
  • “Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed.
  • An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system.
  • Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) RNA, and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.
  • “Homologous” refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position.
  • the percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared X 100. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60% homologous.
  • the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, a comparison is made when two sequences are aligned to give maximum homology.
  • isolated means altered or removed from the natural state.
  • a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.”
  • An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
  • nucleosides nucleobase bound to ribose or deoxyribose sugar via N-glycosidic linkage
  • A refers to adenosine
  • C refers to cytidine
  • G refers to guanosine
  • T refers to thymidine
  • U refers to uridine.
  • moduleating mediating a detectable increase or decrease in the level of a response in a subject compared with the level of a response in the subject in the absence of a treatment or compound, and/or compared with the level of a response in an otherwise identical but untreated subject.
  • the term encompasses perturbing and/or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject.
  • nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns. In addition, the nucleotide sequence may contain modified nucleosides that are capable of being translation by translational machinery in a cell. For example, an mRNA where all of the uridines have been replaced with pseudouridine, 1 -methyl psuedouridine, or another modified nucleoside.
  • operably linked refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter.
  • a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably linked DNA or RNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.
  • nucleotide as used herein is defined as a chain of nucleotides.
  • nucleic acids are polymers of nucleotides.
  • nucleic acids and polynucleotides as used herein are interchangeable.
  • nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides.
  • polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCRTM, and the like, and by synthetic means.
  • recombinant means i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCRTM, and the like, and by synthetic means.
  • the polynucleotide or nucleic acid of the invention is a “nucleoside-modified nucleic acid,” which refers to a nucleic acid comprising at least one modified nucleoside.
  • a “modified nucleoside” refers to a nucleoside with a modification. For example, over one hundred different nucleoside modifications have been identified in RNA (Rozenski, et al., 1999, The RNA Modification Database: 1999 update. Nucl Acids Res 27: 196-197).
  • “pseudouridine” refers, in another embodiment, to m l acp3 (l-methyl-3-(3-amino-3-carboxypropyl) pseudouridine.
  • the term refers to m l (1-methylpseudouridine).
  • the term refers to i (2'-O-methylpseudouridine).
  • the term refers to m5D (5- methyldihydrouridine).
  • the term refers to m3T (3- methylpseudouridine).
  • the term refers to a pseudouridine moiety that is not further modified.
  • the term refers to a monophosphate, diphosphate, or triphosphate of any of the above pseudouridines.
  • the term refers to any other pseudouridine known in the art. Each possibility represents a separate embodiment of the invention.
  • nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.
  • the phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).
  • amino acid refers to any of the twenty naturally occurring amino acids including synthetic amino acids with unnatural side chains and including both D and L optical isomers.
  • peptide As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds.
  • a protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence.
  • Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds.
  • the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types.
  • Polypeptides include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others.
  • the polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.
  • promoter as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence. For example, the promoter that is recognized by bacteriophage RNA polymerase and is used to generate the mRNA by in vitro transcription.
  • transfected or transformed or transduced refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell.
  • a “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.
  • under transcriptional control or “operatively linked” as used herein means that the promoter is in the correct location and orientation in relation to a polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.
  • terapéutica means a treatment and/or prophylaxis.
  • a therapeutic effect is obtained by suppression, diminution, remission, or eradication of at least one sign or symptom of a disease or disorder state.
  • therapeutic compound As used herein, the terms “therapeutic compound”, “therapeutic agent”, “drug”, “active pharmaceutical”, and “active pharmaceutical ingredient” are used interchangeably to refer to chemical entities that display certain pharmacological effects in a body and are administered for such purpose.
  • therapeutic agents include, but are not limited to, hydrophilic therapeutic agents, hydrophobic therapeutic agents, antibiotics, antibodies, small molecules, anti-cancer agents, chemotherapeutic agents, immunomodulatory agents, RNA molecules, siRNA molecules, DNA molecules, gene editing agents, genesilencing agents, CRISPR-associated agents (e.g., guide RNA molecules, endonucleases, and variants thereof), analgesics, vaccines, anticonvulsants; anti-diabetic agents, antifungal agents, antineoplastic agents, anti-parkinsonian agents, anti-rheumatic agents, appetite suppressants, biological response modifiers, cardiovascular agents, central nervous system stimulants, contraceptive agents, dietary supplements, vitamins, minerals, lipids, saccharides,
  • active ingredients suitable for use in the pharmaceutical formulations and methods of the present invention include: hydrophilic, lipophilic, amphiphilic or hydrophobic, and that can be solubilized, dispersed, or partially solubilized and dispersed, on or about the nanocluster.
  • the active agent-nanocluster combination may be coated further to encapsulate the agent-nanocluster combination and may be directed to a target by functionalizing the nanocluster with, e.g., aptamers and/or antibodies.
  • an active ingredient may also be provided separately from the solid pharmaceutical composition, such as for co-administration.
  • Such active ingredients can be any compound or mixture of compounds having therapeutic or other value when administered to an animal, particularly to a mammal, such as drugs, nutrients, cosmeceuticals, nutraceuticals, diagnostic agents, nutritional agents, and the like.
  • the active agents described herein may be found in their native state, however, they will generally be provided in the form of a salt.
  • the active agents described herein include their isomers, analogs and derivatives.
  • an “effective amount” as used herein means an amount which provides a therapeutic or prophylactic benefit.
  • therapeutically effective amount refers to the amount of the subject compound that will elicit the biological or medical response of a tissue, system, or subject that is being sought by the researcher, veterinarian, medical doctor or other clinician.
  • therapeutically effective amount includes that amount of a compound that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the signs or symptoms of the disorder or disease being treated.
  • the therapeutically effective amount will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated.
  • a “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate.
  • a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.
  • To “treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.
  • treating a disease or disorder means reducing the frequency with which a symptom of the disease or disorder is experienced by a patient.
  • Disease and disorder are used interchangeably herein.
  • a disease or disorder is “alleviated” if the severity of at least one sign or symptom of the disease or disorder, the frequency with which such at least one sign or symptom is experienced by a patient, or both, is reduced.
  • moduleating mediating a detectable increase or decrease in the level of a response in a subject compared with the level of a response in the subject in the absence of a treatment or compound, and/or compared with the level of a response in an otherwise identical but untreated subject.
  • the term encompasses perturbing and/or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject, such as, a human.
  • parenteral administration of a composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrastemal injection, or infusion techniques.
  • Ranges throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
  • the present invention is based, in part, on the unexpected results that nanoparticles comprising at least one ionizable amphiphilic Janus dendrimer having the structure of Formula (I) or Formula (II) effectively and efficiently delivered an agent to a target of interest.
  • the present invention relates to an ionizable amphiphilic Janus dendrimer having the structure of Formula (I) or Formula (II).
  • the present invention relates to a nanoparticle comprising at least one ionizable amphiphilic Janus dendrimer of the present invention.
  • the nanoparticle further comprises at least one agent.
  • the nanoparticle further comprises at least one agent that is encapsulated by the ionizable amphiphilic Janus dendrimer of the present invention.
  • the present invention relates to a composition comprising at least one ionizable amphiphilic Janus dendrimer of the present invention or a nanoparticle thereof.
  • the composition is a vaccine.
  • the present invention relates to compositions for delivering an agent to a target of interest using at least one ionizable amphiphilic Janus dendrimer (IAJD) of the present invention or a nanoparticle or a composition thereof, and method of use of the IAJD for targeted delivery of therapeutic agents to the lung.
  • the present invention relates to methods of preventing or treating a lung disease or disorder in a subject using at least one ionizable amphiphilic Janus dendrimer of the present invention or a nanoparticle or a composition thereof.
  • the present invention relates to methods of reducing lung inflammation in a subject using at least one ionizable amphiphilic Janus dendrimer of the present invention or a nanoparticle or a composition thereof.
  • the amphiphilic Janus dendrimer is an amphiphilic Janus dendrimer 34 (IAJD34) having a structure of:
  • the amphiphilic Janus dendrimer is an amphiphilic Janus dendrimer 33 (IAJD33) having a structure of:
  • the invention relates to nanoparticles comprising at least one amphiphilic Janus dendrimer of the present invention.
  • the nanoparticle is a one-component nanoparticle.
  • the nanoparticle is a racemic ionizable amphiphilic Janus dendrimer.
  • the nanoparticle is a dendrimersome nanoparticle (DNP).
  • DNP dendrimersome nanoparticle
  • the nanoparticle comprises at least two amphiphilic Janus dendrimers.
  • the nanoparticle comprises a first ionizable amphiphilic Janus dendrimer and a second ionizable amphiphilic Janus dendrimer.
  • the first ionizable amphiphilic Janus dendrimer has a different structure than the second ionizable amphiphilic Janus dendrimer.
  • the nanoparticles further comprise at least one amphiphilic Janus dendrimer disclosed in Wang, et al., J. Am. Chem. Soc. 2020, 142, 9525-9536; Xiao et al., J. Am. Chem. Soc. 2016, 138, 12655-12663; Torre et al., Proc. Natl. Acad. Sci. U.S.A. 2019, 116, 15378-15385; Percec et al., J. Am. Chem. Soc. 2021, 143, 17724-17743; Wilson et al., J. Polym. Sci.
  • the nanoparticle has a mean diameter of from about 10 nm to about 100,000 nm, about 30 nm to about 1000 nm, about 30 nm to about 500 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm,
  • the nanoparticle is substantially non-toxic.
  • the nanoparticle is biodegradable.
  • the nanoparticle comprises at least one cargo.
  • the invention is not limited to any particular cargo or otherwise agent for which the nanoparticle is able to carry or transport. Rather, the invention includes any agent that can be carried by the nanoparticle.
  • agents that can be carried by the nanoparticle of the invention include, but are not limited to, diagnostic agents, detectable agents, and therapeutic agents.
  • the nanoparticle comprises at least one agent.
  • the nanoparticle encapsulates at least one agent.
  • the weight ratio of the amphiphilic Janus dendrimer : the at least one agent is between about 1 : 1 to about 10,000 : 1.
  • the nanoparticle comprises, or encapsulates, at least one agent.
  • the weight ratio of the amphiphilic Janus dendrimer : the at least one agent is between about 2 : 1 to about 1,000 : 1.
  • the nanoparticle comprises, or encapsulates, at least one agent.
  • the weight ratio of the amphiphilic Janus dendrimer : the at least one agent is between about 3 : 1 to about 10 : 1.
  • the nanoparticle comprises, or encapsulates, at least one agent.
  • the weight ratio of the amphiphilic Janus dendrimer : the at least one agent is between about 4 : 1 to about 10 : 1.
  • the nanoparticle comprises, or encapsulates, at least one agent.
  • the weight ratio of the amphiphilic Janus dendrimer : the at least one agent is between about 5 : 1 to about 10 : 1.
  • the nanoparticle comprises, or encapsulates, at least one agent.
  • the weight ratio of the amphiphilic Janus dendrimer : the at least one agent is between about 6 : 1 to about 10 : 1.
  • the nanoparticle comprises, or encapsulates, at least one agent.
  • the weight ratio of the amphiphilic Janus dendrimer : the at least one agent is between about 7 : 1 to about 10 : 1.
  • the nanoparticle comprises, or encapsulates, at least one agent.
  • the weight ratio of the amphiphilic Janus dendrimer : the at least one agent is between about 8 : 1 to about 10 : 1.
  • the nanoparticle comprises, or encapsulates, at least one agent.
  • the weight ratio of the amphiphilic Janus dendrimer : the at least one agent is between about 9 : 1 to about 10 : 1.
  • the nanoparticle comprises, or encapsulates, at least one agent.
  • the weight ratio of the amphiphilic Janus dendrimer : the at least one agent is between about 9.5 : 1 to about 10 : 1.
  • the nanoparticle is suitable for delivering at least one cargo to a cell of interest.
  • the cargo is at least one agent comprising a diagnostic agent, detectable agent, therapeutic agent, nucleic acid molecule, gene editing agent, vaccine, composition for protein replacement therapy, or any combination thereof.
  • the at least one agent is selected from an mRNA, siRNA, microRNA, CRISPR- Cas9, sgRNA, small molecule, protein, antibody, peptide, protein, or any combination thereof.
  • the at least one agent comprises a nucleic acid molecule.
  • the nucleic acid molecule encodes at least one selected from an antigen, antibody, gene editing molecule, chimeric antigen receptor (CAR), or any combination thereof.
  • the nucleic acid molecule is a DNA molecule or an RNA molecule. In some embodiments, the nucleic acid molecule is selected from cDNA, cRNA, CirRNA, mRNA, miRNA, siRNA, sgRNA, modified RNA, tRNA, antagomir, antisense molecule, targeted nucleic acid, or any combination thereof.
  • the modified RNA is a nucleoside-modified RNA. In some embodiments, the nucleoside-modified RNA comprises pseudouridine. In some embodiments, the nucleoside-modified RNA comprises pseudouridine plus 5-methyl-cytosine. In some embodiments, the nucleoside-modified RNA comprises 5- methyl-uridine. In some embodiments, the nucleoside-modified RNA comprises 1-methyl- pseudouridine.
  • the nanoparticles may be used for the delivery of nucleoside-modified RNA to a subject in need thereof.
  • delivery of a nucleoside-modified RNA to a subject comprises mixing the nucleoside-modified RNA with at least one nanoparticle comprising an LAID of Formula (I) or Formula (II) prior to the step of contacting.
  • a method of invention further comprises administering nucleoside-modified RNA together with at least one nanoparticle comprising an IAJD of Formula (I) or Formula (II).
  • the composition provides for targeted delivery of the encapsulated agent to the lungs.
  • the composition provides for delivery of mRNA molecules encoding anti-inflammatory drugs to the lungs.
  • agents that can be delivered include, but are not limited to, TGF-0, Alpha- 1 -antitrypsin (A1AT), soluble IL-1R, CFTR, IL-4, IL-13, Surfactant Protein D fragment (SP-D fragment), surfactant protein B (SP- B) or surfactant protein C (SP-C) or a fragment or variant thereof.
  • the composition provides for delivery of mRNA molecules encoding TGF-P to the lungs.
  • the nucleoside-modified RNA when present in the nanoparticles, is resistant in aqueous solution to degradation with a nuclease.
  • the therapeutic agent is an isolated nucleic acid.
  • the isolated nucleic acid molecule is one of a DNA molecule or an RNA molecule.
  • the isolated nucleic acid molecule is a cDNA, mRNA, siRNA, shRNA or miRNA molecule.
  • the isolated nucleic acid molecule encodes a therapeutic peptide such a thrombomodulin, endothelial protein C receptor (EPCR), anti -thrombotic proteins including plasminogen activators and their mutants, antioxidant proteins including catalase, superoxide dismutase (SOD) and iron-sequestering proteins.
  • the therapeutic agent is an siRNA, miRNA, shRNA, or an antisense molecule, which inhibits a targeted nucleic acid including those encoding proteins that are involved in aggravation of the pathological processes.
  • the nucleic acid comprises a promoter/regulatory sequence such that the nucleic acid is capable of directing expression of the nucleic acid.
  • the invention encompasses expression vectors and methods for the introduction of exogenous nucleic acid into cells with concomitant expression of the exogenous nucleic acid in the cells such as those described, for example, in Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York) and as described elsewhere herein.
  • siRNA is used to decrease the level of a targeted protein.
  • RNA interference is a phenomenon in which the introduction of double-stranded RNA (dsRNA) into a diverse range of organisms and cell types causes degradation of the complementary mRNA.
  • dsRNA double-stranded RNA
  • Dicer ribonuclease
  • the siRNAs subsequently assemble with protein components into an RNA-induced silencing complex (RISC), unwinding in the process.
  • RISC RNA-induced silencing complex
  • Activated RISC then binds to complementary transcript by base pairing interactions between the siRNA antisense strand and the mRNA.
  • RNA Interference Nuts & Bolts of RNAi Technology, DNA Press, Eagleville, PA (2003); and Gregory J. Hannon, Ed., RNAi A Guide to Gene Silencing, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2003). Soutschek et al.
  • siRNAs that aids in intravenous systemic delivery.
  • Optimizing siRNAs involves consideration of overall G/C content, C/T content at the termini, Tm and the nucleotide content of the 3’ overhang. See, for instance, Schwartz et al., 2003, Cell, 115: 199- 208 and Khvorova et al., 2003, Cell 115:209-216. Therefore, the invention also includes methods of decreasing levels of PTPN22 using RNAi technology.
  • the invention includes a vector comprising an siRNA or an antisense polynucleotide.
  • the siRNA or antisense polynucleotide is capable of inhibiting the expression of a target polypeptide.
  • the expression vectors described herein encode a short hairpin RNA (shRNA) therapeutic agents.
  • shRNA molecules are well known in the art and are directed against the mRNA of a target, thereby decreasing the expression of the target.
  • the encoded shRNA is expressed by a cell, and is then processed into siRNA.
  • the cell possesses native enzymes (e.g., dicer) that cleave the shRNA to form siRNA.
  • the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification of expressing cells from the population of cells sought to be transfected or infected using a the delivery vehicle of the invention.
  • the selectable marker may be carried on a separate piece of DNA and also be contained within the delivery vehicle. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells.
  • Useful selectable markers are known in the art and include, for example, antibioticresistance genes, such as neomycin resistance and the like.
  • the delivery vehicle may contain a vector, comprising the nucleotide sequence or the construct to be delivered.
  • the vector of the invention is an expression vector.
  • Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells.
  • the expression vector is selected from the group consisting of a viral vector, a bacterial vector and a mammalian cell vector.
  • Prokaryote- and/or eukaryote-vector based systems can be employed for use with the invention to produce polynucleotides, or their cognate polypeptides. Many such systems are commercially and widely available.
  • the vector in which the nucleic acid sequence is introduced can be a plasmid, which is or is not integrated in the genome of a host cell when it is introduced in the cell.
  • Illustrative, non-limiting examples of vectors in which the nucleotide sequence of the invention or the gene construct of the invention can be inserted include a tet-on inducible vector for expression in eukaryote cells.
  • the vector may be obtained by conventional methods known by persons skilled in the art (Sambrook et al., 2012).
  • the vector is a vector useful for transforming animal cells.
  • the recombinant expression vectors may also contain nucleic acid molecules, which encode a peptide or peptidomimetic.
  • a promoter may be one naturally associated with a gene or polynucleotide sequence, as may be obtained by isolating the 5’ non-coding sequences located upstream of the coding segment and/or exon. Such a promoter can be referred to as “endogenous.”
  • an enhancer may be one naturally associated with a polynucleotide sequence, located either downstream or upstream of that sequence.
  • certain advantages will be gained by positioning the coding polynucleotide segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a polynucleotide sequence in its natural environment.
  • a recombinant or heterologous enhancer refers also to an enhancer not normally associated with a polynucleotide sequence in its natural environment.
  • Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers not “naturally occurring,” i.e., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression.
  • sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including PCRTM, in connection with the compositions disclosed herein (U.S. Patent 4,683,202, U.S. Patent 5,928,906).
  • control sequences that direct transcription and/or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well.
  • promoter and/or enhancer that effectively directs the expression of the DNA segment in the cell type, organelle, and organism chosen for expression.
  • Those of skill in the art of molecular biology generally know how to use promoters, enhancers, and cell type combinations for protein expression, for example, see Sambrook et al. (2012).
  • the promoters employed may be constitutive, tissue-specific, inducible, and/or useful under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins and/or peptides.
  • the promoter may be heterologous or endogenous.
  • the recombinant expression vectors may also contain a selectable marker gene, which facilitates the selection of host cells.
  • Suitable selectable marker genes are genes encoding proteins such as G418 and hygromycin, which confer resistance to certain drugs, P- galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or an immunoglobulin or portion thereof such as the Fc portion of an immunoglobulin such as IgG.
  • the selectable markers may be introduced on a separate vector from the nucleic acid of interest.
  • the siRNA polynucleotide will have certain characteristics that can be modified to improve the siRNA as a therapeutic compound. Therefore, the siRNA polynucleotide may be further designed to resist degradation by modifying it to include phosphorothioate, or other linkages, methylphosphonate, sulfone, sulfate, ketyl, phosphorodithioate, phosphoramidate, phosphate esters, and the like (see, e.g., Agrawal et al., 1987, Tetrahedron Lett. 28:3539-3542; Stec et al., 1985 Tetrahedron Lett.
  • Any polynucleotide may be further modified to increase its stability in vivo. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends; the use of phosphorothioate or 2' O-methyl rather than phosphodiester linkages in the backbone; and/or the inclusion of nontraditional bases such as inosine, queuosine, and wybutosine and the like, as well as acetyl- methyl-, thio- and other modified forms of adenine, cytidine, guanine, thymine, and uridine.
  • an antisense nucleic acid sequence which is expressed by a plasmid vector is used as a therapeutic agent to inhibit the expression of a target protein.
  • the antisense expressing vector is used to transfect a mammalian cell or the mammal itself, thereby causing reduced endogenous expression of the target protein.
  • Antisense molecules and their use for inhibiting gene expression are well known in the art (see, e.g., Cohen, 1989, In: Oligodeoxyribonucleotides, Antisense Inhibitors of Gene Expression, CRC Press).
  • Antisense nucleic acids are DNA or RNA molecules that are complementary, as that term is defined elsewhere herein, to at least a portion of a specific mRNA molecule (Weintraub, 1990, Scientific American 262:40). In the cell, antisense nucleic acids hybridize to the corresponding mRNA, forming a double-stranded molecule thereby inhibiting the translation of genes.
  • antisense methods to inhibit the translation of genes is known in the art, and is described, for example, in Marcus-Sakura (1988, Anal. Biochem. 172:289).
  • Such antisense molecules may be provided to the cell via genetic expression using DNA encoding the antisense molecule as taught by Inoue, 1993, U.S. Patent No. 5,190,931.
  • antisense molecules of the invention may be made synthetically and then provided to the cell.
  • Antisense oligomers of between about 10 to about 30, or 15 nucleotides are useful in some embodiments since they are easily synthesized and introduced into a target cell.
  • Synthetic antisense molecules contemplated by the invention include oligonucleotide derivatives known in the art which have improved biological activity compared to unmodified oligonucleotides (see U.S. Patent No. 5,023,243).
  • a ribozyme is used as a therapeutic agent to inhibit expression of a target protein.
  • Ribozymes useful for inhibiting the expression of a target molecule may be designed by incorporating target sequences into the basic ribozyme structure, which are complementary, for example, to the mRNA sequence encoding the target molecule. Ribozymes targeting the target molecule, may be synthesized using commercially available reagents (Applied Biosystems, Inc., Foster City, CA) or they may be genetically expressed from DNA encoding them.
  • the therapeutic agent may comprise one or more components of a CRISPR-Cas system, where a guide RNA (gRNA) targeted to a gene encoding a target molecule, and a CRISPR-associated (Cas) peptide form a complex to induce mutations within the targeted gene.
  • gRNA guide RNA
  • Cas CRISPR-associated peptide
  • the therapeutic agent comprises a gRNA or a nucleic acid molecule encoding a gRNA.
  • the therapeutic agent comprises a Cas peptide or a nucleic acid molecule encoding a Cas peptide.
  • the agent comprises a miRNA or a mimic of a miRNA. In one embodiment, the agent comprises a nucleic acid molecule that encodes a miRNA or mimic of a miRNA.
  • MiRNAs are small non-coding RNA molecules that are capable of causing post- transcriptional silencing of specific genes in cells by the inhibition of translation or through degradation of the targeted mRNA.
  • a miRNA can be completely complementary or can have a region of noncomplementarity with a target nucleic acid, consequently resulting in a "bulge" at the region of non-complementarity.
  • a miRNA can inhibit gene expression by repressing translation, such as when the miRNA is not completely complementary to the target nucleic acid, or by causing target RNA degradation, which is believed to occur only when the miRNA binds its target with perfect complementarity.
  • the disclosure also can include double-stranded precursors of miRNA.
  • a miRNA or pri-miRNA can be 18- 100 nucleotides in length, or from 18-80 nucleotides in length.
  • Mature miRNAs can have a length of 19-30 nucleotides, or 21-25 nucleotides, particularly 21, 22, 23, 24, or 25 nucleotides.
  • MiRNA precursors typically have a length of about 70-100 nucleotides and have a hairpin conformation.
  • miRNAs are generated in vivo from pre- miRNAs by the enzymes Dicer and Drosha, which specifically process long pre- miRNA into functional miRNA.
  • the hairpin or mature microRNAs, or pri-microRNA agents featured in the disclosure can be synthesized in vivo by a cell-based system or in vitro by chemical synthesis.
  • the agent comprises an oligonucleotide that comprises the nucleotide sequence of a disease-associated miRNA.
  • the oligonucleotide comprises the nucleotide sequence of a disease-associated miRNA in a pre - microRNA, mature or hairpin form.
  • a combination of oligonucleotides comprising a sequence of one or more disease-associated miRNAs, any pre -miRNA, any fragment, or any combination thereof is envisioned.
  • MiRNAs can be synthesized to include a modification that imparts a desired characteristic. For example, the modification can improve stability, hybridization thermodynamics with a target nucleic acid, targeting to a particular tissue or cell -type, or cell permeability, e.g., by an endocytosis-dependent or -independent mechanism.
  • Modifications can also increase sequence specificity, and consequently decrease off-site targeting. Methods of synthesis and chemical modifications are described in greater detail below. If desired, miRNA molecules may be modified to stabilize the miRNAs against degradation, to enhance half-life, or to otherwise improve efficacy. Desirable modifications are described, for example, in U.S. Patent Publication Nos. 20070213292, 20060287260, 20060035254. 20060008822. and 2005028824, each of which is hereby incorporated by reference in its entirety.
  • the single- stranded oligonucleotide agents featured in the disclosure can include 2'-O-methyl, 2'-fluorine, 2'-O-methoxyethyl, 2'-O-aminopropyl, 2'-amino, and/or phosphorothioate linkages.
  • Inclusion of locked nucleic acids (LNA), ethylene nucleic acids (ENA), e.g., 2'-4'-ethylene- bridged nucleic acids, and certain nucleotide modifications can also increase binding affinity to the target.
  • LNA locked nucleic acids
  • ENA ethylene nucleic acids
  • pyranose sugars in the oligonucleotide backbone can also decrease endonucleolytic cleavage.
  • An oligonucleotide can be further modified by including a 3' cationic group, or by inverting the nucleoside at the 3 '-terminus with a 3 -3' linkage. In another alternative, the 3 '-terminus can be blocked with an aminoalkyl group.
  • Other 3' conjugates can inhibit 3'-5' exonucleolytic cleavage. While not being bound by theory, a 3' may inhibit exonucleolytic cleavage by sterically blocking the exonuclease from binding to the 3' end of the oligonucleotide. Even small alkyl chains, aryl groups, or heterocyclic conjugates or modified sugars (D-ribose, deoxyribose, glucose etc.) can block 3'-5'-exonucleases.
  • the miRNA includes a 2'-modified oligonucleotide containing oligodeoxynucleotide gaps with some or all intemucleotide linkages modified to phosphorothioates for nuclease resistance.
  • the presence of methylphosphonate modifications increases the affinity of the oligonucleotide for its target RNA and thus reduces the IC5Q. This modification also increases the nuclease resistance of the modified oligonucleotide. It is understood that the methods and reagents of the present disclosure may be used in conjunction with any technologies that may be developed to enhance the stability or efficacy of an inhibitory nucleic acid molecule.
  • miRNA molecules include nucleotide oligomers containing modified backbones or non-natural internucleoside linkages. Oligomers having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this disclosure, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone are also considered to be nucleotide oligomers.
  • Nucleotide oligomers that have modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, methyl and other alkyl phosphonates including 3 '-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriest- ers, and boranophosphates.
  • Various salts, mixed salts and free acid forms are also included.
  • a miRNA described herein which may be in the mature or hairpin form, may be provided as a naked oligonucleotide.
  • it may be desirable to utilize a formulation that aids in the delivery of a miRNA or other nucleotide oligomer to cells see, e.g., U.S. Pat. Nos. 5,656,61 1, 5,753,613, 5,785,992, 6,120,798, 6,221,959, 6,346,613, and 6,353,055, each of which is hereby incorporated by reference).
  • the miRNA composition is at least partially crystalline, uniformly crystalline, and/or anhydrous (e.g., less than 80, 50, 30, 20, or 10% water).
  • the miRNA composition is in an aqueous phase, e.g., in a solution that includes water.
  • the aqueous phase or the crystalline compositions can be incorporated into a delivery vehicle, e.g., a liposome (particularly for the aqueous phase), or a particle (e.g., a microparticle as can be appropriate for a crystalline composition).
  • the miRNA composition is formulated in a manner that is compatible with the intended method of administration.
  • a miRNA composition can be formulated in combination with another agent, e.g., another therapeutic agent or an agent that stabilizes an oligonucleotide agent, e.g., a protein that complexes with the oligonucleotide agent.
  • another agent e.g., another therapeutic agent or an agent that stabilizes an oligonucleotide agent, e.g., a protein that complexes with the oligonucleotide agent.
  • Still other agents include chelators, e.g., EDTA (e.g., to remove divalent cations such as Mg), salts, and RNAse inhibitors (e.g., a broad specificity RNAse inhibitor).
  • the miRNA composition includes another miRNA, e g., a second miRNA composition (e.g., a microRNA that is distinct from the first).
  • the composition comprises an oligonucleotide composition that mimics the activity of a miRNA.
  • the composition comprises oligonucleotides having nucleobase identity to the nucleobase sequence of a miRNA, and are thus designed to mimic the activity of the miRNA.
  • the oligonucleotide composition that mimics miRNA activity comprises a double-stranded RNA molecule which mimics the mature miRNA hairpins or processed miRNA duplexes.
  • the oligonucleotide shares identity with endogenous miRNA or miRNA precursor nucleobase sequences.
  • An oligonucleotide selected for inclusion in a composition of the invention may be one of a number of lengths. Such an oligonucleotide can be from 7 to 100 linked nucleosides in length.
  • an oligonucleotide sharing nucleobase identity with a miRNA may be from 7 to 30 linked nucleosides in length.
  • An oligonucleotide sharing identity with a miRNA precursor may be up to 100 linked nucleosides in length.
  • an oligonucleotide comprises 7 to 30 linked nucleosides.
  • an oligonucleotide comprises 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 28, 29, or 30 linked nucleotides. In certain embodiments, an oligonucleotide comprises 19 to 23 linked nucleosides. In certain embodiments, an oligonucleotide is from 40 up to 50, 60, 70, 80, 90, or 100 linked nucleosides in length.
  • an oligonucleotide has a sequence that has a certain identity to a miRNA or a precursor thereof.
  • Nucleobase sequences of mature miRNAs and their corresponding stem-loop sequences described herein are the sequences found in miRBase, an online searchable database of miRNA sequences and annotation. Entries in the miRBase Sequence database represent a predicted hairpin portion of a miRNA transcript (the stem-loop), with information on the location and sequence of the mature miRNA sequence.
  • the miRNA stem-loop sequences in the database are not strictly precursor miRNAs (pre-miRNAs), and may in some instances include the pre-miRNA and some flanking sequence from the presumed primary transcript.
  • the miRNA nucleobase sequences described herein encompass any version of the miRNA, including the sequences described in Release 10.0 of the miRBase sequence database and sequences described in any earlier Release of the miRBase sequence database.
  • a sequence database release may result in the re-naming of certain miRNAs.
  • a sequence database release may result in a variation of a mature miRNA sequence.
  • the compositions of the invention encompass oligomeric compound comprising oligonucleotides having a certain identity to any nucleobase sequence version of a miRNAs described herein.
  • an oligonucleotide has a nucleobase sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the miRNA over a region of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases. Accordingly, in certain embodiments the nucleobase sequence of an oligonucleotide may have one or more non-identical nucleobases with respect to the miRNA.
  • the composition comprises a nucleic acid molecule encoding a miRNA, precursor, mimic, or fragment thereof.
  • the composition may comprise a viral vector, plasmid, cosmid, or other expression vector suitable for expressing the miRNA, precursor, mimic, or fragment thereof in a desired mammalian cell or tissue.
  • the invention includes a nanoparticle comprising or encapsulating one or more nucleic acid molecule.
  • the nucleic acid molecule is a nucleoside-modified mRNA molecule.
  • the nucleoside- modified mRNA molecule encodes a therapeutic agent.
  • the nucleoside- modified mRNA molecule encodes a plurality of therapeutic agents.
  • the nucleoside-modified mRNA molecule encodes an anti-inflammatory agent.
  • nucleotide sequences encoding a therapeutic agent can alternatively comprise sequence variations with respect to an original nucleotide sequence, for example, substitutions, insertions and/or deletions of one or more nucleotides, with the condition that the resulting polynucleotide encodes a polypeptide according to the invention.
  • nucleotide sequences that encode amino acid sequences that are substantially homologous to the amino acid sequences recited herein and preserve the immunogenic function of the original amino acid sequence.
  • an amino acid sequence is “substantially homologous” to any of the amino acid sequences described herein when its amino acid sequence has a degree of identity with respect to the amino acid sequence of at least 60%, at least 70%, at least 85%, at least 95%, or greater than 95%.
  • the identity between two amino acid sequences is determined by using the BLASTN algorithm (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S sharp et al., J. Mol. Biol. 215: 403-410 (1990)).
  • the invention relates to a construct, comprising a nucleotide sequence encoding a therapeutic agent.
  • the construct comprises a plurality of nucleotide sequences encoding a plurality of therapeutic agents.
  • the construct encodes 1 or more, 2 or more, 5 or more, 10 or more, 15 or more, or 20 or more therapeutic agents.
  • Exemplary therapeutic agents that can be delivered include, but are not limited to, TGF-P, Alpha- 1 -antitrypsin (A1AT), soluble IL-1R, CFTR, IL-4, IL-13, Surfactant Protein D fragment (SP-D fragment), surfactant protein B (SP-B) or surfactant protein C (SP-C) or a fragment or variant thereof.
  • A1AT Alpha- 1 -antitrypsin
  • SP-D fragment Surfactant Protein D fragment
  • SP-B surfactant protein B
  • SP-C surfactant protein C
  • the therapeutic agent comprises a nucleotide sequence encoding TGFp, or a fragment or variant thereof.
  • the nucleotide sequence encoding TGFP comprises:
  • the therapeutic agent comprises a nucleotide sequence encoding A1AT isoform 1 (AATl-isol), or a fragment or variant thereof.
  • the nucleotide sequence encoding A1AT comprises:
  • the therapeutic agent comprises a nucleotide sequence encoding A1AT isoform 2 (AATl-iso2), or a fragment or variant thereof.
  • the nucleotide sequence encoding A1AT comprises:
  • ATCTTCCTGGGCAAGGTGGTGGACCCCACCCACAAGTAA (SEQ ID NO: 5), or an RNA molecule comprising an RNA sequence corresponding to SEQ ID NO:5.
  • the therapeutic agent comprises a nucleotide sequence encoding IL-1 soluble Receptor isoform 1 (ILlra-isol), or a fragment or variant thereof.
  • the nucleotide sequence encoding ILlra-isol comprises: ATGACCGCCGCCCAGGCCGAGGCCGCCTGCCGCCCCTCCGGCAAGCG
  • GGCTGGTTCCTGTGCACCACCCTGGAGGCCGACCGCCCCGTGTCCCTGACCAACAC CCCCGAGGAGCCCCTGATCGTGACCAAGTTCTACTTCCAGGAGGACCAGTAA SEQ ID N0:7, or an RNA molecule comprising an RNA sequence corresponding to SEQ ID NO:7.
  • the therapeutic agent comprises a nucleotide sequence encoding IL-1 soluble Receptor isoform 2 (ILlra-iso2), or a fragment or variant thereof.
  • the nucleotide sequence encoding ILlra-iso2 comprises:
  • the therapeutic agent comprises a nucleotide sequence encoding IL-1 soluble Receptor isoform 3 (ILlra-iso3), or a fragment or variant thereof.
  • the nucleotide sequence encoding ILlra-iso3 comprises:
  • the composition comprises a plurality of constructs, each construct encoding at least one therapeutic agent. In certain embodiments, the composition comprises 1 or more, 2 or more, 5 or more, 10 or more, 15 or more, or 20 or more constructs. In one embodiment, the composition comprises a first construct, comprising a nucleotide sequence encoding a first therapeutic agent; and a second construct, comprising a nucleotide sequence encoding a second agent. In some embodiments, the second agent is a second therapeutic agent.
  • the construct is operatively bound to a translational control element.
  • the construct can incorporate an operatively bound regulatory sequence for the expression of the nucleotide sequence of the invention, thus forming an expression cassette.
  • nucleic acid sequences encapsulated in the nanoparticle of the invention can be obtained using recombinant methods known in the art, such as, for example by screening libraries from cells expressing the gene, by deriving the gene from a vector known to include the same, or by isolating directly from cells and tissues containing the same, using standard techniques.
  • the nucleic acid molecule of interest can be produced synthetically.
  • the nucleic acid can be cloned into a number of types of vectors.
  • the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid.
  • Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, sequencing vectors and vectors optimized for in vitro transcription.
  • nucleic acid molecule comprises a nucleoside-modified RNA.
  • Nucleoside-modified mRNA have particular advantages over non-modified mRNA, including for example, increased stability, low or absent innate immunogenicity, and enhanced translation.
  • Nucleoside-modified mRNA useful in the invention is further described in U.S. Patent No. 8,278,036, which is incorporated by reference herein in its entirety.
  • nucleoside-modified mRNA does not activate any pathophysiologic pathways, translates very efficiently and almost immediately following delivery, and serve as templates for continuous protein production in vivo lasting for several days (Kariko et al., 2008, Mol Ther 16: 1833-1840; Kariko et al., 2012, Mol Ther 20:948-953).
  • the amount of mRNA required to exert a physiological effect is small and that makes it applicable for human therapy.
  • expressing a protein by delivering the encoding mRNA has many benefits over methods that use protein, plasmid DNA or viral vectors.
  • the coding sequence of the desired protein is the only substance delivered to cells, thus avoiding all the side effects associated with plasmid backbones, viral genes, and viral proteins.
  • the mRNA does not carry the risk of being incorporated into the genome and protein production starts immediately after mRNA delivery. For example, high levels of circulating proteins have been measured within 15 to 30 minutes of in vivo injection of the encoding mRNA.
  • using mRNA rather than the protein also has many advantages.
  • the nucleoside-modified RNA comprises the naturally occurring modified-nucleoside pseudouridine.
  • inclusion of pseudouridine makes the mRNA more stable, non-immunogenic, and highly translatable (Kariko et al., 2008, Mol Ther 16: 1833-1840; Anderson et al., 2010, Nucleic Acids Res 38:5884-5892; Anderson et al., 2011, Nucleic Acids Research 39:9329-9338; Kariko et al., 2011, Nucleic Acids Research 39:el42; Kariko et al., 2012, Mol Ther 20:948-953; Kariko et al., 2005, Immunity 23:165-175).
  • RNA containing pseudouridines suppress their innate immunogenicity (Kariko et al., 2005, Immunity 23: 165-175). Further, protein-encoding, in vitro-transcribed RNA containing pseudouridine can be translated more efficiently than RNA containing no or other modified nucleosides (Kariko et al., 2008, Mol Ther 16: 1833-1840).
  • the invention encompasses RNA, oligoribonucleotide, and polyribonucleotide molecules comprising pseudouridine or a modified nucleoside.
  • the composition comprises an isolated nucleic acid encoding a therapeutic agent, wherein the nucleic acid comprises a pseudouridine or a modified nucleoside.
  • the nucleoside-modified RNA of the invention is IVT RNA.
  • the nucleoside-modified RNA is synthesized by T7 phage RNA polymerase.
  • the nucleoside-modified mRNA is synthesized by SP6 phage RNA polymerase.
  • the nucleoside-modified RNA is synthesized by T3 phage RNA polymerase.
  • the modified nucleoside is m 1 acp 3v P (l-methyl-3-(3-amino- 3 -carboxypropyl) pseudouridine.
  • the modified nucleoside is m ll P (1- methylpseudouridine).
  • the modified nucleoside is Tm (2'-O- methylpseudouridine.
  • the modified nucleoside is nr D (5- methyldihydrouridine).
  • the modified nucleoside is m 3 (3- methylpseudouridine).
  • the modified nucleoside is a pseudouridine moiety that is not further modified.
  • the modified nucleoside is a monophosphate, diphosphate, or triphosphate of any of the above pseudouridines.
  • the modified nucleoside is any other pseudouridine-like nucleoside known in the art.
  • the nucleoside that is modified in the nucleoside- modified RNA the invention is uridine (U).
  • the modified nucleoside is cytidine (C).
  • the modified nucleoside is adenosine (A).
  • the modified nucleoside is guanosine (G).
  • the modified nucleoside of the invention is m 5 C (5- methylcytidine). In another embodiment, the modified nucleoside is irf U (5-methyluridine). In another embodiment, the modified nucleoside is m 6 A (N 6 -methyladenosine). In another embodiment, the modified nucleoside is s 2 U (2 -thiouridine). In another embodiment, the modified nucleoside is T (pseudouridine). In another embodiment, the modified nucleoside is Um (2'-O-methyluridine).
  • the modified nucleoside is nfA (1 -methyladenosine); m 2 A (2-methyladenosine); Am (2'-O-methyladenosine); ms 2 m 6 A (2-methylthio-N 6 - methyladenosine); i 6 A (N 6 -isopentenyladenosine); ms 2 i6A (2-methylthio- N 6 isopentenyladenosine); io 6 A (N 6 -(cis-hydroxyisopentenyl)adenosine); ms 2 io 6 A (2- methylthio-N 6 -(cis-hydroxyisopentenyl) adenosine); g 6 A (N 6 -glycinylcarbamoyladenosine); t 6 A (N 6 -threonylcarbamoyladenosine); ms 2 t 6 A (2-methylthio-N 6
  • a nucleoside-modified RNA of the invention comprises a combination of 2 or more of the above modifications. In another embodiment, the nucleoside- modified RNA comprises a combination of 3 or more of the above modifications. In another embodiment, the nucleoside-modified RNA comprises a combination of more than 3 of the above modifications.
  • the fraction of modified residues is 0.2%. In another embodiment, the fraction is 0.3%. In another embodiment, the fraction is 0.4%. In another embodiment, the fraction is 0.5%. In another embodiment, the fraction is 0.6%. In another embodiment, the fraction is 0.8%. In another embodiment, the fraction is 1%. In another embodiment, the fraction is 1.5%. In another embodiment, the fraction is 2%. In another embodiment, the fraction is 2.5%. In another embodiment, the fraction is 3%. In another embodiment, the fraction is 4%.
  • the fraction is 5%. In another embodiment, the fraction is 6%. In another embodiment, the fraction is 8%. In another embodiment, the fraction is 10%. In another embodiment, the fraction is 12%. In another embodiment, the fraction is 14%. In another embodiment, the fraction is 16%. In another embodiment, the fraction is 18%. In another embodiment, the fraction is 20%. In another embodiment, the fraction is 25%. In another embodiment, the fraction is 30%. In another embodiment, the fraction is 35%. In another embodiment, the fraction is 40%. In another embodiment, the fraction is 45%. In another embodiment, the fraction is 50%. In another embodiment, the fraction is 60%. In another embodiment, the fraction is 70%. In another embodiment, the fraction is 80%. In another embodiment, the fraction is 90%. In another embodiment, the fraction is 100%.
  • the fraction is less than 5%. In another embodiment, the fraction is less than 3%. In another embodiment, the fraction is less than 1%. In another embodiment, the fraction is less than 2%. In another embodiment, the fraction is less than 4%. In another embodiment, the fraction is less than 6%. In another embodiment, the fraction is less than 8%. In another embodiment, the fraction is less than 10%. In another embodiment, the fraction is less than 12%. In another embodiment, the fraction is less than 15%. In another embodiment, the fraction is less than 20%. In another embodiment, the fraction is less than 30%. In another embodiment, the fraction is less than 40%. In another embodiment, the fraction is less than 50%. In another embodiment, the fraction is less than 60%. In another embodiment, the fraction is less than 70%.
  • 0.1% of the residues of a given nucleoside are modified.
  • the fraction of the given nucleotide that is modified is 0.2%.
  • the fraction is 0.3%.
  • the fraction is 0.4%.
  • the fraction is 0.5%.
  • the fraction is 0.6%.
  • the fraction is 0.8%.
  • the fraction is 1%.
  • the fraction is 1.5%.
  • the fraction is 2%.
  • the fraction is 2.5%.
  • the fraction is 3%.
  • the fraction is 4%.
  • the fraction is 5%. In another embodiment, the fraction is 6%. In another embodiment, the fraction is 8%. In another embodiment, the fraction is 10%. In another embodiment, the fraction is 12%. In another embodiment, the fraction is 14%. In another embodiment, the fraction is 16%. In another embodiment, the fraction is 18%. In another embodiment, the fraction is 20%. In another embodiment, the fraction is 25%. In another embodiment, the fraction is 30%. In another embodiment, the fraction is 35%. In another embodiment, the fraction is 40%. In another embodiment, the fraction is 45%. In another embodiment, the fraction is 50%. In another embodiment, the fraction is 60%. In another embodiment, the fraction is 70%. In another embodiment, the fraction is 80%. In another embodiment, the fraction is 90%. In another embodiment, the fraction is 100%.
  • the fraction of the given nucleotide that is modified is less than 8%. In another embodiment, the fraction is less than 10%. In another embodiment, the fraction is less than 5%. In another embodiment, the fraction is less than 3%. In another embodiment, the fraction is less than 1%. In another embodiment, the fraction is less than 2%. In another embodiment, the fraction is less than 4%. In another embodiment, the fraction is less than 6%. In another embodiment, the fraction is less than 12%. In another embodiment, the fraction is less than 15%. In another embodiment, the fraction is less than 20%. In another embodiment, the fraction is less than 30%. In another embodiment, the fraction is less than 40%. In another embodiment, the fraction is less than 50%. In another embodiment, the fraction is less than 60%. In another embodiment, the fraction is less than 70%.
  • a nucleoside-modified RNA of the invention is translated in the cell more efficiently than an unmodified RNA molecule with the same sequence.
  • the nucleoside-modified RNA exhibits enhanced ability to be translated by a target cell.
  • translation is enhanced by a factor of 2- fold relative to its unmodified counterpart.
  • translation is enhanced by a 3-fold factor.
  • translation is enhanced by a 5-fold factor.
  • translation is enhanced by a 7-fold factor.
  • translation is enhanced by a 10-fold factor.
  • translation is enhanced by a 15-fold factor.
  • translation is enhanced by a 20-fold factor.
  • translation is enhanced by a 50-fold factor. In another embodiment, translation is enhanced by a 100-fold factor. In another embodiment, translation is enhanced by a 200-fold factor. In another embodiment, translation is enhanced by a 500-fold factor. In another embodiment, translation is enhanced by a 1000-fold factor. In another embodiment, translation is enhanced by a 2000-fold factor. In another embodiment, the factor is 10-1000-fold. In another embodiment, the factor is 10-100-fold. In another embodiment, the factor is 10-200- fold. In another embodiment, the factor is 10-300-fold. In another embodiment, the factor is 10- 500-fold. In another embodiment, the factor is 20-1000-fold. In another embodiment, the factor is 30-1000-fold. In another embodiment, the factor is 50-1000-fold. In another embodiment, the factor is 100-1000-fold. In another embodiment, the factor is 200-1000-fold. In another embodiment, translation is enhanced by any other significant amount or range of amounts.
  • the therapeutic agent includes an isolated peptide that modulates a target.
  • the peptide of the invention inhibits or activates a target directly by binding to the target thereby modulating the normal functional activity of the target.
  • the peptide of the invention modulates the target by competing with endogenous proteins.
  • the peptide of the invention modulates the activity of the target by acting as a transdominant negative mutant.
  • the variants of the polypeptide therapeutic agents may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (such as a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, (ii) one in which there are one or more modified amino acid residues, e.g., residues that are modified by the attachment of substituent groups, (iii) one in which the polypeptide is an alternative splice variant of the polypeptide of the invention, (iv) fragments of the polypeptides and/or (v) one in which the polypeptide is fused with another polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification (for example, His-tag) or for detection (for example, Sv5 epitope tag).
  • a conserved or non-conserved amino acid residue such as a conserved amino acid residue
  • substituted amino acid residue may or may
  • the fragments include polypeptides generated via proteolytic cleavage (including multisite proteolysis) of an original sequence. Variants may be post-translationally, or chemically modified. Such variants are deemed to be within the scope of those skilled in the art from the teaching herein.
  • the nanoparticles may further comprise any lipid capable of forming a particle to which the one or more nucleic acid molecules are attached, or in which the one or more nucleic acid molecules are encapsulated.
  • lipid refers to a group of organic compounds that are derivatives of fatty acids (e.g., esters) and are generally characterized by being insoluble in water but soluble in many organic solvents.
  • Lipids are usually divided in at least three classes: (1) “simple lipids” which include fats and oils as well as waxes; (2) “compound lipids” which include phospholipids and glycolipids; and (3) “derived lipids” such as steroids.
  • the nanoparticles do not further comprise any lipid capable of forming a particle to which the one or more nucleic acid molecules are attached, or in which the one or more nucleic acid molecules are encapsulated.
  • the nanoparticles do not further comprise any or all of a simple lipid, a compound lipid, or a derived lipid.
  • the nanoparticle comprises a cationic lipid.
  • cationic lipid refers to a lipid that is cationic or becomes cationic (protonated) as the pH is lowered below the pK of the ionizable group of the lipid, but is progressively more neutral at higher pH values. At pH values below the pK, the lipid is then able to associate with negatively charged nucleic acids.
  • the cationic lipid comprises a zwitterionic lipid that assumes a positive charge on pH decrease.
  • the nanoparticle does not comprise a cationic lipid.
  • the cationic lipid which is optionally present or not present in the nanoparticles comprises any of a number of lipid species which carry a net positive charge at a selective pH, such as physiological pH.
  • lipids include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC); N-(2,3- dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); N,N-distearyl-N,N- dimethylammonium bromide (DDAB); N-(2, 3 -di oleoyloxy )propyl)-N,N,N- trimethylammonium chloride (DOTAP); 3-(N — (N',N'-dimethylaminoethane)- carbamoyl)cholesterol (DC-Chol), N-(l-(2,3-dioleoyloxy)propyl)
  • DODAC N,N
  • cationic lipids are available which can be used in the invention. These include, for example, LIPOFECTIN® (commercially available cationic liposomes comprising DOTMA and l,2-dioleoyl-sn-3 -phosphoethanolamine (DOPE), from GIBCO/BRL, Grand Island, N.Y.); LIPOFECT AMINE® (commercially available cationic liposomes comprising N-(l-(2,3-dioleyloxy)propyl)-N-(2- (sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE), from GIBCO/BRL); and TRANSFECTAM® (commercially available cationic lipids comprising dioctadecylamidoglycyl carboxyspermine (DOGS) in ethanol from Promega Corp., Madison, Wis.).
  • LIPOFECTIN® commercially available cationic liposome
  • lipids are cationic and have a positive charge at below physiological pH: DODAP, DODMA, DMDMA, l,2-dilinoleyloxy-N,N- dimethylaminopropane (DLinDMA), 1 ,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA).
  • the cationic lipid is an amino lipid.
  • amino lipids include those described in WO 2012/016184, incorporated herein by reference in its entirety.
  • Representative amino lipids include, but are not limited to, l,2-dilinoleyoxy-3- (dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoley oxy-3 -morpholinopropane (DLin- MA), l,2-dilinoleoyl-3 -dimethylaminopropane (DLinDAP), l,2-dilinoleylthio-3- dimethylaminopropane (DLin-S-DMA), l-linoleoyl-2-linoleyloxy-3 -dimethylaminopropane (DLin-2-DMAP), l,2-dilinoleyloxy-3 -trimethylaminopropane chloride salt (DLin-TMA.Cl), l,2-dilinoley
  • the nanoparticles further comprise a steroid or steroid analogue.
  • a “steroid” is a compound comprising the following carbon skeleton: [0186]
  • the steroid or steroid analogue is cholesterol.
  • the nanoparticles do not comprise a steroid or steroid analogue. In one embodiment, the nanoparticles do not comprise cholesterol.
  • the nanoparticles further comprise a stabilizer.
  • the stabilizer comprises oligooxyetylenes.
  • the stabilizer comprises a water soluble macromolecule.
  • the stabilizer comprises a water soluble oligomer.
  • the stabilizer comprises a carbohydrate.
  • the nanoparticle comprises one or more targeting moieties which are capable of targeting the nanoparticle to a cell or cell population.
  • the targeting moiety is a ligand which directs the nanoparticle to a receptor found on a cell surface.
  • the nanoparticle comprises one or more internalization domains.
  • the nanoparticle comprises one or more domains which bind to a cell to induce the internalization of the nanoparticle.
  • the one or more internalization domains bind to a receptor found on a cell surface to induce receptor-mediated uptake of the nanoparticle.
  • the nanoparticle is capable of binding a biomolecule in vivo, where the nanoparticle-bound biomolecule can then be recognized by a cell-surface receptor to induce internalization.
  • the nanoparticle binds systemic ApoE, which leads to the uptake of the nanoparticle and associated cargo.
  • the present invention relates to compositions comprising at least one amphiphilic Janus dendrimer of the present invention and/or nanoparticle thereof.
  • the composition further comprises at least one agent described herein.
  • the invention also relates to compositions comprising at least one compound of Formula (I) or Formula (II) and methods of use thereof for delivering an encapsulated agent to the lung parenchyma.
  • agents that can be encapsulated in the compositions of the invention include, but are not limited to, diagnostic agents, detectable agents, and therapeutic agents.
  • the composition comprises nanoparticles comprising a compound of Formula (I) or Formula (II) and at least one agent encapsulated by the nanoparticle.
  • the encapsulated agent comprises an agent for reducing inflammation in a subject.
  • the composition may be prepared by injection of a mixture comprising a compound described herein into a suitable solution, such as a solution comprising the agent to be encapsulated.
  • the composition of the invention comprises in vitro transcribed (IVT) RNA molecule.
  • IVVT in vitro transcribed
  • the composition of the invention comprises IVT RNA molecule which encodes an agent.
  • the IVT RNA molecule of the present composition is a nucleoside-modified mRNA molecule.
  • the nucleoside-modified mRNA molecule encodes an anti-inflammatory agent.
  • the nucleoside-modified mRNA molecule encodes TGF-P, Alpha- 1 -antitrypsin (A1AT), soluble IL-1R, CFTR, IL-4, IL-13, Surfactant Protein D fragment (SP-D fragment), surfactant protein B (SP-B) or surfactant protein C (SP- C), or a fragment or variant thereof.
  • the nucleoside-modified mRNA molecule encodes TGF-0, Alpha- 1 -antitrypsin (A1AT) or soluble IL-1R.
  • the nucleic acid molecule encoding TGF-P comprises SEQ ID NO: 1, SEQ ID NO:2. In one embodiment, the nucleic acid molecule encoding TGF-P comprises an mRNA molecule comprising a sequence corresponding to the sequence of SEQ ID NO: 1. In one embodiment, the nucleic acid molecule encoding Al AT comprises an mRNA transcribed from SEQ ID NO:2. In one embodiment, the nucleic acid molecule encoding A1AT comprises SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6.
  • the nucleic acid molecule encoding A1AT comprises an mRNA molecule comprising a sequence corresponding to the sequence of SEQ ID NO:3 (AlAT-isol) or SEQ ID NO:5 (AlAT-iso2).
  • the nucleic acid molecule encoding A1AT comprises an mRNA transcribed from 3_2560_pUC-ccTEV-co.m.AATl-isol(AAC28869.1)-A101 (SEQ ID NO:4) or 6_2560_pUC-ccTEV-co.m.AATl-iso2(NP_001239498.1)-A101 (SEQ ID NO:6).
  • the nucleic acid molecule encoding soluble IL-1R comprises SEQ ID NO:7, SEQ ID NO 8, SEQ ID NOV, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12. In one embodiment, the nucleic acid molecule encoding soluble IL-1R comprises an mRNA molecule comprising a sequence corresponding to the sequence of SEQ ID NO:7 (ILlra-isol), SEQ ID N0:9 (ILlra-iso2) or SEQ ID NO: 11 (ILlra-iso3).
  • the nucleic acid molecule encoding soluble IL-1R comprises an mRNA transcribed from 3_2560_pUC-ccTEV- co.m.ILlra-isol-AlOl (SEQ ID N0:8), 6_2560_pUC-ccTEV-co.m.ILlra-iso2p-A101 (SEQ ID NO: 10) or 9_2560_pUC-ccTEV-co.m.ILlra-iso3-A101 (SEQ ID NO: 12).
  • the invention is not limited to any particular agent or combination of agents.
  • the nucleoside-modified mRNA molecule encodes SEQ ID NO: 13 (alpha-1 protease inhibitor 1), SEQ ID NO: 14 (alpha- 1 -antitrypsin 1-1 isoform 2), SEQ ID NO: 15 (Interleukin- 1 receptor antagonist protein isoform 2 precursor), SEQ ID NO: 16 (Interleukin- 1 receptor antagonist protein isoform 3), or SEQ ID NO: 17 (Interleukin- 1 receptor antagonist protein isoform 1), or a fragment or variant thereof.
  • the composition comprises at least one nucleoside-modified RNA molecule encoding a combination of at least two agents. In one embodiment, the composition comprises a combination of two or more nucleoside-modified RNA molecules encoding a combination of two or more agents.
  • the method comprises the systemic administration of the composition into the subject, including for example intradermal administration. In certain embodiments, the method comprises administering a plurality of doses to the subject. In another embodiment, the method comprises administering a single dose of the composition, where the single dose is effective in inducing a therapeutic response.
  • the composition of the invention comprises a combination of agents described herein.
  • a composition comprising a combination of agents described herein has an additive effect, wherein the overall effect of the combination is approximately equal to the sum of the effects of each individual agent.
  • a composition comprising a combination of agents described herein has a synergistic effect, wherein the overall effect of the combination is greater than the sum of the effects of each individual agent.
  • a composition comprising a combination of agents comprises individual agents in any suitable ratio.
  • the composition comprises a 1 : 1 ratio of two individual agents.
  • the combination is not limited to any particular ratio. Rather any ratio that is shown to be effective is encompassed.
  • compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology.
  • preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.
  • compositions are principally directed to pharmaceutical compositions which are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as non-human primates, cattle, pigs, horses, sheep, cats, and dogs.
  • compositions that are useful in the methods of the invention may be prepared, packaged, or sold in formulations suitable for ophthalmic, oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, intravenous, intracerebroventricular, intradermal, intramuscular, or another route of administration.
  • Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunogenic-based formulations.
  • a pharmaceutical composition of the invention may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses.
  • a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
  • the amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
  • compositions of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered.
  • the composition may comprise between 0.1% and 100% (w/w) active ingredient.
  • composition of the invention may further comprise one or more additional pharmaceutically active agents.
  • Controlled- or sustained-release formulations of a pharmaceutical composition of the invention may be made using conventional technology.
  • Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like.
  • parenteral administration is contemplated to include, but is not limited to, intraocular, intravitreal, subcutaneous, intraperitoneal, intramuscular, intradermal, intrastemal injection, intratumoral, intravenous, intracerebroventricular and kidney dialytic infusion techniques.
  • Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations.
  • Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents.
  • the active ingredient is provided in dry (i.e. powder or granular) form for reconstitution with a suitable vehicle (e.g. sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.
  • compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution.
  • This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein.
  • Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example.
  • Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides.
  • compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.
  • a pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for pulmonary administration via the buccal cavity.
  • a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 to about 7 nanometers, or from about 1 to about 6 nanometers.
  • Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant may be directed to disperse the powder or using a self-propelling solvent/powder-dispensing container such as a device comprising the active ingredient dissolved or suspended in a low-boiling propellant in a sealed container.
  • such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. In some embodiments, at least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 nanometers.
  • Dry powder compositions may include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.
  • Low boiling propellants generally include liquid propellants having a boiling point of below 65°F at atmospheric pressure.
  • the propellant may constitute 50 to 99.9% (w/w) of the composition, and the active ingredient may constitute 0.1 to 20% (w/w) of the composition.
  • the propellant may further comprise additional ingredients such as a liquid non-ionic or solid anionic surfactant or a solid diluent (sometimes having a particle size of the same order as particles comprising the active ingredient).
  • Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations.
  • Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents.
  • the active ingredient is provided in dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.
  • the pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution.
  • This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein.
  • Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example.
  • Other acceptable diluents and solvents include, but are not limited to, Ringer’s solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides.
  • compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.
  • additional ingredients include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials.
  • compositions of the invention are known in the art and described, for example in Remington's Pharmaceutical Sciences (1985, Genaro, ed., Mack Publishing Co., Easton, PA), which is incorporated herein by reference.
  • the invention provides methods of delivering an agent to a lung cell, or tissue of a subject.
  • the agent is a therapeutic agent for the treatment or prevention of a disease or disorder.
  • the agent is an anti-inflammatory agent for the treatment or prevention of inflammation in the lung.
  • the antiinflammatory agent is TGF-0, Alpha- 1 -antitrypsin (A1AT), soluble IL-1R, CFTR, IL-4, IL-13, Surfactant Protein D fragment (SP-D fragment), surfactant protein B (SP-B) or surfactant protein C (SP-C) or a fragment or variant thereof.
  • the anti-inflammatory agent is TGF-p.
  • the anti-inflammatory agent is an mRNA molecule comprising SEQ ID NO: 1.
  • the anti-inflammatory agent is an mRNA molecule comprising SEQ ID NO:2.
  • the invention provides methods for treating or preventing a disease or disorder comprising administering an effective amount of a composition comprising at least one therapeutic agent.
  • the composition is administered to a subject having inflammation of the lung.
  • the composition is administered to a subject at risk for inflammation of the lung.
  • the composition may be co-administered to a subject who is being treated with a therapeutic agent known to be associated with an increased risk of inflammation of the lung.
  • co-administration of the composition and at least one additional therapeutic agent prevents or reduces drug-induced inflammation.
  • the composition is administered to a subject who has increased likelihood, though genetic factors, environmental factors, or the like, of developing lung inflammation.
  • the method prevents or treats asthma, chronic obstructive pulmonary disease (COPD), emphysema, acute respiratory distress syndrome (ARDS), lung cancer, sarcoidosis, asbestosis, chronic cough, pneumothorax, pulmonary embolism, pleural effusion, rheumatoid lung disease, pulmonary fibrosis, bronchiectasis, tuberculosis, bronchitis, pneumonia, acute lung injury (ALI), emphysema, cystic fibrosis or idiopathic fibrosis.
  • the lung cancer is non-small cell lung cancer.
  • the method reduces at least one symptom of asthma, chronic obstructive pulmonary disease (COPD), emphysema, acute respiratory distress syndrome (ARDS), lung cancer, sarcoidosis, asbestosis, chronic cough, pneumothorax, pulmonary embolism, pleural effusion, rheumatoid lung disease, pulmonary fibrosis, bronchiectasis, tuberculosis, bronchitis, pneumonia, acute lung injury (ALI), emphysema, cystic fibrosis or idiopathic fibrosis.
  • the lung cancer is non-small cell lung cancer.
  • the method comprises administering a composition comprising at least one nucleoside-modified nucleic acid molecule encoding at least one therapeutic agent. In one embodiment, the method comprises administering a composition comprising a first nucleoside-modified nucleic acid molecule encoding at least one therapeutic agent and a second therapeutic agent. In one embodiment, the method comprises administering a first composition comprising at least one nucleoside-modified nucleic acid molecules encoding at least one therapeutic agent and administering a second composition comprising at least one nucleoside-modified nucleic acid molecule encoding at least one additional therapeutic agent.
  • the method comprises administering to subject a plurality of nucleoside-modified nucleic acid molecules encoding a plurality of therapeutic agents.
  • the method of the invention allows for sustained expression of the therapeutic agent for at least several days following administration.
  • the method in certain embodiments, also provides for transient expression, as in certain embodiments, the nucleic acid is not integrated into the subject genome.
  • the method comprises administering nucleoside- modified RNA which provides stable expression of the therapeutic agent.
  • the method of the invention comprises systemic administration of the subject, including for example enteral or parenteral administration.
  • the method comprises intradermal delivery of the composition.
  • the method comprises intravenous delivery of the composition.
  • the method comprises intramuscular delivery of the composition.
  • the method comprises subcutaneous delivery of the composition.
  • the method comprises inhalation of the composition.
  • the method comprises intranasal delivery of the composition.
  • composition of the invention may be administered to a subject either alone, or in conjunction with another agent.
  • the therapeutic and prophylactic methods of the invention thus encompass the use of pharmaceutical compositions comprising a nanoparticle encapsulating an RNA molecule encoding a therapeutic agent alone or in combination with at least one additional therapeutic agent.
  • the pharmaceutical compositions useful for practicing the invention may be administered to deliver a dose of from ng/kg/day and 100 mg/kg/day.
  • the invention envisions administration of a dose which results in a concentration of the compound of the invention from lOnM and 10 pM in a mammal.
  • dosages which may be administered in a method of the invention to a mammal range in amount from 0.01 pg to about 50 mg per kilogram of body weight of the mammal, while the precise dosage administered will vary depending upon any number of factors, including but not limited to, the type of mammal and type of disease state being treated, the age of the mammal and the route of administration.
  • the dosage of the compound will vary from about 0.1 pg to about 10 mg per kilogram of body weight of the mammal.
  • the dosage will vary from about 1 pg to about 1 mg per kilogram of body weight of the mammal.
  • composition may be administered to a mammal as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less.
  • the frequency of the dose will be readily apparent to the skilled artisan and will depend upon any number of factors, such as, but not limited to, the type and severity of the disease being treated, the type and age of the mammal, etc.
  • administration of an composition or vaccine of the invention may be performed by single administration or boosted by multiple administrations.
  • the invention includes a method comprising administering one or more compositions encoding at least one therapeutic agent described herein.
  • the method has an additive effect, wherein the overall effect of the administering the combination is approximately equal to the sum of the effects of administering each therapeutic agent.
  • the method has a synergistic effect, wherein the overall effect of administering the combination is greater than the sum of the effects of administering each therapeutic agent.
  • Example 1 Targeted delivery of TGF-B mRNA to murine lung parenchyma using one- component ionizable amphiphilic Janus Dendrimers
  • IAJD34 is a single component lipid particle that can perform targeted delivery of mRNA to the lung (Zhang et al., 2021, Journal of the American Chemical Society 143, 17975- 17982).
  • the current studies have shown that IAJD34 can successfully deliver luciferase mRNA to the lung, specifically targeting lung parenchyma. This is of particular therapeutic advantage as targeting the smaller airways, specifically the more vascular regions at airway zones 16 and below, is necessary to treat most lung injury and disease (Johnson et al., 2010, J. Aerosol Med. Pulm. Drug Deliv. 23, 243-252; Matthay et al., 2005, Am. J. Respir. Cell Mol. Biol.
  • TGF-P mRNA-IAJD34 was able to deliver dose-dependent levels of TGF-B mRNA to the lung with limited inflammation and toxicity observed. Once delivered, TGF-P protein was produced, processed, and mediated downstream cytokine signaling. Delivery of TGF-P was transiently expressed over the course of 48 h, which is important as long-term delivery of TGF-P can lead to significant fibrosis.
  • TGF-P mRNA-IAJD34 was used to treat ITB effects on the lung 3 days postinjury showed only mild improvements in lung histology and barrier function. Although the use of the day 3 timepoint in BALB/ c mice meant that these factors were only mildly affected by ITB, our intention was to demonstrate that delivery of TGF-P mRNA-IAJD34 to the lung would reduce inflammatory signaling. Indeed, there was a significant TGF-P mRNA-IAJD34 effect that correlated with TGF-P expression. These results indicate that TGF-P was successfully delivered to the lower lung and a significant signaling effect was observed. Furthermore, this effect appears to be transitory in nature as would be required in a therapeutic setting.
  • TGF-P mRNA-IAJD34 As a therapeutic in this ALI model, the effect of TGF-P at 7 days or longer post-injury should be characterized, which may require multiple dosing regimens.
  • This study establishes the potential use of IAJD34 to treat ALI and other pulmonary ailments that currently require targeted clinical interventions and demonstrates the potential of mRNA delivery for therapeutic use in the lung.
  • Codon optimized sequences of TGF-P or Luc were synthesized and cloned into an mRNA plasmid (Pardi et al., 2015, J. Control. Release 217, 345-351; Martinez et al., 2021, Science 373, 991-998). Briefly, nucleoside-modified mRNAs were transcribed to contain 101 nucleotide-long poly(A) tails. mRNAs were modified with ml'P-5'-triphosphate (Trilink, # N- 1081) instead of UTP and capped co-transcriptionally using the trinucleotide capl analog, CleanCap (Trilink, # N-7413).
  • mRNA was purified by cellulose (Sigma-Aldri ch, # 11363-250 G) purification 25 All mRNA were analyzed for quality control through agarose gel electrophoresis, dsRNA, endotoxin, and IFN-a assays and in vitro transfection. mRNA was stored at -20 °C until ready for use.
  • HEK 293 cells were seeded into a 24-well cell culture plate at a density of 150,000 cells/well in Dulbecco’s Modified Enriched Medium (DMEM) containing fetal bovine serum (10%), L-Glutamine, and penicillin/streptomycin. Cells were allowed to grow for 24 hrs and then TGF-P mRNA (500 n per/well) was transfected into cells in triplicate using lipofectamine MessengerMax (Life Technologies, Carlsbad, CA) and OptiMEM serum-free medium (ThermoFisher Scientific, # 31985-062, Rockford, IL) according to manufacturer guidelines.
  • DMEM Modified Enriched Medium
  • OptiMEM serum-free medium ThermoFisher Scientific, # 31985-062, Rockford, IL
  • Membranes were stripped and re-probed with anti-GAPDH (Cell Signaling Technology,# 21185, Danvers, MA) for 1 h at room temperature at a 1 : 1000 dilution, and a secondary goat anti-rabbit IgG HRP (Bio-Rad,# 170-6515 Bio-Rad, Hercules, CA) at a 1 : 10,000 for 1 h at room temperature.
  • Membranes were washed in ECL Prime Western Blotting Detection Reagent (Amersham Biosciences, Amersham, UK,# RPN2232) prior to visualization using an Amersham Cytiva imager (Amersham Biosciences, Amersham, UK).
  • TGF-P protein expression in lung tissue was performed on collected lung tissue. Tissue from the accessory lobe was mechanically homogenized on dry ice and digested in lysis buffer with protease inhibitors (100 pL/0.33 g tissue). The tissues were sonicated, centrifuged (2 min, 1000 x g), and supernatants were assessed for protein concentration using a PierceTM BCA Protein Assay. Equal amounts of protein from lung tissue samples (45 pg per well) were analyzed for TGF-P protein expression as described above. Antibodies for TGF-P were used at 1 : 5000, and goat anti-rabbit HRP at 1 : 5000. Membranes were washed in ECL Prime Western Blotting Detection Reagent prior to visualization on x-ray film. Uncropped gel images are available in the Source Data file.
  • IAJD34 was synthesized as previously described (Zhang et al., 2021, J. Am. Chem. Soc. 143, 12315-12327) and the purity and structural identity of final products and intermediates were determined using various techniques, including thin-layer chromatography (TLC), high-pressure liquid chromatography (HPLC), 1 H and 13 C NMR, and Electrospray Ionization Time-of-Flight (ESI TOF) mass spectrometry (Zhang et al., 2021, J. Am. Chem. Soc. 143, 12315-12327). Co-assembly of IAID34 and mRNA was per formed (Zhang et al., 2021, J. Am. Chem. Soc.
  • Nucleoside-modified mRNA encoding Luc mRNA or TGF-P mRNA was dissolved at a concentration of 4 mg/ml in UltraPure DNase/RNase-free PCR-certified water (Teknova, # W3440). IAJD34 was dissolved in ethanol at a concentration of 80 mg/ml.
  • the mRNA was mixed with 10 mM acetate buffer (pH 4.0), and this solution was rapidly mixed with IAFD34 in ethanol at an IAJD34/mRNA weight-to-weight ratio of 40 and vortexed for 5 s.
  • the prepared formulation was analyzed for size (nm), poly dispersity index (POI), and zeta potential using dynamic light scattering (DLS) prior to injection of empty-1 AJD34, Luc mRNA-IAJD34, and TGF-P mRNA- IAJD34.
  • mRNA encapsulation efficiency defined as the amount of mRNA encapsulated versus free, was determined using a Quant-iTTM RiboGreenTM RNA Assay Kit (Invitrogen) according to manufacturer instructions. Briefly, to measure nonencapsulated mRNA (free mRNA), mRNA-IAJD formulations were diluted with Tris-EDTA buffer (TE) and treated with the RiboGreenTM reagent. To measure total mRNA, mRNA-IAJD formulations were disrupted by treatment with 2% Triton X-100 in TE buffer and treated with RiboGreenTM reagent. Both conditions were per- formed in triplicate to ensure proper release of encapsulated mRNA.
  • TE Tris-EDTA buffer
  • RiboGreenTM reagent To measure total mRNA, mRNA-IAJD formulations were disrupted by treatment with 2% Triton X-100 in TE buffer and treated with RiboGreenTM reagent. Both conditions were per- formed in trip
  • ESI- TOF mass spectrometry was performed on a Thermo Fisher Scientific ExactiveTM. IAJD 34 was dis- solved in dichloromethane (3 mg/ml). The report contains the ionization method (ESI in positive mode), chemical formula, [ion]charge, mass to charge ratio (m/z): calculated value, found value.
  • mice Male and female wild-type BALB/ c (8-10 weeks) mice were used for all experiments. Mice were housed under standard conditions with food and water provided ad libitum.
  • mice Female or male 6-8-week-old BALB/ c mice were administered 10 pg of Luc mRNA-IAJD34 in alOO pL volume via retro-orbital intravenous injection. Four hours post-injection, mice were injected intraperitoneally (i.p.) with D-luciferin (Regis Technologies) at a dose of 150 mg/kg of body weight. The mice were anesthetized in a ventilated anesthesia chamber with 3% isoflurane (Piramal Healthcare Limited) in oxygen and imaged 10 min post D-luciferin inj ection using an in vivo imaging system (IVIS, PerkinElmer, Waltham, MA).
  • mice were sacrificed, organs were collected immediately, and bioluminescence imaging was performed .
  • Bioluminescence was quantified as proton flux (photons/ seconds) in each region of interest using Living Image software v3.2 (PerkinElmer)( Zhang et al., 2022, J. Am. Chem. Soc. 144, 4746-4753).
  • TGF-P mRNA was co-assembled with IAJD34 as described above. The formulation was then concentrated using a Vivaspin ultrafiltration spin column MWCO 10,000 (Cytiva # 28-9322-47) according to manufacture guidelines. Mice were randomly assigned to control or treated. Mice were anesthetized with isoflurane and received a single retro- orbital intravenous injection of 10 pg, 20 pg, or 30 pg of TGF-P mRNA co-assembled with IAJD34 or empty IAJD34. The following criteria to assess the toxicity of formulated TGF-P mRNA-IAJDs was utilized: mice behavior, serum and BAL fluid cytokine analysis, liver enzyme activities, and main organs' histological score.
  • mice were sacrificed 24 h post-injections, and their serum samples and organs were collected. Organs were fixed in 4% paraformaldehyde and further stained with H&E as described below. The liver, spleen, kidney, and lungs were inspected for signs of inflammation and deviations from normal histology.
  • ELISAs for aspartate aminotransferase (AST; Abeam ab263882) and alanine aminotransferase (ALT; Abeam ab282882) were performed in serum in a 1 :80 dilution according to manufacture guidelines, lnterleukin-6 (IL-6; Abeam ab222503) was used to measure serum and first wash BAL fluid levels in accordance with manufacturer guidelines.
  • AST aspartate aminotransferase
  • ALT alanine aminotransferase
  • IL-6 lnterleukin-6
  • Cytokine in the first 1ml of BAL fluid was performed by the University of Pennsylvania Human Immunology Core (RRID: SCR_022380) using a Milliplex Max Mouse Cytokine/Chemokine Magnetic Bead Panel - Premixed 32 Plex - Immunology Multiplex Assay (MCYTMAG-70K- PX32; MilliporeSigma, Burlington, MA). Samples were run in duplicate, and cytokine and chemokine concentrations were determined from a matched standard curve.
  • mice were randomly assigned to treatment groups. Mice were anesthetized with isoflurane and received a single retro-orbital intravenous injection of 10 pg of TGF-0 mRNA formulated with IAJD34 or empty IAJD34. while still under anesthesia, mice immediately received a single intratracheal instillation of either 50 pLPBS or 50 pL of bleomycin (3U/kg of body weight) (Santa Cruz Biotechnology, Inc., Dallas, TX; # sc- 200134B) diluted in PBS (Stevenson et al., 2022, J. Pharmacol. Exp. Ther. 382, 356-365).
  • mice Following injections and treatment, mice were observed to ensure full recovery from anesthesia, and that dose was successfully administered. Animals were weighed daily and provided supportive care when necessary. Mice were sacrificed 3 days post intratracheal administration of bleomycin via a single intraperitoneal injection of ketamine (135 mg/kg of body weight) and xylazine (30 mg/kg of body weight) (Fort Dodge Ani mal Health, Fort Dodge, IA).
  • Lungs were lavaged with 1ml of ice-cold PBS, followed by five 1 ml washes of ice-cold PBS through a 20-g auge canula inserted into the trachea. Collected BAL fluid was centrifuged at 300 * g for 8 mins. The cell-free supernatant from the first wash was collected for protein and cytokine analysis, and the cell-free supernatant from the five subsequent washes (5 ml) was collected for phospholipid analysis. Cell pellets from both washes were combined and resuspended in 1 ml of staining buffer (5%FBS in PBS, 0.
  • Lung tissue digest [0251] Lung tissue from the right lobes was incubated at 37 °C for 30 min with intermittent shaking in 5 ml of collagenase buffer (2 mg/ml colagenase type IV (Sigma Aldrich, St. Louis, MO) in RPMI 1640 (ThermoFisher Scientific, Rock ford , IL) with 5% FBS (ThermoFisher Scientific, Rockford, IL)). The digested tissue was filtered through a 70 pm strainer, washed with RPMI with 5% FBS, and centrifuged (6 min, 400 xg). The cell pellet was lysed with Red Blood Cell Lysis Buffer (Sigma Aldrich, St. Louis, MO) for 5 min.
  • the purified cell pellet was resuspended at a concentration of! x 10 8 cells/ml PBS with 2% FBS and 1 mM EDTA.
  • CD45 + leukocytes were isolated using the EasySepTM Mouse CD45 Positive Selection Kit (Stemcell Technologies, Cam- bridge, MA) and prepared for flow cytometry.
  • Positively selected CD45 lung digest cells were plated at 200,000 cells per well in a poly -D-lysine coated Seahorse XF96 Cell Culture Micro- plate (Agilent, Santa Clara, C A) and incubated at 37 °C, 5% CO2 for 1 h to allow for cell adhesion.
  • the cells were fed with EC AR medium (DMEM media, pH 7.4 (Agilent # 103575-100) with 2mM L- glutamine) or OCR medium ( DMEM media, pH 7.4 with 25 mM glucose, ImM pyruvate, and 2 mM L-glutamine).
  • the extracellular acidification rate (EC AR) and oxygen consumption rate (OCR) were measured using a Seahorse XF96 Analyzer (Agilent Technologies, Santa Clara, CA).
  • ECAR extracellular acidification rate
  • OCR oxygen consumption rate
  • CD45 + cells were sequentially treated with 25 mM glucose, 4 pM oligomycin, and 50 mM 2-deoxy-d-glucose (2-DG).
  • OCR measurement the cells were sequentially treated with 4 pM oligomy- cin, 1 pM carbonyl cyanide ptrifluoromethoxyphenylhydrazone (FCCP), 0.5 pM rotenone, and 0.5 pM antimycin A.
  • FCCP carbonyl cyanide ptrifluoromethoxyphenylhydrazone
  • FCCP carbonyl cyanide ptrifluoromethoxyphenylhydrazone
  • FCCP carbonyl cyanide ptrifluoromethoxyphenylhydra
  • the left lung lobe was inflation fixed in 3% paraformaldehyde and embedded in paraffin. Liver, spleen, and kidney tissue were also fixed in 3% paraformaldehyde and embedded in paraffin. Four-micrometer sections were cut, slide-mounted, and left unstained for IHC or stained with hematoxylin and eosin (H&E) to observe histological changes.
  • H&E hematoxylin and eosin
  • tissues were blindly scored by a board-certified pathologist to determine overt toxicological pathology.
  • scans were blindly scored and quantified via ImageJ (NIH) .
  • Tissues were incubated at 4 °C for 18 h with anti -firefly luciferase (Abeam ab238448; Waltham, MA 1: 100) or TGF-0 (Abeam ab215715; Waltham, MA 1 : 100) antibody in a blocking buffer along with IgG controls (Pro-Sci 3703; Fort Collins, CO, matched concentrations). Sections were washed in decreasing concentrations of Tween -PB S ( l%-0.5 %) and incubated with biotin-conj ugated secondary antibody (Vector Laboratories Vectastain Rabbit Kit; Newark, CA) for 1 h at ambient temperature.
  • biotin-conj ugated secondary antibody Vector Laboratories Vectastain Rabbit Kit; Newark, CA
  • Antibody binding was vi sualized beneath a microscope using a DAB Peroxidase Substrate Kit (Vector Laboratories, Newark, CA). Slides were scanned at40X magnification using a VS 120 Virtual Slide Microscope (Olympus, Waltham, MA) and viewed with Oly VIA software (Olympus, Waltham , MA) at 400X magnification.
  • Results are reported as means ⁇ SE unless otherwise indicated. Data were tested for normal distribution using a Shapiro -Wilks test. If normally distributed, statistical significance for multiple group comparisons was determined using a one-way ANOVA with Tukey's post-hoc test or Sidak's multiple comparison test. If not normally distributed, statistical significance for multiple comparisons was determined using a Kruskal-Walli's test with a Dunn's multiple comparison test if needed. For parametric single comparisons, statistical significance was determined using an unpaired t-test with Welch's correction compared to control groups as indicated in figure legends If single comparisons were nonparametric, statistical significance was determined using a Mann-Whitney U test. All P-values ⁇ 0.05 were considered statistically significant.
  • n 3- 18 animals /group and is further indicated in figure legends. Studies are represented as at least 2 independent studies.
  • mice were injected with the Luc mRNA-IAJD34 formulation at a dose of 10 pg per mouse.
  • Live mice were imaged at various time points (4, 24, 48, and 72 h) post-injection, and the luciferase intensity was quantified as a whole-body flux (p/s) (Figure 2C).
  • Luciferin intensity peaked at 4 h postinjection (3.12 x 10 7 ⁇ 9.15 x 10 6 ), by 24 h, the flux intensity decreased but remained elevated by 37%.
  • FIG 48 and 72-h post-injection there was a more significant drop to 3% and 1.9%, respectively (Figure 2D and Figure 3).
  • Luc mRNA-IAJD34 was injected at 10, 20, and 30 pg doses per mouse and imaged at 24 h post-injection. There was a dose-dependent increase in lung-specific luciferase expression, reaching 9.3 x 10 8 at 30 pg per mouse ( Figure 2E, Figure 5 and Figure 6). Immunohistochemical staining of lung tissue with the 30 pg dose revealed that expression of luciferase protein was well distributed throughout the alveolar epithelium, with minimal staining in the upper airway compared to the control ( Figure 2F). The mRNA delivery to the alveolar level (i.e., below the 16th generati on of the airway tree) had previously been unsuccessful, the expression of luciferase in these lower respiratory zones suggests our approach may serve as a promising therapeutic.
  • TGF-P a prominent anti-inflammatory cytokine associated with the resolution of ALI
  • the production and quality of TGF-P mRNA were validated (Figure 7 A). Protein expression of TGF-P was confirmed in Human epithelial kidney (HEK293) cells following transfection with TGF-P mRNA ( Figure 7B).
  • TGF-P mRNA-IAJD34 bronchoalveolar lavage fluid
  • BAL bronchoalveolar lavage fluid
  • AM alveolar macrophage
  • TGF-P protein exists in an unprocessed form with a latency-associated peptide that is cleaved to form an active protein, which can then initiate signal transduction (Lyons et al., 1988, J. Cell Biol. 106, 1659-1665; Bottinger et al., 1996, Proc. Natl. Acad. Sci. USA 93, 5877-5882).
  • TGF-P mRNA- IAJD34 was reaching the lung and being processed
  • TGF-P protein expression was confirmed in lung tissue digest using western blotting. Cleaved TGF-P was significantly increased at all doses compared to the control, whereas unprocessed and total TGF-P were only significantly increased in the 20 pg and 30 pg groups ( Figure 14).
  • G-CSF Granulocytecolony stimulating factor
  • IL-la interleukin-1 alpha
  • IL 9 interleukin 9
  • TNFa tumor necrosis factor
  • TGF-P protein expression alters the downstream signaling of pro- and anti-inflammatory cytokines.
  • OCR oxygen consumption rate
  • TGF-P mRNA-IAJD34 Evaluating pulmonary delivery of TGF-P mRNA-IAJD34 over time [0267] Due to the lack of toxicity, limited alterations in cell phenotype, and efficient expression of TGF-P protein and downstream signals, the 10 pg dose of TGF-P mRNA-IAJD34 was selected for further development.
  • TGF-P mRNA IAJD34 can deliver robust and transient TGF-P to the lung without significant signs of inflammation or toxicity.
  • Transient expression is therapeutically beneficial as prolonged TGF-P expression can lead to fibrin deposition and fibrosis (Meng et al., 2016, Nat. Rev. Nephrol. 12, 325).
  • TGF-P mRNA-IAJD34 can limit pulmonary injury following exposure to bleomycin
  • Intratracheal bleomycin is a laboratory model of ALI, characterized by acute pulmonary inflammation occurring over the first 7 days, transitioning to fibrotic development around 14 days, and resolving at 21-28 days post initial exposure (Stevenson et al., 2022, 1. Pharmacol. Exp. Ther. 382, 356-365; Wilkinson et al., 2020, Toxicol. Appl. Pharmacol. 407, 115236; Izbicki et al., 2002, Int. J. Exp. Pathol. 83, 111-119).
  • mice were treated with a 10 pg dose of TGF-P mRNA- IAJD34 or empty-lAJD34 concurrently to instillation of bleomycin or PBS control.
  • TGF-P mRNA- IAJD34 mice were euthanized 3 days post ITB or PBS exposure.
  • ITB causes weight loss in mice over the course of the first 7 days51 « Substantial weight loss was observed in groups exposed to ITB, where treatment with TGF-P mRNA-IAJD34 had no impact on ITB-induced decreases in percent body weight (Figure 22).
  • ITB is also associated with increases in alveolar thickness, immune cell infiltration, and tissue consolidation that variably develop over the first 7 days post-exposure (Stevenson et al., 2022, J. Pharmacol. Exp. Ther. 382, 356-365;
  • BALB/c mice are slightly more resistant to ITB expo- sure as compared to C57BL6/J mice (Pottier et al., 2007, Am. J. Respir. Crit. Care Med. 176, 1098-1107; Schrier et al., 1983, Am. Rev. Respir. Dis. 127, 63-66; Gur et al., 2000, Exp. Lung Res. 26, 521-534).
  • mice were chosen for this study as IAJD targeting and development has primarily been characterized in this strain of mice (Zhang et al., 2021, J. Am. Chem. Soc. 143, 12315-12327; Zhang et al., 2022, J. Am. Chem. Soc. 144, 4746-4753; Zhang et al., 2021, J. Am. Chem. Soc. 143, 17975-17982).
  • the 3-day timepoint was chosen as the peak of inflammatory signaling post ITB injury. Successful delivery of TGF-P is predicted to have its greatest effect on inflammatory cellular phenotype at this time point.
  • ITB-induced ALI is also associated with increases in pulmonary epithelial injury, marked by increased BAL fluid protein and disrupted epithelial lipid barriers (Allawz et al., 2019, Curr. Opin. Toxicol. 13, 68-73). Exposure to ITB caused a significant increase in BAL fluid protein content as compared to control. TGF-P mRNA-IAJD34 treatment reduced the protein level within the BAL but not significantly ( Figure 24A). Neither ITB nor TGF-P mRNA-IATD34 had an effect on BAL fluid phospholipid levels, hypothesized to be a limitation of the 3-day time-point ( Figure 24B).
  • ITB induces a loss of BAL cells, a consequence of early pulmonary inflammation and cell death.
  • TGF-P mRNA-IAJD34 TGF-P mRNA-IAJD34
  • AMs viable CD45+ Siglec F+ F4/80+ BAL cells
  • CD1 lc+ CD1 lb- resident (CD1 lc+ CD1 lb- ), recruited (CD1 lc- CD1 lb+ ), or migratory macrophages (CD1 lc+ CD1 lb+ ).
  • exposure to bleomycin reduced resident AMs and increased recruited AMs compared to PBS control ( Figure 24D, 24E) (Stevenson et al., 2022, J. Pharmacol. Exp. Ther. 382, 356-365; Wilkinson et al., 2020, Toxicol. Appl. Pharmacol.
  • TGF-P mRNA-IAJD34 Treatment with TGF-P mRNA-IAJD34 helped mitigate some AM alterations but had no effect on IMs at this time point. Innate TGF-P expression is expressed more highly in human AMs when compared to other lung cell types (Yu et al., 2017, Immunity 47, 903- 912). There is some evidence that TGF-P plays a less significant role in IM cell maturation and activity, but this remains largely speculative (Yu et al., 2017, Immunity 47, 903-912). Further model development may be beneficial to understanding the role that TGF-P plays in modulating ITB-induced AM and IM cell characteristics.
  • TGF-P mRNA-IAJD34 Treatment with TGF-P mRNA-IAJD34 prevented ITB-induced increases in all three of these pro-inflammatory cytokines, consistent with the anti-inflammatory signaling of TGF-P ( Figure 25A- 25C). Exposure to ITB caused a reduction in IL-lex and IL-2 compared to PBS control, irrespective of treatment with TGF-P mRNA-IAJD34 ( Figure 25D, 25E). Both IL-1 a and IL-2 are also pro- inflammatory cytokines, and it is unclear why ITB exposure decreased these cytokines, though it may be a result of AM resident cell loss. Thus, treatment with TGF-P mRNA-IAJD34 can prevent components of ITB-induced pulmonary inflammatory signaling.
  • Example 2 Alpha- 1 -Antitrypsin mRNA treatment is useful for the treatment of genetically induced chronic obstructive pulmonary disease in a genetic knockout model.
  • FIG. 27 shows representative IVIS images of whole-body mice and organs were taken 4 hours post-injection and show a strong luciferase signal in the lung of C57B1/6 mice.
  • FIG. 28 shows representative IVIS images of whole-body mice and organs were taken 4 hours post-injection and show a strong luciferase signal in the lung of C57B1/6 mice.
  • FIG. 29 shows representative IVIS images of whole-body mice and organs were taken 4 hours post-injection and show a strong luciferase signal in the lung of BALB/c mice
  • FIG. 30 shows representative IVIS images of whole-body mice and organs were taken 4 hours post-injection and show a strong luciferase signal in the lung of BALB/c mice.

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Abstract

L'invention concerne des procédés d'utilisation de dendrimères Janus amphiphiles ciblant les poumons qui forment des nanoparticules permettant la délivrance d'agents thérapeutiques au parenchyme pulmonaire.
PCT/US2025/024306 2024-04-12 2025-04-11 Délivrance ciblée d'agents thérapeutiques à base d'arnm au parenchyme pulmonaire à l'aide d'un système de délivrance à un composant pour acides nucléiques Pending WO2025217538A2 (fr)

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