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WO2024092086A1 - Compositions et procédés pour régénérer l'endothélium cornéen - Google Patents

Compositions et procédés pour régénérer l'endothélium cornéen Download PDF

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WO2024092086A1
WO2024092086A1 PCT/US2023/077845 US2023077845W WO2024092086A1 WO 2024092086 A1 WO2024092086 A1 WO 2024092086A1 US 2023077845 W US2023077845 W US 2023077845W WO 2024092086 A1 WO2024092086 A1 WO 2024092086A1
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pro
lnp
growth factor
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nucleoside
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Panteleimon ROMPOLAS
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The Trustees Of The University Of Pennsylvania
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0048—Eye, e.g. artificial tears

Definitions

  • Corneal endothelial disorders are a leading cause of reduced vision and blindness, affecting approximately 4% of the population over 40 [Whitcher, et al. Bull World Health Organ. 2001 ;79(3):214-21 ].
  • a progressive decline in the number of corneal endothelial cells due to aging or other factors can lead to corneal edema and loss of vision, due to a decompensated ion pumping capacity [Eghrari, et al. Prog Mol Biol Transl Sci. 2015;134:79-97],
  • the comeal endothelium is a tissue with limited documented regenerative activity [Joyce, Exp Eye Res. 2012 Feb;95(l): 16-23], Corneal endothelial cells regulate the optical transparency of the cornea by maintaining the stroma in a state of relative dehydration, through their ion pumping activity and barrier function [Tuft, Coster, Eye (Lond). 1990;4 (Pt 3):389-424; Maurice, J Physiol.
  • Corneal endothelial cells are developmentally derived from the neural crest and establish a tissue monolayer in the posterior corneal surface, which becomes mitotically inactive postnatally and into adulthood, through well studied mechanisms of contact inhibition and insulation from growth signals [Murphy, et al. Invest Ophthalmol Vis Sci. 1984 Mar;25(3):312-22; Bourne, et al. Invest Ophthalmol Vis Sci. 1997 Mar;38(3):779-82; Yee, et al. Curr Eye Res. 1985 Jun;4(6):671-8],
  • RNA interference RNA interference
  • CRISPR clustered regularly interspaced short palindromic repeats
  • dCas9 deactivated CRISPR-associated protein 9
  • the invention provides a lipid nanoparticle (LNP), wherein the LNP comprises:
  • RNA nucleoside-modified ribonucleic acid
  • the at least one pro-growth factor comprises any one or more of cyclin-dependent kinase 4 (CDK4), cyclin DI (CCND1), sex determining region Y-box 2 (SOX2), c-myc transcription factor (MYC), and yes-associated protein 1 (YAP).
  • CDK4 cyclin-dependent kinase 4
  • CCND1 cyclin DI
  • SOX2 sex determining region Y-box 2
  • MYC c-myc transcription factor
  • YAP yes-associated protein 1
  • the LNP further comprises at least one surface-linked antibody, wherein the at least one surface-linked antibody targets a corneal endothelium cell surface marker.
  • the at least one nucleoside-modified RNA is messenger RNA (mRNA).
  • mRNA messenger RNA
  • the at least one nucleoside-modified RNA comprises pseudouridine and/or 1-methyl-pseudouridine.
  • the at least one nucleoside-modified RNA is in vitro transcribed (IVT) RNA.
  • the at least one pro-growth factor comprises CDK4, CCND1, SOX2, MYC, and YAP.
  • the at least one pro-growth factor consists of CDK4, CCND1, SOX2, MYC, and YAP.
  • the corneal endothelium cell surface marker is selected from N- cadherin, NCAM (CD56), connexin 43, integrin a3pi, integrin b5, VDAC3, aquaporin 1, chloride channel 3, SLC4A4, CD44, GPC4, and any combination thereof.
  • the at least one ionizable lipid encapsulates the at least one nucleoside-modified RNA.
  • the at least one ionizable lipid is a cationic lipid.
  • the at least one pro-growth factor comprises or consists of CDK4, and further wherein the CDK4 comprises SEQ ID NO: 2 or SEQ ID NO: 12;
  • the at least one pro-growth factor comprises or consists of CCND1, and further wherein the CCND1 comprises SEQ ID NO: 4 or SEQ ID NO: 14;
  • the at least one pro-growth factor comprises or consists of SOX2, and further wherein the SOX2 comprises SEQ ID NO: 6 or SEQ ID NO: 16;
  • the at least one pro-growth factor comprises or consists of MYC, and further wherein the MYC comprises SEQ ID NO: 8 or SEQ ID NO: 18;
  • the at least one pro-growth factor comprises or consists of YAP, further wherein the YAP comprises SEQ ID NO: 10 or SEQ ID NO: 20.
  • regenerating corneal endothelium in the subject comprises inducing mitosis in at least 5%, at least 10%, at least 15%, or at least 20% of corneal endothelium cells in the subject.
  • the subject has vision loss due to a corneal endothelium disease or disorder (e.g., age-related corneal degeneration, Fuchs’ endothelial corneal dystrophy, and/or cataract surgical injury).
  • a corneal endothelium disease or disorder e.g., age-related corneal degeneration, Fuchs’ endothelial corneal dystrophy, and/or cataract surgical injury.
  • regenerating corneal endothelium in the subject treats the vision loss in the subject.
  • the subject is a human.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the LNP described herein and at least one pharmaceutically acceptable carrier, diluent, and/or excipient.
  • the invention provides the LNP described herein for use in a method of regenerating corneal endothelium in the subject.
  • the invention provides a method of regenerating corneal endothelium in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a lipid nanoparticle (LNP) and at least one pharmaceutically acceptable carrier, diluent, and/or excipient, wherein the LNP comprises:
  • RNA nucleoside-modified ribonucleic acid
  • the administering comprises intracameral injection.
  • the at least one pro-growth factor comprises any one or more of cyclin-dependent kinase 4 (CDK4), cyclin DI (CCND1), sex determining region Y-box 2 (SOX2), c-myc transcription factor (MYC), and yes-associated protein 1 (YAP).
  • CDK4 cyclin-dependent kinase 4
  • CCND1 cyclin DI
  • SOX2 sex determining region Y-box 2
  • MYC c-myc transcription factor
  • YAP yes-associated protein 1
  • the LNP further comprises at least one surface-linked antibody, wherein the at least one surface-linked antibody targets a corneal endothelium cell surface marker.
  • the at least one nucleoside-modified RNA is messenger RNA (mRNA).
  • mRNA messenger RNA
  • the at least one nucleoside-modified RNA comprises pseudouridine and/or 1 -methyl -pseudouridine. In certain embodiments, the at least one nucleoside-modified RNA is in vitro transcribed (IVT) RNA.
  • IVTT in vitro transcribed
  • the at least one pro-growth factor comprises CDK4, CCND1, SOX2, MYC, and YAP.
  • the at least one pro-growth factor consists of CDK4, CCND1, SOX2, MYC, and YAP.
  • the corneal endothelium cell surface marker is selected from N- cadherin, NCAM (CD56), connexin 43, integrin ct3pi, integrin b5, VDAC3, aquaporin 1, chloride channel 3, SLC4A4, CD44, GPC4, and any combination thereof.
  • the at least one ionizable lipid encapsulates the at least one nucleoside-modified RNA.
  • the at least one ionizable lipid is a cationic lipid.
  • the at least one pro-growth factor comprises or consists of CDK4, and further wherein the CDK4 comprises SEQ ID NO: 2 or SEQ ID NO: 12;
  • the at least one pro-growth factor comprises or consists of CCND1, and further wherein the CCND1 comprises SEQ ID NO: 4 or SEQ ID NO: 14;
  • the at least one pro-growth factor comprises or consists of SOX2, and further wherein the SOX2 comprises SEQ ID NO: 6 or SEQ ID NO: 16;
  • the at least one pro-growth factor comprises or consists of MYC, and further wherein the MYC comprises SEQ ID NO: 8 or SEQ ID NO: 18;
  • the at least one pro-growth factor comprises or consists of YAP, further wherein the YAP comprises SEQ ID NO: 10 or SEQ ID NO: 20.
  • regenerating corneal endothelium in a subject in need thereof comprises inducing mitosis in at least 5%, at least 10%, at least 15%, or at least 20% of corneal endothelium cells in the subject.
  • the administering comprises administering a first dose.
  • the administering further comprises administering one or more subsequent doses.
  • the subject has vision loss due to a corneal endothelium disease or disorder (e.g., age-related corneal degeneration, Fuchs’ endothelial comeal dystrophy, and/or cataract surgical injury).
  • a corneal endothelium disease or disorder e.g., age-related corneal degeneration, Fuchs’ endothelial comeal dystrophy, and/or cataract surgical injury.
  • regenerating corneal endothelium in the subject treats the vision loss in the subject.
  • the subject is a human.
  • the invention provides a kit for regenerating corneal endothelium in a subject in need thereof, the kit comprising:
  • a pharmaceutical composition comprising a lipid nanoparticle (LNP) and at least one pharmaceutically acceptable carrier, diluent, and/or excipient, wherein the LNP comprises: (a) at least one nucleoside-modified ribonucleic acid (RNA) encoding at least one pro-growth factor, wherein each pro-growth factor is encoded by a distinct nucleoside-modified RNA; and (b) an ionizable lipid; wherein the LNP is capable of regenerating corneal endothelium in the subject; and
  • instructional material(s) for intracameral injection of a therapeutically effective amount of the pharmaceutical composition to the subject (ii) instructional material(s) for intracameral injection of a therapeutically effective amount of the pharmaceutical composition to the subject.
  • the at least one pro-growth factor comprises any one or more of cyclin-dependent kinase 4 (CDK4), cyclin DI (CCND1), sex determining region Y-box 2 (SOX2), c-myc transcription factor (MYC), and yes-associated protein 1 (YAP).
  • CDK4 cyclin-dependent kinase 4
  • CCND1 cyclin DI
  • SOX2 sex determining region Y-box 2
  • MYC c-myc transcription factor
  • YAP yes-associated protein 1
  • the LNP further comprises at least one surface-linked antibody, wherein the at least one surface-linked antibody targets a corneal endothelium cell surface marker.
  • the at least one nucleoside-modified RNA is messenger RNA (mRNA).
  • mRNA messenger RNA
  • the at least one nucleoside-modified RNA comprises pseudouridine and/or 1-methyl-pseudouridine.
  • the at least one nucleoside-modified RNA is in vitro transcribed (IVT) RNA.
  • the at least one pro-growth factor comprises CDK4, CCND1, SOX2, MYC, and YAP.
  • the at least one pro-growth factor consists of CDK4, CCND1, SOX2, MYC, and YAP.
  • the corneal endothelium cell surface marker is selected from N- cadherin, NCAM (CD56), connexin 43, integrin a3pi, integrin b5, VDAC3, aquaporin 1, chloride channel 3, SLC4A4, CD44, GPC4, and any combination thereof.
  • the at least one ionizable lipid encapsulates the at least one nucleoside-modified RNA.
  • the at least one ionizable lipid is a cationic lipid.
  • the at least one pro-growth factor comprises or consists of CDK4, and further wherein the CDK4 comprises SEQ ID NO: 2 or SEQ ID NO: 12;
  • the at least one pro-growth factor comprises or consists of CCND1, and further wherein the CCND1 comprises SEQ ID NO: 4 or SEQ ID NO: 14;
  • the at least one pro-growth factor comprises or consists of SOX2, and further wherein the SOX2 comprises SEQ ID NO: 6 or SEQ ID NO: 16;
  • the at least one pro-growth factor comprises or consists of MYC, and further wherein the MYC comprises SEQ ID NO: 8 or SEQ ID NO: 18;
  • the at least one pro-growth factor comprises or consists of YAP, further wherein the YAP comprises SEQ ID NO: 10 or SEQ ID NO: 20.
  • regenerating corneal endothelium in a subject in need thereof comprises inducing mitosis in at least 5%, at least 10%, at least 15%, or at least 20% of corneal endothelium cells in the subject.
  • the subject has vision loss due to a corneal endothelium disease or disorder (e.g., age-related corneal degeneration, Fuchs’ endothelial comeal dystrophy, and/or cataract surgical injury).
  • a corneal endothelium disease or disorder e.g., age-related corneal degeneration, Fuchs’ endothelial comeal dystrophy, and/or cataract surgical injury.
  • regenerating corneal endothelium in the subject treats the vision loss in the subject.
  • the subject is a human.
  • FIGs. 1A - 1 J relate to intravital imaging of and age-related morphological and geneexpression changes in the murine corneal endothelium.
  • FIG. 1A shows the experimental approach to directly analyze the physiology and regeneration of the mouse corneal endothelium at the single-cell level, by intravital imaging. Schematic shows optical sectioning of the live mouse corneal endothelium, imaged by two-photon microscopy.
  • FIG. IB high magnification images of the mouse corneal endothelium from a 3 -month and 22-month old mouse, using membrane- or nuclear-localized in vivo fluorescent reporters. Yellow arrows indicate guttae-like structures between corneal endothelial cells.
  • FIG. 1A shows the experimental approach to directly analyze the physiology and regeneration of the mouse corneal endothelium at the single-cell level, by intravital imaging. Schematic shows optical sectioning of the live mouse corneal endothelium, imaged by two-photon microscopy.
  • FIG. 1J 3-dimensional rendering of corneal endothelial nuclei from young and aged corneas.
  • FIG. 2A is a schematic of the mammalian eye and a magnified view of the structural organization of the cornea.
  • FIG. 2B is a schematic of the experimental approach for deconstructing the cornea in vivo by optical sectioning with two-photon microscopy.
  • FIG. 2C shows representative low (left) and high (right) magnification images of the epithelium, stroma and endothelium, from a young and old mouse, taken at the indicated depths from the surface of the cornea.
  • a globally expressed, membrane-localized fluorescent reporter (Rosa26-mTomato ⁇ was used to resolve the fine tissue morphology in vivo.
  • FIG. 3 shows high resolution live imaging of the basal and apical surfaces of the corneal endothelium which reveals difference in young and aged mice. Images were acquired using 2 pm thick serial optical sections. Corresponding models illustrate the distinct cell-cell contacts and membrane organization of the basal and apical surfaces of the corneal endothelium (left panels; adapted from He et al. Sci Rep 2016). A globally expressed, membrane-localized fluorescent reporter (Rosa26-mTomato) was used to resolve the fine tissue morphology in vivo. Scale bar 50 pm.
  • FIGs. 4A - 4B show that aging corneal endothelial cells lose their identity. Unbiased clustering of integrated samples across three ages (2-, 6- and 11 -month-old) in wild type mice revealed 8 cell populations of the cornea.
  • FIG. 4A Dot plot demonstrating cell type specific marker expression patterns.
  • FIG. 4B Violin plots of select differentially expressed genes of biological significance by maturity demonstrate decreased endothelial markers and increased epithelial, fibroblast, and senescence markers with age.
  • FIGs. 5A - 5F illustrate the finding that injury captures the regenerative potential of the corneal endothelium.
  • FIG. 5A Representative time points from injury repair of the corneal endothelium captured by longitudinal live imaging. Panels show en face view of the corneal endothelium generated by maximum projection of serial optical sections. Scale bar 200 pm.
  • FIG. 5B Representative high magnification views from the same eye of the central and peripheral corneal endothelium, imaged before and after injury. Scale bar 50 pm.
  • FIG. 5C Quantification of cell density in the central and peripheral regions of the corneal epithelium.
  • FIG. 5D Representative high magnification views of the corneal endothelium taken before and two days after injury.
  • a globally expressed red fluorescent reporter with nuclear localization Rosa26-nTomato is used to visualize all corneal endothelial cells in the tissue.
  • Actively proliferating cells arrows are visualized by green fluorescence from expression of the in vivo cell cycle reporter (CyclinBl- GFP).
  • FIG. 5 E Examples of cell proliferation in the corneal endothelium, two days after scrape injury, in Young (3-month old) and Aged (15-month old) mice. Scale bar 50 pm.
  • FIG. 5F Examples of cell proliferation in the corneal endothelium, two days after scrape or laser injury.
  • FIG. 6 shows representative low and high magnification maximal projections of the endothelial layers (bottom panels) and corresponding side views of the entire thickness of the cornea (top panels), taken at the indicated time points before and after injury.
  • a globally expressed, nuclear-localized red fluorescent reporter (Rosa26-nTomato) was used to resolve cell numbers and density in vivo.
  • the stromal extracellular matrix is visualized by Second Harmonic Generation (SHG) and depicted in cyan.
  • SHG Second Harmonic Generation
  • FIGs. 7A - 7B illustrate that wound healing re-establishes the corneal endothelial layer but results in reduced cell density.
  • FIG. 7A shows representative low magnification maximal projections of the endothelial layers (en face view) and corresponding high magnification views of the central and peripheral corneal endothelium (bottom panels, en face view), taken at the indicated time points before and after injury.
  • a globally expressed, membrane-localized red fluorescent reporter (Rosa26-mTomato) was used to resolve tissue and cell morphology in vivo.
  • the stromal extracellular matrix is visualized by Second Harmonic Generation (SHG) and depicted in green (top panel). Scale bars 200 pm.
  • FIG. 7B shows a schematic of the corneal endothelial repair process after mechanical injury, resulting in a decrease in endothelial cell density.
  • FIGs. 8A - 8H illustrate that longitudinal live imaging reveals the cellular and tissue dynamics of corneal endothelial injury response.
  • FIG. 8A Validation of the cell cycle reporter (CyclinBl-GFP) used to capture corneal endothelial proliferation in vivo. Top left panel shows the sequence elements of the transgenic reporter. Expression of GFP recapitulates the kinetics of the endogenous cyclin Bl enzyme, with a peak signal intensity during the G2/M phases of the cell cycle.
  • FIG. 8B Panels show representative frames from a time sequence, which depict actively cycling cells in the live corneal epithelium. Red arrows show cells that complete mitosis, in whose daughter cells the GFP signal quickly becomes undetectable.
  • FIG. 8A Validation of the cell cycle reporter (CyclinBl-GFP) used to capture corneal endothelial proliferation in vivo. Top left panel shows the sequence elements of the transgenic reporter. Expression of GFP recapitulates the kinetics of the endogenous
  • FIG. 8C Representative low magnification en face views of the corneal endothelium taken at indicated timepoints during scrape injury repair. Proliferating corneal endothelial cells can be visualized by green fluorescence (CyclinBl-GFP).
  • FIG. 8D Corresponding side views of the entire thickness of the cornea taken at the indicated time points before and after scrape injury. The stromal extracellular matrix is visualized by Second Harmonic Generation (SHG; XZ views).
  • FIG. 8E Graph showing changes in stromal thickness during scrape injury repair.
  • FIG. 8F Representative low magnification en face views of the corneal endothelium taken at indicated timepoints during femtosecond laser injury repair.
  • FIG. 8G Corresponding side views of the entire thickness of the cornea taken at the indicated time points before and after laser injury. The stromal extracellular matrix is visualized by Second Harmonic Generation (SHG; XZ views).
  • FIG. 8H Graph showing changes in stromal thickness during laser injury repair. Scale bars 200 pm.
  • FIGs. 9A - 9E illustrate that corneal endothelial cells are equipotent in their proliferative capacity.
  • FIG. 9A Experimental design for exogenous protein expression in corneal endothelial cells in vivo, via intracameral injection of encapsulated modified mRNA.
  • FIG. 9B Example of GFP expression in corneal endothelial cells 24 hours after mRNA injection into the mouse eye.
  • FIG. 9C Experimental design of in vivo lineage tracing of corneal endothelial cells via in situ delivery of Cre-recombinase encoding mRNA, followed by longitudinal live imaging.
  • FIG. 9A Experimental design for exogenous protein expression in corneal endothelial cells in vivo, via intracameral injection of encapsulated modified mRNA.
  • FIG. 9B Example of GFP expression in corneal endothelial cells 24 hours after mRNA injection into the mouse eye.
  • FIG. 9C Experimental design of in vivo lineage tracing
  • FIG. 9D Examples of multi-color in vivo lineage tracing of corneal endothelial cells, using the Rosa26- confetti reporter.
  • the panel shows low and high magnification images of the same mouse eye imaged after mRNA injection (1st Timepoint) and re-imaged after one year.
  • FIG. 9E Lineage tracing of corneal endothelial cell activity during injury repair. Left panels show low and high magnification views of the wound margins, taken at one and ten days after injury. Arrows show representative single corneal endothelial cells at the wound margin that divided during this timeframe. Right panels show equivalent areas of the same corneas located distally from the wound margin and illustrate the absence of cell divisions in these areas during wound healing. Scale bars 50 pm.
  • FIGs. 10A - 10C show that in vivo injection of encapsulated modified mRNA modulates protein expression in the murine corneal endothelium.
  • FIG. 10A Validation of efficacy and specificity of protein expression in the corneal endothelium by modified mRNA delivered in vivo, by injection into the anterior chamber of the eye (intracameral). A GFP encoding modified mRNA was injected into the left eye of a live mouse and the same eye was imaged 24 hours after injection. Expression of GFP was detected in the corneal endothelium of the injected. No GFP signal was detected in other cellular compartments of the cornea, the lens or anywhere in the uninjected contralateral eye.
  • FIG. 10B Representative low magnification en face views of the same corneas taken at the indicated timepoints. Expression of GFP wanes after 24 hours and detectable signal persists up to seven days from injection of the mRNA.
  • FIG. IOC Co-expression of GFP and mCherry in corneal endothelial cells after injection of a cocktail consisting of equal amounts of mRNA encoding for each fluorescent protein. Scale bar 50 pm.
  • FIGs. 11A - 11B show that cell migration contributes to wound healing of the corneal endothelium.
  • FIG. 11A Experimental design to visualize the activity of single endothelial cells and analyze their contribution to injury repair by in vivo lineage tracing. Clonal labeling of single corneal endothelial cells is achieved with a single injection of modified mRNA encoding for Cre-recombinase. Cells that express the enzyme are permanently labeled with the Cre reporter (mGFP) and their activity is tracked by longitudinal live imaging.
  • FIG. 11B Representative low (top panels) and high (bottom panels) magnification views of the wounded corneal endothelium taken at the indicated time points during injury repair.
  • Cre-recombinase encoding mRNA was injected before the injury to mark corneal endothelial cells and track their activity during repair.
  • a globally expressed, membrane-localized cre reporter (Rosa26-mTmG) was used to resolve tissue and cell morphology in vivo and to mark cells which express the Cre- recombinase with mGFP. Blue arrowheads indicate filopodia-like structures of migrating corneal endothelial cells during wound healing. Scale bars 200 pm.
  • FIGs. 12A - 12B show that multicolor in vivo lineage tracing reveals the distinct activities of corneal endothelial cells during wound healing.
  • FIG. 12A Validation of multi-color labeling of corneal endothelial cells. Diagram shows the sequence elements of the Rosa26- confetti reporter and the possible recombination events that result in ten unique color combinations when mice homozygous for the two confetti alleles are used. Imaging of the corneal endothelium after injection of Cre-encoding modified mRNA confirmed these theoretical color labels expressed stochastically in single corneal endothelial cells. FIG.
  • FIG. 12B Representative low (top panels) and high (bottom panels) magnification views of the corneal endothelium taken at the indicated time points before and after injury. Cre-recombinase encoding mRNA was injected before the injury to mark corneal endothelial cells and track their activity during repair. Note that labeled cells remain quiescent for at least two months before injury (yellow squares), but only those proximal to the wound margin become activated and proliferate after injury (red squares). Scale bars 200 pm.
  • FIGs. 13A - 13B show that unbiased in vivo lineage tracing captures exclusive contribution of corneal endothelial cells at the wound edge.
  • FIG. 13A Experimental strategy for un-biased lineage tracing of corneal endothelial cells by in vivo photo-labeling and longitudinal live imaging.
  • FIG. 13B Representative low (top panels) and high (bottom panels) magnification views of the corneal endothelium taken at the indicated time points after injury. Equivalent groups of cells were photo-labeled at various distances from the center of the cornea (1-3) as well as proximal to the wound margin (4).
  • Imaging at 850 nm was used to visualize the preactivated state of the globally expressed PAGFP reporter and was used as a counterstain to visualize unlabeled cells in the tissue. Imaging at 930 nm illuminates only cells in which the PAGFP was irreversibly photo-activated. The same corneas were re-imaged to track changes in the labeled cells after injury. Note that labeled cells distal to wound edge remain quiescent and do not directly contribute to injury repair. Scale bars 500 pm.
  • FIGs. 14A - 14B relate to modified mRNA constructs.
  • FIG. 14A A schematic of sequence elements of exemplary mRNAs used herein.
  • FIG. 14B Western blot analysis of protein expression in 392T cells transfected with the indicated mRNAs.
  • FIGs. 15A - 15J relate to mRNA-mediated cell cycle activation of corneal endothelial cells in vivo.
  • FIG. 15A Experimental design for reprogramming and induced proliferation of corneal endothelial cells via intracameral injection of CCMSY mRNAs.
  • a Cre-encoding mRNA is used to label cells that uptake the mRNAs in the corneas of a Cre-reporter mouse line (Rosa26-nTnG). Cells that incorporate the mRNAs switch from a nuclear Tomato to a nuclear GFP signal and their fate can be traced longitudinally. The activity of corneal endothelial cells is tracked over time by longitudinal live imaging.
  • FIG. 15B Representative examples from a lineage tracing experiment following mRNA injections. Cells that receive only the Cre mRNA remain quiescent while those that also receive the CCMSY mRNAs undergo cell divisions.
  • FIG. 15C Whole mount immunolabeling with the endothelial marker ZO-1, of corneas from Rosa26-nTnG mice collected ten days after injection with mRNA encoding for Cre plus the cocktail of progrowth proteins. Asterisks show cells that have incorporated and expressed the mRNA as indicated but the switch from a nuclear-Tomato to a nuclear-GFP signal (Rosa26-nTnG reporter). FIG.
  • FIG. 15D Example of a lineage tracing experiment using the in vivo cell cycle reporter (Cycling Bl -GFP) in combination with the Cre-reporter (Rosa26-tdTomato).
  • Cells that receive the Cre + CCMSY mRNAs are the only ones that show positive GFP signal, indicating selective activation of the cell cycle in these cells.
  • FIG. 15E Graph showing the regenerative effect of the CCMSY mRNAs treatment. The average cell density of the corneal endothelium in 12-month-old mice injected with the CCMSY mRNAs increased after treatment to levels equivalent to those of 3 -month-old mice. For quantification of cell density as a function of age see Figure 1C.
  • FIG. 15H Growth curves of the corneal endothelium in mice injected with the Cre-recombinase alone (Control), or plus the CCMSY mRNAs. Note that CCMSY induce only transient proliferation in the tissue.
  • FIG. 151) Whole mount immunolabeling with the endothelial markers zonula-occludens-1 (ZO-1) and N-cadherin, of corneas collected two months after injection with CCMSY mRNAs and compared non-injected.
  • FIG. 15J Schematic of the mRNA-mediated proliferation of corneal endothelial cells.
  • Endothelial cells that incorporate the mRNAs after the intracameral injection are shown in red. After treatment, endothelial cells that express the proteins encoded by the CCMSY mRNAs undergo a limited round of proliferation which leads to a stable increase in endothelial cell density. Scale bars 200 pm.
  • FIGs. 16A - 16C illustrate the finding that in vivo lineage tracing demonstrates efficacy of mRNAs delivered in situ, to elicit transient cell proliferation in the corneal endothelium.
  • FIG. 16A Experimental design for reprogramming and induced proliferation of corneal endothelial cells via in vivo injection of mRNAs encoding for pro-growth factors (CCMSY: Cdk4 / Ccndl / Myc / Sox2 / Yap).
  • a Cre-encoding mRNA is used to label cells that uptake the mRNAs in the corneas of a Cre-reporter mouse line (Rosa26-nTnG).
  • FIG. 16B Panels show representative low and high magnification en face views of the corneal endothelium at the indicated timepoints from mRNA injection of Cre-recombinase alone (Control), or plus the CCMSY mRNAs encoding for pro-growth proteins.
  • Cells that incorporate the Cre-mRNA switch from a nuclear Tomato to a nuclear GFP signal (Rosa26-nTnG) and their fate is traced longitudinally by live imaging. Red squares indicate the same cells imaged during the time course and depicted in high magnification in the bottom panels.
  • FIG. 16C Longterm follow-up represented in the time-series of the corneal endothelium taken at the indicated timepoints from mRNA injection with CCMSY+Cre mRNAs. Note that after the initial spur of proliferation the tissue remains stable for at least two months after the time of injection. Scale bars 200 pm.
  • FIGs. 17A - 17B show that an in vivo cell cycle reporter is specifically activated in corneal endothelial cells that uptake the mRNAs.
  • FIG. 17A Experimental design for reprogramming and induced proliferation of corneal endothelial cells via in vivo injection of mRNAs encoding for pro-growth factors (CCMSY : Cdk4 / Ccndl / Myc / Sox2 / Yap). The mice used in these experiments express an in vivo cell cycle reporter (CyclingBl-GFP).
  • CyclingBl-GFP A Cre- encoding mRNA is used to label cells that uptake the mRNAs in the corneas of a Cre-reporter mouse line (Rosa26-nTomato).
  • FIG. 17B Panels show representative low and high magnification en face views of the corneal endothelium at the indicated timepoints from CCMSY+Cre mRNAs injection.
  • White squares indicate the same cells imaged during the time course and depicted in high magnification in panels 1-3.
  • Yellow arrowheads indicate one example of a cell that is marked with tdTomato after the uptake and expression of Cre mRNA (+CCMSY mRNAs), also showing positive Cyclin Bl-GFP signal. Scale bars 200 pm.
  • FIGs. 18A - 18B illustrate mRNA-mediated corneal endothelial proliferation in a murine model of early onset Fuchs’ dystrophy (FEDC).
  • FIG. 18B Fluorescence microscopy image taken two days after CCMSY + mCherry mRNA injection.
  • the present invention relates generally to compositions, methods and kits for regenerating corneal endothelium to treat vision loss in a subject.
  • the invention includes a lipid nanoparticle (LNP) harboring nucleoside-modified RNA(s) encoding at least one pro-growth factor, and methods and kits comprising the LNP for regenerating corneal endothelium to treat vision loss in a subject.
  • LNP lipid nanoparticle
  • the invention includes a lipid nanoparticle (LNP) comprising: (a) at least one nucleoside-modified ribonucleic acid (RNA) encoding at least one pro-growth factor, wherein each pro-growth factor is encoded by a distinct nucleoside-modified RNA; and (b) an ionizable lipid; wherein the LNP is capable of regenerating corneal endothelium in a subject in need thereof.
  • LNP lipid nanoparticle
  • the at least one pro-growth factor comprises any one or more of cyclin-dependent kinase 4 (CDK4), cyclin DI (CCND1), sex determining region Y-box 2 (SOX2), c-myc transcription factor (MYC), and yes-associated protein 1 (YAP).
  • CDK4 cyclin-dependent kinase 4
  • CCND1 cyclin DI
  • SOX2 sex determining region Y-box 2
  • MYC c-myc transcription factor
  • YAP yes-associated protein 1
  • the at least one pro-growth factor comprises any one or more of CDK4, CCND1, SOX2, MYC, YAP, NANOG, OCT4, SIRT1, an activating ligand of SIRT1, CTNND1, CTNNB1, ZONAB, E2F2, FGF1, FGF10, IGF1, IGF2, Col8al, Col8a2, Col4a3, Col4a4, SLC4al 1, Aqpl, and WWTR1.
  • the LNP further comprises at least one surface-linked antibody, wherein the at least one surface-linked antibody targets a corneal endothelium cell surface marker.
  • the invention includes a method of enhancing regeneration of corneal endothelium in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a lipid nanoparticle (LNP) and at least one pharmaceutically acceptable carrier, diluent, and/or excipient, wherein the LNP comprises: (a) at least one nucleoside-modified ribonucleic acid (RNA) encoding at least one pro-growth factor, wherein each pro-growth factor is encoded by a distinct nucleosi demodified RNA; and (b) an ionizable lipid; wherein the LNP regenerates corneal endothelium in the subject.
  • RNA nucleoside-modified ribonucleic acid
  • the at least one pro-growth factor comprises any one or more of cyclin-dependent kinase 4 (CDK4), cyclin DI (CCND1), sex determining region Y-box 2 (SOX2), c-myc transcription factor (MYC), and yes-associated protein 1 (YAP).
  • CDK4 cyclin-dependent kinase 4
  • CCND1 cyclin DI
  • SOX2 sex determining region Y-box 2
  • MYC c-myc transcription factor
  • YAP yes-associated protein 1
  • the at least one pro-growth factor comprises any one or more of CDK4, CCND1, SOX2, MYC, YAP, NANOG, OCT4, SIRT1, an activating ligand of SIRT1, CTNND1, CTNNB1, ZONAB, E2F2, FGF1, FGF10, IGF1, IGF2, Col8al, Col8a2, Col4a3, Col4a4, SLC4al 1, Aqpl, and WWTR1.
  • the LNP further comprises at least one surface-linked antibody, wherein the at least one surface-linked antibody targets a corneal endothelium cell surface marker.
  • the administering comprises intracameral injection.
  • the invention includes a kit for regenerating corneal endothelium in a subject in need thereof, the kit comprising:
  • a pharmaceutical composition comprising a lipid nanoparticle (LNP) and at least one pharmaceutically acceptable carrier, diluent, and/or excipient, wherein the LNP comprises: (a) at least one nucleoside-modified ribonucleic acid (RNA) encoding at least one pro-growth factor, wherein each pro-growth factor is encoded by a distinct nucleoside-modified RNA; and (b) an ionizable lipid; wherein the LNP is capable of regenerating corneal endothelium in the subject; and
  • instructional material(s) for intracameral injection of a therapeutically effective amount of the pharmaceutical composition to the subject (ii) instructional material(s) for intracameral injection of a therapeutically effective amount of the pharmaceutical composition to the subject.
  • the at least one pro-growth factor comprises any one or more of cyclin- dependent kinase 4 (CDK4), cyclin DI (CCND1), sex determining region Y-box 2 (SOX2), c- myc transcription factor (MYC), and yes-associated protein 1 (YAP).
  • CDK4 cyclin- dependent kinase 4
  • CCND1 cyclin DI
  • SOX2 sex determining region Y-box 2
  • MYC c- myc transcription factor
  • YAP yes-associated protein 1
  • the at least one pro-growth factor comprises any one or more of CDK4, CCND1, SOX2, MYC, YAP, NANOG, OCT4, SIRT1, an activating ligand of SIRT1, CTNND1, CTNNB1, ZONAB, E2F2, FGF1, FGF10, IGF1, IGF2, Col8al, Col8a2, Col4a3, Col4a4, SLC4al 1, Aqpl, and WWTR1.
  • the LNP further comprises at least one surface-linked antibody, wherein the at least one surface-linked antibody targets a corneal endothelium cell surface marker. Definitions
  • an element means one element or more than one element.
  • antibody refers to an immunoglobulin molecule, which specifically binds with an antigen or with a cell surface marker.
  • Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules.
  • the antibodies in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, monospecific antibodies, bispecific antibodies, multi-specific antibodies, as well as single chain variable fragment (scFv) antibodies and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et ak, 1988, Science 242:423-426).
  • antibody fragment refers to a portion of an intact antibody comprising the antigenic determining variable regions of an intact antibody.
  • antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.
  • Antibodies and antibody fragments may be generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage or by a synthetic DNA or RNA molecule encoding the antibody.
  • Said DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art.
  • the RNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the RNA has been obtained by transcribing DNA (synthetic or cloned) or other technology, which is available and well known in the art.
  • antigen or “Ag” as used herein is defined as a molecule that provokes an adaptive immune response. This immune response may involve either antibody production, or the activation of specific immunogenically-competent cells, or both.
  • antigens can be derived from recombinant or genomic DNA or RNA.
  • any DNA or RNA which comprises a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an adaptive immune response therefore encodes an “antigen” as that term is used herein.
  • an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated or synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid.
  • 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.
  • an “effective amount” as used herein means an amount which provides a therapeutic or prophylactic benefit.
  • Encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an RNA (such as 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 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 non-coding 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.
  • “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.
  • “Instructional material(s)” as used herein includes a publication, a recording, a diagram, or any other medium of expression that can be used to communicate the usefulness of a composition and/or compound of the invention in a kit.
  • the instructional material may describe a method of using the composition and/or compound of the invention in a method of the invention.
  • the instructional material of the kit may, for example, be affixed to a container that contains the compound and/or composition of the invention or be shipped together with a container that contains the compound and/or composition.
  • the instructional material may be shipped separately from the container with the intention that the recipient uses the instructional material and the compound cooperatively. Delivery of the instructional material may be, for example, by physical delivery of the publication or other medium of expression communicating the usefulness of the kit, or may alternatively be achieved by electronic transmission, for example by means of a computer, such as by electronic mail, or download from a website.
  • 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.
  • modulating 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.
  • 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 translated 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.
  • patient refers to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein.
  • the patient, subject, or individual is a human.
  • 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” or “nucleoside modified RNA” which refers to a nucleic acid or RNA molecule comprising at least one modified nucleoside.
  • a “modified nucleoside” refers to a nucleoside with a modification compared to a reference nucleoside. 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 to the natural product which is a C-glycosyl pyrimidine that consists of uracil having a beta-D-ribofuranosyl residue attached at position 5 (i.e., 5-(beta- D-Ribofuranosyl)uracil).
  • the term refers to m 1 acp 3 ⁇
  • the term refers to m lv P (1- methylpseudouridine).
  • the term refers to ⁇
  • the term refers to m 5 D (5-methyldihydrouridine). In another embodiment, the term refers to 3-methylpseudouridine). In another embodiment, the term refers to a pseudouridine moiety that is not further modified. In another embodiment, the term refers to a monophosphate, diphosphate, or triphosphate of any of the above pseudouridines. In another embodiment, the term refers to any other pseudouridine known in the art. Each possibility represents a separate embodiment of the present invention.
  • 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.
  • the promoter that is recognized by bacteriophage RNA polymerase and is used to generate the mRNA by in vitro transcription.
  • an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample.
  • an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more other species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific.
  • an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific.
  • the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody.
  • a particular structure e.g., an antigenic determinant or epitope
  • terapéutica as used herein 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.
  • 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.
  • To “treat” a disease or disorder 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.
  • transfected or “transformed” or “transduced” as used herein 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.
  • 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.
  • 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.
  • compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present invention that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present invention that consist essentially of, or consist of, the recited processing steps.
  • the present invention relates to compositions and methods for regenerating corneal endothelium in a subject in need thereof.
  • the subject has vision loss due to a corneal endothelium disease or disorder, such as age-related corneal degeneration, Fuchs’ endothelial corneal dystrophy, and/or cataract surgical injury.
  • the invention provides a lipid nanoparticle (LNP), pharmaceutical compositions comprising the LNP and uses thereof in methods and kits for regenerating corneal endothelium in a subject in need thereof.
  • the LNP comprises one or more nucleic acid molecules described herein.
  • the LNP comprises (a) at least one nucleoside-modified RNA molecule encoding at least one pro-growth factor, and (b) at least one ionizable lipid.
  • the at least one pro-growth factor consists of one pro-growth factor.
  • the at least one pro-growth factor consists of two pro-growth factors.
  • the at least one pro-growth factor consists of three pro-growth factors.
  • the at least one pro-growth factor consists of four pro-growth factors.
  • the at least one pro-growth factor consists of five pro-growth factors.
  • the invention provides a lipid nanoparticle (LNP) that is capable of regenerating corneal endothelium in a subject in need thereof.
  • the LNP comprises at least one nucleoside-modified ribonucleic acid (RNA) encoding at least one pro-growth factor. Each progrowth factor is encoded by a distinct nucleoside-modified RNA.
  • the LNP comprises at least one ionizable lipid.
  • the at least one pro-growth factor consists of one pro-growth factor.
  • the at least one pro-growth factor consists of two pro-growth factors.
  • the at least one pro-growth factor consists of three pro-growth factors.
  • the at least one pro-growth factor consists of four pro-growth factors.
  • the at least one pro-growth factor consists of five pro-growth factors.
  • the at least one nucleoside-modified RNA is messenger RNA (mRNA). In some embodiments, the at least one nucleoside-modified RNA comprises pseudouridine and/or 1-methyl-pseudouridine. In some embodiments, the at least one nucleoside- modified RNA is in vitro transcribed (IVT) RNA. In certain embodiments, the at least one nucleoside-modified RNA is IVT mRNA comprising pseudouridine and/or 1-methyl- pseudouridine.
  • mRNA messenger RNA
  • the at least one nucleoside-modified RNA comprises pseudouridine and/or 1-methyl-pseudouridine. In some embodiments, the at least one nucleoside- modified RNA is in vitro transcribed (IVT) RNA. In certain embodiments, the at least one nucleoside-modified RNA is IVT mRNA comprising pseudouridine and/or 1-methyl- pseudouridine.
  • the at least one ionizable lipid encapsulates the at least one nucleoside-modified RNA. In certain embodiments, the at least one ionizable lipid is a cationic lipid. Non-limiting examples of cationic lipids are described herein.
  • the LNP further comprises at least one surface-linked antibody, wherein the at least one surface-linked antibody targets a corneal endothelium cell surface marker.
  • the corneal endothelium cell surface marker is selected from N-cadherin, NCAM (CD56), connexin 43, integrin a3[31, integrin b5, VDAC3, aquaporin 1, chloride channel 3, SLC4A4, CD44, GPC4, and any combination thereof.
  • regenerating corneal endothelium in a subject in need thereof comprises inducing mitosis in at least 5%, at least 10%, at least 15%, or at least 20% of corneal endothelium cells in the subject.
  • the subject has vision loss due to a corneal endothelium disease or disorder (e.g., age-related corneal degeneration, Fuchs’ endothelial corneal dystrophy, and/or cataract surgical injury).
  • regenerating corneal endothelium in the subject treats the vision loss in the subject.
  • the subject is a human.
  • pro-growth factor(s) refers to key enzymes and transcription factors which, either alone or in combination, activate relevant signaling pathways to allow the corneal endothelial cells to undergo mitosis.
  • pro-growth factors include cyclin-dependent kinase 4 (CDK4), cyclin DI (CCND1), sex determining region Y-box 2 (SOX2), c-myc transcription factor (MYC), yes-associated protein 1 (YAP), NANOG transcription factor (NANOG), octamer-binding transcription factor 4 (OCT4), Sirtuin 1 (SIRT1) and activating ligands thereof, catenin delta-1 (CTNND1), beta-catenin (CTNNB1), Y-box binding protein 3 (YBX3) aka DNA-binding protein A (CSDA) aka ZONAB, transcription factor E2F2 (E2F2), FGF1, FGF10, IGF1, IGF2, Col8al,
  • CDK4 and CCND1 form a complex that promotes progression of the cell cycle [Tchakarska and Sola, Cell Cycle. 2020 Jan; 19(2): 163-178], Sox2 and Myc, two of the Yamanaka factors for somatic cell re-programming, are known to promote proliferation in various cell types, including in corneal endothelial cells [Takahashi and Yamanaka, Cell. 2006 Aug 25; 126(4):663-76; Chang, et al., Stem Cells. 2018 Dec;36(12): 1851-1862], Yap is a downstream effector of the Hippo pathway, that is critical for mediating contact inhibition growth [Joyce, et al., Invest Ophthalmol Vis Sci.
  • FGF1, FGF10, IGF1 and IGF2 are growth factors that play crucial roles in various cellular processes.
  • FGF1 is a member of the fibroblast growth factor (FGF) family and is involved in various processes, including cell growth, morphogenesis, tissue repair.
  • FGF 10 plays a significant role in embryonic development, tissue repair.
  • IGF1 plays a pivotal role in cell growth, differentiation, and survival. It has potent mitogenic and cell survival activities.
  • IGF2 is involved in cell growth and development, especially during embryogenesis.
  • Aqpl and Slc4al l are involved in the regulation of corneal hydration.
  • Aqpl encodes for the water channel protein Aquaporin-1. This protein facilitates the rapid movement of water across cell membranes.
  • Slc4al 1 encodes a bicarbonate transporter protein that is involved in the transport of ions and water across the corneal endothelium. It plays a vital role in maintaining corneal clarity by helping regulate the hydration of the cornea. Mutations in the Slc4al 1 gene have been linked to corneal endothelial dystrophy, corneal clouding, and sensorineural deafness. While most commonly associated with another corneal dystrophy called congenital hereditary endothelial dystrophy (CHED), mutations in Slc4al 1 have also been implicated in FECD.
  • CHED congenital hereditary endothelial dystrophy
  • the at least one pro-growth factor comprises any one or more of cyclin-dependent kinase 4 (CDK4), cyclin DI (CCND1), sex determining region Y-box 2 (SOX2), c-myc transcription factor (MYC), and yes-associated protein 1 (YAP).
  • CDK4 cyclin-dependent kinase 4
  • CCND1 cyclin DI
  • SOX2 sex determining region Y-box 2
  • MYC c-myc transcription factor
  • YAP yes-associated protein 1
  • the at least one pro-growth factor comprises CDK4, CCND1, SOX2, MYC, and YAP.
  • the at least one pro-growth factor consists of CDK4, CCND1, SOX2, MYC, and YAP.
  • the at least one pro-growth factor comprises any one or more of CDK4, CCND1, SOX2, MYC, YAP, NANOG, OCT4, SIRT1, an activating ligand of SIRT1, CTNND1, CTNNB1, ZONAB, E2F2, FGF1, FGF10, IGF1, IGF2, Col8al, Col8a2, Col4a3, Col4a4, SLC4al l, Aqpl, and WWTR1.
  • the invention includes all such isoforms and splice variants of CDK4, CCND1, SOX2, MYC, YAP, NANOG, OCT4, SIRT1, an activating ligand of SIRT1, CTNND1, CTNNB1, ZONAB, E2F2, FGF1, FGF10, IGF1, IGF2, Col8al, Col8a2, Col4a3, Col4a4, SLC4al l, Aqpl, and WWTR1.
  • Table 1 provides exemplary nucleotide and amino acid sequences of certain pro-growth factors of the invention.
  • Table 1 Exemplary nucleotide and amino acid sequences.
  • the at least one pro-growth factor comprises or consists of CDK4.
  • the CDK4 comprises SEQ ID NO: 2 or SEQ ID NO: 12.
  • the CDK4 comprises an amino acid sequence that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 12.
  • the at least one pro-growth factor comprises or consists of CCND1.
  • the CCND1 comprises SEQ ID NO: 4 or SEQ ID NO: 14.
  • the CCND1 comprises an amino acid sequence that has at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 4 or SEQ ID NO: 14.
  • the at least one pro-growth factor comprises or consists of SOX2.
  • the SOX2 comprises SEQ ID NO: 6 or SEQ ID NO: 16.
  • the SOX2 comprises an amino acid sequence that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 6 or SEQ ID NO: 16.
  • the at least one pro-growth factor comprises or consists of MYC.
  • the MYC comprises SEQ ID NO: 8 or SEQ ID NO: 18.
  • the MYC comprises an amino acid sequence that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 8 or SEQ ID NO: 18.
  • the at least one pro-growth factor comprises or consists of YAP.
  • the YAP comprises SEQ ID NO: 10 or SEQ ID NO: 20.
  • the YAP comprises an amino acid sequence that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 10 or SEQ ID NO: 20.
  • the at least one pro-growth factor comprises or consists of NANOG.
  • the at least one pro-growth factor comprises or consists of OCT4.
  • the at least one pro-growth factor comprises or consists of
  • the at least one pro-growth factor comprises or consists of an activating ligand of SIRT1.
  • the at least one pro-growth factor comprises or consists of CTNND1.
  • the at least one pro-growth factor comprises or consists of CTNNB1.
  • the at least one pro-growth factor comprises or consists of ZONAB.
  • the at least one pro-growth factor comprises or consists of E2F2.
  • the at least one pro-growth factor comprises or consists of FGF1.
  • the at least one pro-growth factor comprises or consists of
  • the at least one pro-growth factor comprises or consists of IGF 1.
  • the at least one pro-growth factor comprises or consists of IGF2.
  • the at least one pro-growth factor comprises or consists of
  • the at least one pro-growth factor comprises or consists of Col8a2.
  • the at least one pro-growth factor comprises or consists of Col4a3.
  • the at least one pro-growth factor comprises or consists of Col4a4.
  • the at least one pro-growth factor comprises or consists of SLC4al l.
  • the at least one pro-growth factor comprises or consists of Aqpl.
  • the at least one pro-growth factor comprises or consists of WWTR1.
  • the pro-growth factor comprises more than one domain or subunit (e.g., two or more subunits) of the pro-growth factor.
  • the two or more subunits of the pro-growth factor can be expressed as a fusion protein.
  • the nucleoside-modified RNA encodes a first subunit of the pro-growth factor and a second subunit of the pro-growth factor, wherein the first subunit and the second subunit are linked to each other via a flexible linker, such as a glycine serine linker.
  • the LNP comprises a distinct nucleoside-modified RNA encoding each individual subunit of the pro-growth factor.
  • the nucleotide sequence(s) encoding the pro-growth factor may be derived from any animal which expresses the pro-growth factor. Non-limiting examples include a mouse, a rat, a pig, a simian, and a human.
  • the LNP comprises a nucleoside modified RNA encoding a pro-growth factor from a mammal (e.g., a human), and said LNP is used in a method of regenerating corneal endothelium in the same mammal (e.g., a human).
  • the LNP comprises a nucleoside modified RNA encoding a pro-growth factor from a mammal (e.g., a human), and said LNP is used in a method of regenerating corneal endothelium in a different mammal (e.g., a mouse).
  • the pro-growth factor is an engineered and/or variant version of a naturally-occurring pro-growth factor.
  • engineered pro-growth factors include recombinant, edited, tagged, and/or fusion pro-growth factors.
  • the pro-growth factor is a variant of a naturally occurring progrowth factor.
  • the progrowth factor or a subunit thereof comprises an amino acid sequence that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any naturally-occurring or known reference amino acid sequence of the pro-growth factor or subunit thereof.
  • the pro-growth factor or a subunit thereof is encoded by a nucleotide sequence that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any naturally-occurring or known reference nucleotide sequence encoding the pro-growth factor or subunit thereof.
  • lipid 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 lipids, for example a lipid of Formula (I), (II) or (III).
  • lipid nanoparticles are included in a formulation comprising a nucleoside-modified RNA as described herein.
  • such lipid nanoparticles comprise a cationic lipid (e.g., a lipid of Formula (I), (II) or (III)) and one or more excipient selected from neutral lipids, charged lipids, steroids and polymer conjugated lipids (e g., a pegylated lipid such as a pegylated lipid of structure (IV), such as compound IVa).
  • a cationic lipid e.g., a lipid of Formula (I), (II) or (III)
  • excipient selected from neutral lipids, charged lipids, steroids and polymer conjugated lipids
  • a pegylated lipid such as a pegylated lipid of structure (IV), such as compound IVa
  • the nucleoside-modified RNA is encapsulated in the lipid portion of the lipid nanoparticle or an aqueous space enveloped by some or all of the lipid portion of the lipid 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.
  • the lipid nanoparticles have a mean diameter of from about 30 nm to about 150 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, 100 nm, 105 nm, 110 nm, 115 nm, 120 n
  • the LNP may 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 LNP comprises at least one ionizable lipid.
  • the ionizable lipid is a cationic lipid.
  • the LNP comprises one or more cationic lipids, and one or more stabilizing lipids.
  • Stabilizing lipids include neutral lipids and pegylated lipids.
  • the LNP 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 cationic lipid 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-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); 3-(N — (N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N- (l-(2,3-dioleoyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-
  • cationic lipids are available which can be used in the present invention. These include, for example, LIPOFECTIN® (commercially available cationic liposomes comprising DOTMA and 1,2- 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 liposomes comprising
  • 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.
  • Suitable amino lipids useful in the invention include those described in WO 2012/016184, incorporated herein by reference in its entirety.
  • Representative amino lipids include, but are not limited to, 1, 2-dilinoley oxy-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), 1,2- dil
  • Suitable amino lipids include those having the formula: wherein Ri and R2 are either the same or different and independently optionally substituted C10-C24 alkyl, optionally substituted C10-C24 alkenyl, optionally substituted C10-C24 alkynyl, or optionally substituted C10-C24 acyl;
  • R3 and R4 are either the same or different and independently optionally substituted Ci-Ce alkyl, optionally substituted C2-C6 alkenyl, or optionally substituted C2-C6 alkynyl or R3 and Rr may join to form an optionally substituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms chosen from nitrogen and oxygen;
  • Rs is either absent or present and when present is hydrogen or Ci-Cs alkyl; m, n, and p are either the same or different and independently either 0 or 1 with the proviso that m, n, and p are not simultaneously 0; q is 0, 1, 2, 3, or 4; and
  • Y and Z are either the same or different and independently O, S, or NH.
  • Ri and R2 are each linoleyl, and the amino lipid is a dilinoleyl amino lipid. In some embodiments, the amino lipid is a dilinoleyl amino lipid.
  • a representative useful dilinoleyl amino lipid has the formula:
  • n 0, 1, 2, 3, or 4.
  • the cationic lipid is a DLin-K-DMA. In some embodiments, the cationic lipid is DLin-KC2-DMA (DLin-K-DMA above, wherein n is 2)-
  • the cationic lipid component of the LNPs has the structure of Formula (I):
  • R la and R lb are, at each occurrence, independently either (a) H or C1-C12 alkyl, or (b) R la is H or C1-C12 alkyl, and R lb together with the carbon atom to which it is bound is taken together with an adjacent R lb and the carbon atom to which it is bound to form a carbon-carbon double bond;
  • R 2a and R 2b are, at each occurrence, independently either (a) H or C1-C12 alkyl, or (b) R 2a is H or C1-C12 alkyl, and R 2b together with the carbon atom to which it is bound is taken together with an adjacent R 2b and the carbon atom to which it is bound to form a carbon-carbon double bond;
  • R 3a and R 3b are, at each occurrence, independently either (a) H or C1-C12 alkyl, or (b) R 3a is H or Ci-C 12 alkyl, and R 3b together with the carbon atom to which it is bound is taken together with an adjacent R 3b and the carbon atom to which it is bound to form a carbon-carbon double bond;
  • R 4a and R 4b are, at each occurrence, independently either (a) H or C1-C12 alkyl, or (b) R 4a is H or C1-C12 alkyl, and R 4b together with the carbon atom to which it is bound is taken together with an adjacent R 4b and the carbon atom to which it is bound to form a carbon-carbon double bond;
  • R 5 and R 6 are each independently methyl or cycloalkyl
  • R 7 is, at each occurrence, independently H or C1-C12 alkyl
  • R 8 and R 9 are each independently C1-C12 alkyl; or R 8 and R 9 , together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring comprising one nitrogen atom; a and d are each independently an integer from 0 to 24; b and c are each independently an integer from 1 to 24; and e is 1 or 2.
  • R la and R lb are not isopropyl when a is 6 or n-butyl when a is 8.
  • R la and R lb are not isopropyl when a is 6 or n-butyl when a is 8.
  • R 8 and R 9 are each independently unsubstituted Ci- C12 alkyl; or R 8 and R 9 , together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring comprising one nitrogen atom;
  • carbon-carbon double bond refers to one of the following structures: wherein R a and R b are, at each occurrence, independently H or a substituent.
  • R a and R b are, at each occurrence, independently H, C1-C12 alkyl or cycloalkyl, for example H or C1-C12 alkyl.
  • the lipid compounds of Formula (I) have the following structure (la):
  • lipid compounds of Formula (I) have the following structure (lb):
  • the lipid compounds of Formula (I) have the following structure (Ic):
  • a, b, c and d are each independently an integer from 2 to 12 or an integer from 4 to 12.
  • a, b, c and d are each independently an integer from 8 to 12 or 5 to 9.
  • a is 0.
  • a is 1.
  • a is 2.
  • a is 3.
  • a is 4.
  • a is 5.
  • a is 6.
  • a is 7.
  • a 8. In some embodiments, a is 9.
  • a is 10. In more embodiments, a is 11. In yet other embodiments, a is 12. In some embodiments, a is 13. In other embodiments, a is 14. In more embodiments, a is 15. In yet other embodiments, a is 16.
  • b is 1. In other embodiments, b is 2. In more embodiments, b is 3. In yet other embodiments, b is 4. In some embodiments, b is 5. In other embodiments, b is 6. In more embodiments, b is 7. In yet other embodiments, b is 8. In some embodiments, b is 9. In other embodiments, b is 10. In more embodiments, b is 11. In yet other embodiments, b is 12. In some embodiments, b is 13. In other embodiments, b is 14. In more embodiments, b is 15. In yet other embodiments, b is 16. In some more embodiments of Formula (I), c is 1 . In other embodiments, c is 2. In more embodiments, c is 3.
  • c is 4. In some embodiments, c is 5. In other embodiments, c is 6. In more embodiments, c is 7. In yet other embodiments, c is 8. In some embodiments, c is 9. In other embodiments, c is 10. In more embodiments, c is 11. In yet other embodiments, c is 12. In some embodiments, c is 13. In other embodiments, c is 14. In more embodiments, c is 15. In yet other embodiments, c is 16.
  • d is 0. In some embodiments, d is 1. In other embodiments, d is 2. In more embodiments, d is 3. In yet other embodiments, d is 4. In some embodiments, d is 5. In other embodiments, d is 6.
  • d is 7. In yet other embodiments, d is 8. In some embodiments, d is 9. In other embodiments, d is 10. In more embodiments, d is 11. In yet other embodiments, d is 12. In some embodiments, d is 13. In other embodiments, d is 14. In more embodiments, d is 15. In yet other embodiments, d is 16.
  • a and d are the same.
  • b and c are the same. In some other specific embodiments, a and d are the same and b and c are the same.
  • a and b and the sum of c and d in Formula (I) are factors which may be varied to obtain a lipid of Formula (I) having the desired properties.
  • a and b are chosen such that their sum is an integer ranging from 14 to 24.
  • c and d are chosen such that their sum is an integer ranging from 14 to 24.
  • the sum of a and b and the sum of c and d are the same.
  • the sum of a and b and the sum of c and d are both the same integer which may range from 14 to 24.
  • a. b, c and d are selected such the sum of a and b and the sum of c and d is 12 or greater.
  • e is 1. In other embodiments, e is 2.
  • R la , R 2a , R 3a and R 4a of Formula (I) are not particularly limited.
  • R la , R 2a , R 3a and R 4a are H at each occurrence.
  • at least one of R la , R 2a , R 3a and R 4a is C1-C12 alkyl.
  • at least one of R la , R 2a , R 3a and R 4a is Ci-Cs alkyl.
  • at least one of R la , R 2a , R 3a and R 4a is Ci-Ce alkyl.
  • the Ci-Cs alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.
  • R la , R lb , R 4a and R 4b are C1-C12 alkyl at each occurrence.
  • At least one of R la , R lb , R 4a and R 4b is H or R la , R lb , R 4a and R 4b are H at each occurrence.
  • R lb together with the carbon atom to which it is bound is taken together with an adjacent R lb and the carbon atom to which it is bound to form a carbon-carbon double bond.
  • R 4b together with the carbon atom to which it is bound is taken together with an adjacent R 4b and the carbon atom to which it is bound to form a carbon-carbon double bond.
  • R 5 and R 6 of Formula (I) are not particularly limited in the foregoing embodiments. In certain embodiments one or both of R 5 or R 6 is methyl.
  • R 5 or R 6 is cycloalkyl for example cyclohexyl.
  • the cycloalkyl may be substituted or not substituted.
  • the cycloalkyl is substituted with C1-C12 alkyl, for example tertbutyl.
  • R 7 are not particularly limited in the foregoing embodiments of Formula (I). In certain embodiments, at least one R 7 is H. In some other embodiments, R 7 is H at each occurrence. In certain other embodiments R 7 is C1-C12 alkyl.
  • one of R 8 or R 9 is methyl. In other embodiments, both R 8 and R 9 are methyl.
  • R 8 and R 9 together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring.
  • R 8 and R 9 together with the nitrogen atom to which they are attached, form a 5-membered heterocyclic ring, for example a pyrrolidinyl ring.
  • the lipid of Formula (I) has one of the structures set forth in Table 1 below.
  • the LNPs comprise a lipid of Formula (I), at least one nucleoside- modified ribonucleic acid (RNA) encoding at least one pro-growth factor, wherein each progrowth factor is encoded by a distinct nucleoside-modified RNA, and one or more excipients selected from neutral lipids, steroids and pegylated lipids.
  • RNA nucleoside- modified ribonucleic acid
  • each progrowth factor is encoded by a distinct nucleoside-modified RNA
  • excipients selected from neutral lipids, steroids and pegylated lipids.
  • the lipid of Formula (I) is compound 1-5.
  • the lipid of Formula (I) is compound 1-6.
  • the cationic lipid component of the LNPs has the structure of Formula (II): or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein:
  • G 3 is Ci-Ce alkylene
  • R a is H or C1-C12 alkyl
  • R la and R lb are, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R la is H or C1-C12 alkyl, and R lb together with the carbon atom to which it is bound is taken together with an adjacent R lb and the carbon atom to which it is bound to form a carbon-carbon double bond;
  • R 2a and R 2b are, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R 2a is H or C1-C12 alkyl, and R 2b together with the carbon atom to which it is bound is taken together with an adjacent R 2b and the carbon atom to which it is bound to form a carbon-carbon double bond;
  • R 3a and R 3b are, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R 3a is H or C1-C12 alkyl, and R 3b together with the carbon atom to which it is bound is taken together with an adjacent R 3b and the carbon atom to which it is bound to form a carbon-carbon double bond;
  • R 4a and R 4b are, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R 4a is H or C1-C12 alkyl, and R 4b together with the carbon atom to which it is bound is taken together with an adjacent R 4b and the carbon atom to which it is bound to form a carbon-carbon double bond;
  • R 5 and R 6 are each independently H or methyl
  • R 7 is C4-C20 alkyl
  • R 8 and R 9 are each independently C1-C12 alkyl; or R 8 and R 9 , together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring; a, b, c and d are each independently an integer from 1 to 24; and x is 0, 1 or 2.
  • the lipid compound has one of the following structures (IIA) or (IIB):
  • the lipid compound has structure (IIA). In other embodiments, the lipid compound has structure (IIB).
  • one of L 1 or L 2 is a direct bond.
  • a “direct bond” means the group (e.g., L 1 or L 2 ) is absent.
  • each of L 1 and L 2 is a direct bond.
  • R la is H or C1-C12 alkyl
  • R lb together with the carbon atom to which it is bound is taken together with an adjacent R lb and the carbon atom to which it is bound to form a carbon-carbon double bond.
  • R 4a is H or Ci-C 12 alkyl
  • R 4b together with the carbon atom to which it is bound is taken together with an adjacent R 4b and the carbon atom to which it is bound to form a carbon-carbon double bond.
  • R 2a is H or C1-C12 alkyl
  • R 2b together with the carbon atom to which it is bound is taken together with an adjacent R 2b and the carbon atom to which it is bound to form a carbon-carbon double bond.
  • R 3a and R 3b are H or C1-C12 alkyl
  • R 3a is H or C1-C12 alkyl
  • R 3b together with the carbon atom to which it is bound is taken together with an adjacent R 3b and the carbon atom to which it is bound to form a carbon-carbon double bond.
  • the lipid compound has one of the following structures (IIC) or (IID): wherein e, f, g and h are each independently an integer from 1 to 12.
  • the lipid compound has structure (IIC). In other embodiments, the lipid compound has structure (HD). In various embodiments of structures (IIC) or (IID), e, f, g and h are each independently an integer from 4 to 10.
  • a, b, c and d are each independently an integer from 2 to 12 or an integer from 4 to 12. In other embodiments, a, b, c and d are each independently an integer from 8 to 12 or 5 to 9. In some certain embodiments, a is 0. In some embodiments, a is 1 . Tn other embodiments, a is 2. In more embodiments, a is 3. In yet other embodiments, a is 4. In some embodiments, a is 5. In other embodiments, a is 6. In more embodiments, a is 7. In yet other embodiments, a is 8. In some embodiments, a is 9. In other embodiments, a is 10. In more embodiments, a is 11. In yet other embodiments, a is 12. In some embodiments, a is 13. In other embodiments, a is 14. In more embodiments, a is 15. In yet other embodiments, a is 16.
  • b is 1. In other embodiments, b is 2. In more embodiments, b is 3. In yet other embodiments, b is 4. In some embodiments, b is 5. In other embodiments, b is 6. In more embodiments, b is 7. In yet other embodiments, b is 8. In some embodiments, b is 9. In other embodiments, b is 10. In more embodiments, b is 11. In yet other embodiments, b is 12. In some embodiments, b is 13. In other embodiments, b is 14. In more embodiments, b is 15. In yet other embodiments, b is 16.
  • c is 1. In other embodiments, c is 2. In more embodiments, c is 3. In yet other embodiments, c is 4. In some embodiments, c is 5. In other embodiments, c is 6. In more embodiments, c is 7. In yet other embodiments, c is 8. In some embodiments, c is 9. In other embodiments, c is 10. In more embodiments, c is 11. In yet other embodiments, c is 12. In some embodiments, c is 13. In other embodiments, c is 14. In more embodiments, c is 15. In yet other embodiments, c is 16.
  • d is 0. In some embodiments, d is 1. In other embodiments, d is 2. In more embodiments, d is 3. In yet other embodiments, d is 4. In some embodiments, d is 5. In other embodiments, d is 6.
  • d is 7. In yet other embodiments, d is 8. In some embodiments, d is 9. In other embodiments, d is 10. In more embodiments, d is 11. In yet other embodiments, d is 12. In some embodiments, d is 13. In other embodiments, d is 14. In more embodiments, d is 15. In yet other embodiments, d is 16.
  • e is 1. In other embodiments, e is 2. In more embodiments, e is 3. In yet other embodiments, e is 4. In some embodiments, e is 5. In other embodiments, e is 6. In more embodiments, e is 7. In yet other embodiments, e is 8. In some embodiments, e is 9. In other embodiments, e is 10. In more embodiments, e is 11. In yet other embodiments, e is 12.
  • f is 1. In other embodiments, f is 2. In more embodiments, f is 3. In yet other embodiments, f is 4. In some embodiments, f is 5. In other embodiments, f is 6. In more embodiments, f is 7. In yet other embodiments, f is 8. In some embodiments, f is 9. In other embodiments, f is 10. In more embodiments, f is 11. In yet other embodiments, f is 12.
  • g is 1. In other embodiments, g is 2. In more embodiments, g is 3. In yet other embodiments, g is 4. In some embodiments, g is 5. In other embodiments, g is 6. In more embodiments, g is 7. In yet other embodiments, g is 8. In some embodiments, g is 9. In other embodiments, g is 10. In more embodiments, g is 11. In yet other embodiments, g is 12.
  • h is 1. In other embodiments, e is 2. In more embodiments, h is 3. In yet other embodiments, h is 4. In some embodiments, e is 5. In other embodiments, h is 6. In more embodiments, h is 7. In yet other embodiments, h is 8. In some embodiments, h is 9. In other embodiments, h is 10. In more embodiments, h is 11. In yet other embodiments, h is 12.
  • a and d are the same. In some other embodiments, b and c are the same. In some other specific embodiments and a and d are the same and b and c are the same.
  • the sum of a and b and the sum of c and d of Formula (II) are factors which may be varied to obtain a lipid having the desired properties.
  • a and b are chosen such that their sum is an integer ranging from 14 to 24.
  • c and d are chosen such that their sum is an integer ranging from 14 to 24.
  • the sum of a and b and the sum of c and d are the same.
  • the sum of a and b and the sum of c and d are both the same integer which may range from 14 to 24.
  • a. b, c and d are selected such that the sum of a and b and the sum of c and d is 12 or greater.
  • R la , R 2a , R 3a and R 4a of Formula (II) are not particularly limited.
  • at least one of R la , R 2a , R' a and R 4a is H.
  • R la , R 2a , R 3a and R 4a are H at each occurrence.
  • at least one of R la , R 2a , R 3a and R 4a is C1-C12 alkyl.
  • at least one of R la , R 2a , R 3a and R 4a is Ci-Cs alkyl.
  • At least one of R la , R 2a , R 3a and R 4a is Ci-Ce alkyl.
  • the Ci-Cs alkyl is methyl, ethyl, n-propyl, iso-propyl, n- butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.
  • R la , R lb , R 4a and R 4b are C1-C12 alkyl at each occurrence.
  • At least one of R lb , R 2b , R 3b and R 4b is H or R lb , R 2b , R 3b and R 4b are H at each occurrence.
  • R lb together with the carbon atom to which it is bound is taken together with an adjacent R lb and the carbon atom to which it is bound to form a carbon-carbon double bond.
  • R 4b together with the carbon atom to which it is bound is taken together with an adjacent R 4b and the carbon atom to which it is bound to form a carbon-carbon double bond.
  • R 5 and R 6 of Formula (II) are not particularly limited in the foregoing embodiments.
  • one of R 5 or R 6 is methyl.
  • each of R 5 or R 6 is methyl.
  • R 5 or R 6 is methyl.
  • R b is branched C1-C15 alkyl.
  • R b has one of the following structures:
  • one of R 8 or R 9 is methyl.
  • both R 8 and R 9 are methyl.
  • R 8 and R 9 together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring.
  • R 8 and R 9 together with the nitrogen atom to which they are attached, form a 5-membered heterocyclic ring, for example a pyrrolidinyl ring.
  • R 8 and R 9 together with the nitrogen atom to which they are attached, form a 6-membered heterocyclic ring, for example a piperazinyl ring.
  • G 3 is C2-C4 alkylene, for example C3 alkylene.
  • the lipid compound has one of the structures set forth in Table 2 below.
  • the LNPs comprise a lipid of Formula (II), at least one nucleoside- modified ribonucleic acid (RNA) encoding at least one pro-growth factor, wherein each progrowth factor is encoded by a distinct nucleoside-modified RNA, and one or more excipient selected from neutral lipids, steroids and pegylated lipids.
  • the lipid of Formula (II) is compound II-9.
  • the lipid of Formula (II) is compound II- 10.
  • the lipid of Formula (II) is compound II- 11.
  • the lipid of Formula (II) is compound 11-12.
  • the lipid of Formula (II) is compound 11-32.
  • G 1 and G 2 are each independently unsubstituted C1-C12 alkylene or C1-C12 alkenylene;
  • G 3 is C1-C24 alkylene, C1-C24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene;
  • R a is H or C1-C12 alkyl
  • R 1 and R 2 are each independently C6-C24 alkyl or C6-C24 alkenyl
  • R 4 is C1-C12 alkyl
  • R 3 is H or Ci-Ce alkyl; and x is 0, 1 or 2.
  • the lipid has one of the following structures (IIIA) or (IIIB): wherein:
  • A is a 3 to 8-membered cycloalkyl or cycloalkylene ring
  • R 6 is, at each occurrence, independently H, OH or C1-C24 alkyl; n is an integer ranging from 1 to 15.
  • the lipid has structure (IIIA), and in other embodiments, the lipid has structure (IHB).
  • the lipid has one of the following structures (IIIC) or (HID): wherein y and z are each independently integers ranging from 1 to 12.
  • the lipid has one of the following structures (HIE) or (IIIF):
  • the lipid has one of the following structures
  • n is an integer ranging from 2 to 12, for example from 2 to 8 or from 2 to 4.
  • n is 3, 4, 5 or 6.
  • n is 3.
  • n is 4.
  • n is 5.
  • n is 6.
  • y and z are each independently an integer ranging from 2 to 10.
  • y and z are each independently an integer ranging from 4 to 9 or from 4 to 6.
  • R 6 is H.
  • R 6 is C1-C24 alkyl.
  • R 6 is OH.
  • G 3 is unsubstituted. In other embodiments, G 3 is substituted. In various different embodiments, G 3 is linear C1-C24 alkylene or linear C1-C24 alkenylene. In some other foregoing embodiments of Formula (III), R 1 or R 2 , or both, is C6-C24 alkenyl.
  • R 1 and R 2 each, independently have the following structure: wherein: R 7a and R 7b are, at each occurrence, independently H or C1-C12 alkyl; and a is an integer from 2 to 12, wherein R 7a , R 7b and a are each selected such that R 1 and R 2 each independently comprise from 6 to 20 carbon atoms. For example, in some embodiments a is an integer ranging from 5 to 9 or from 8 to 12.
  • At least one occurrence of R 7a is H.
  • R 7a is H at each occurrence.
  • at least one occurrence of R 711 is C i-Cs alkyl.
  • Ci-Cs alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, tert- butyl, n-hexyl or n-octyl.
  • R 1 or R 2 has one of the following structures:
  • R 4 is methyl or ethyl.
  • the cationic lipid of Formula (III) has one of the structures set forth in Table 3 below.
  • the LNPs comprise a lipid of Formula (III), at least one nucleoside-modified ribonucleic acid (RNA) encoding at least one pro-growth factor, wherein each pro-growth factor is encoded by a distinct nucleoside-modified RNA, and one or more excipient selected from neutral lipids, steroids and pegylated lipids.
  • the lipid of Formula (III) is compound III-3.
  • the lipid of Formula (III) is compound III-7.
  • the cationic lipid is present in the LNP in an amount from about 30 to about 95 mole percent. In some embodiments, the cationic lipid is present in the LNP in an amount from about 30 to about 70 mole percent. In some embodiments, the cationic lipid is present in the LNP in an amount from about 40 to about 60 mole percent. In some embodiments, the cationic lipid is present in the LNP in an amount of about 50 mole percent. In some embodiments, the LNP comprises only cationic lipids. In certain embodiments, the L1 P comprises one or more additional lipids which stabilize the formation of particles during their formation.
  • Suitable stabilizing lipids include neutral lipids and anionic lipids.
  • neutral lipid refers to any one of a number of lipid species that exist in either an uncharged or neutral zwitterionic form at physiological pH.
  • Representative neutral lipids include diacylphosphatidylcholines, diacylphosphatidylethanolamines, ceramides, sphingomyelins, dihydro sphingomyelins, cephalins, and cerebrosides.
  • Exemplary neutral lipids include, for example, di stearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4- (N-maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-
  • the LNPs comprise a neutral lipid selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM.
  • the molar ratio of the cationic lipid (e.g., lipid of Formula (I)) to the neutral lipid ranges from about 2: 1 to about 8:1.
  • the LNPs further comprise a steroid or steroid analogue.
  • a “steroid” is a compound comprising the following carbon skeleton:
  • the steroid or steroid analogue is cholesterol.
  • the molar ratio of the cationic lipid (e ., lipid of Formula (I)) to cholesterol ranges from about 2:1 to 1 :1.
  • anionic lipid refers to any lipid that is negatively charged at physiological pH. These lipids include phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamines, N- succinylphosphatidylethanolamines, N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifying groups joined to neutral lipids.
  • phosphatidylglycerol cardiolipin
  • diacylphosphatidylserine diacylphosphatidic acid
  • N-dodecanoylphosphatidylethanolamines N-dodecanoylphosphatidylethanolamines
  • N- succinylphosphatidylethanolamines N-glutarylphosphatidyl
  • the LNP comprises glycolipids (e.g., monosialoganglioside GMi). In certain embodiments, the LNP comprises a sterol, such as cholesterol.
  • the LNPs comprise a polymer conjugated lipid.
  • polymer conjugated lipid refers to a molecule comprising both a lipid portion and a polymer portion.
  • An example of a polymer conjugated lipid is a pegylated lipid.
  • pegylated lipid refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art and include l-(monomethoxy-polyethyleneglycol)-2,3- dimyristoylglycerol (PEG-s- DMG) and the like.
  • the LNP comprises an additional, stabilizing -lipid which is a polyethylene glycol-lipid (pegylated lipid).
  • Suitable polyethylene glycol-lipids include PEG- modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramides (e g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols.
  • Representative polyethylene glycol-lipids include PEG-c-DOMG, PEG-c-DMA, and PEG-s-DMG.
  • the polyethylene glycol-lipid is N-[(methoxy poly(ethylene glycol)2ooo)carbamyl]-l,2- dimyristyloxlpropyl-3-amine (PEG-c-DMA). In some embodiments, the polyethylene glycol- lipid is PEG-c-DOMG).
  • the LNPs comprise a pegylated diacylglycerol (PEG-DAG) such as l-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG- DMG), a pegylated phosphatidylethanoloamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O-(2’,3’-di(tetradecanoyloxy)propyl-l-O-(O- methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as co-methoxy(polyethoxy)ethyl-N-(2,3- di(tetradecanoxy)propyl)carbamate or 2,3-d
  • the LNPs comprise a pegylated lipid having the following structure (IV): or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein:
  • R 10 and R 11 are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and z has mean value ranging from 30 to 60.
  • R 10 and R 11 are not both n-octadecyl when z is 42.
  • R 10 and R 11 are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 18 carbon atoms.
  • R 10 and R 11 are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 12 to 16 carbon atoms.
  • R 10 and R 11 are each independently a straight or branched, saturated or unsaturated alkyl chain containing 12 carbon atoms.
  • R 10 and R 11 are each independently a straight or branched, saturated or unsaturated alkyl chain containing 14 carbon atoms. In other embodiments, R 10 and R 11 are each independently a straight or branched, saturated or unsaturated alkyl chain containing 16 carbon atoms. In still more embodiments, R 10 and R 11 are each independently a straight or branched, saturated or unsaturated alkyl chain containing 18 carbon atoms. In still other embodiments, R 10 is a straight or branched, saturated or unsaturated alkyl chain containing 12 carbon atoms and R 11 is a straight or branched, saturated or unsaturated alkyl chain containing 14 carbon atoms.
  • z spans a range that is selected such that the PEG portion of (II) has an average molecular weight of about 400 to about 6000 g/mol. In some embodiments, the average z is about 45.
  • the pegylated lipid has one of the following structures:
  • n is an integer selected such that the average molecular weight of the pegylated lipid is about 2500 g/mol.
  • the additional lipid is present in the LNP in an amount from about 1 to about 10 mole percent. In some embodiments, the additional lipid is present in the LNP in an amount from about 1 to about 5 mole percent. In some embodiments, the additional lipid is present in the LNP in about 1 mole percent or about 1.5 mole percent.
  • the LNPs comprise a lipid of Formula (I), at least one nucleosi demodified ribonucleic acid (RNA) encoding at least one pro-growth factor, wherein each progrowth factor is encoded by a distinct nucleoside-modified RNA, a neutral lipid, a steroid and a pegylated lipid.
  • the lipid of Formula (I) is compound 1-6.
  • the neutral lipid is DSPC.
  • the steroid is cholesterol.
  • the pegylated lipid is compound IVa.
  • the LNP comprises one or more targeting moieties, which are capable of targeting the LNP to a cell or cell population.
  • the targeting moiety is a ligand, which directs the LNP to a receptor found on a cell surface.
  • the LNP comprises one or more internalization domains.
  • the LNP comprises one or more domains, which bind to a cell to induce the internalization of the LNP.
  • the one or more internalization domains bind to a receptor found on a cell surface to induce receptor-mediated uptake of the LNP.
  • the LNP is capable of binding a biomolecule in vivo, where the LNP-bound biomolecule can then be recognized by a cell-surface receptor to induce internalization.
  • the LNP binds systemic ApoE, which leads to the uptake of the LNP and associated cargo.
  • Embodiments of the lipid of Formula (I) can be prepared according to General Reaction Scheme 1 (“Method A”), wherein R is a saturated or unsaturated C1-C24 alkyl or saturated or unsaturated cycloalkyl, m is 0 or 1 and n is an integer from 1 to 24.
  • Method A General Reaction Scheme 1
  • compounds of structure A-l can be purchased from commercial sources or prepared according to methods familiar to one of ordinary skill in the art.
  • a mixture of A-l, A-2 and DMAP is treated with DCC to give the bromide A-3.
  • a mixture of the bromide A-3, a base (e.g., N,N-diisopropylethylamine) and the N,N-dimethyldiamine A-4 is heated at a temperature and time sufficient to produce A-5 after any necessary workup and or purification step.
  • a base e.g., N,N-diisopropylethylamine
  • N,N-dimethyldiamine A-4 is heated at a temperature and time sufficient to produce A-5 after any necessary workup and or purification step.
  • Compound B-5 can be prepared according to General Reaction Scheme 2 (“Method B”), wherein R is a saturated or unsaturated C1-C24 alkyl or saturated or unsaturated cycloalkyl, m is 0 or 1 and n is an integer from 1 to 24.
  • Method B General Reaction Scheme 2
  • compounds of structure B-1 can be purchased from commercial sources or prepared according to methods familiar to one of ordinary skill in the art.
  • a solution of B-1 (1 equivalent) is treated with acid chloride B-2 (1 equivalent) and a base (e.g., triethylamine).
  • the crude product is treated with an oxidizing agent (e.g., pyridinum chlorochromate) and intermediate product B-3 is recovered.
  • An oxidizing agent e.g., pyridinum chlorochromate
  • B-3 an acid (e.g., acetic acid), and N,N-dimethylaminoamine B-4 is then treated with a reducing agent (e.g., sodium triacetoxyborohydride) to obtain B-5 after any necessary work up and/or purification.
  • a reducing agent e.g., sodium triacetoxyborohydride
  • starting materials A-l and B-1 are depicted above as including only saturated methylene carbons, starting materials which include carbon-carbon double bonds may also be employed for preparation of compounds which include carbon-carbon double bonds.
  • lipid of Formula (I) e.g., compound C-7 or C9
  • Method C General Reaction Scheme 3
  • R is a saturated or unsaturated C1-C24 alkyl or saturated or unsaturated cycloalkyl
  • m is 0 or 1
  • n is an integer from 1 to 24.
  • compounds of structure C-l can be purchased from commercial sources or prepared according to methods familiar to one of ordinary skill in the art.
  • Embodiments of the compound of Formula (II) can be prepared according to General Reaction Scheme 4 (“Method D”), wherein R la , R lb , R 2a , R 2b , R 3a , R 3b , R 4a , R 4b , R 3 , R 6 , R 8 , R 9 , L 1 , L 2 , G 1 , G 2 , G 3 , a, b, c and d are as defined herein, and R 7 represents R 7 or a C3-C19 alkyl.
  • Method D General Reaction Scheme 4
  • D-l and D-2 can be purchased from commercial sources or prepared according to methods familiar to one of ordinary skill in the art.
  • a solution of D-l and D-2 is treated with a reducing agent (e.g., sodium triacetoxyborohydride) to obtain D-3 after any necessary work up.
  • a solution of D-3 and a base e.g. trimethylamine, DMAP
  • acyl chloride D-4 or carboxylic acid and DCC
  • D-5 can be reduced with LiAlH4 D-6 to give D-7 after any necessary work up and/or purification.
  • Embodiments of the lipid of Formula (II) can be prepared according to General Reaction Scheme 5 (“Method E”), wherein R la , R lb , R 2a , R 2b , R 3a , R 3b , R 4a , R 4b , R 5 , R 6 , R 7 , R 8 , R 9 , L 1 , L 2 , G 3 , a, b, c and d are as defined herein.
  • compounds of structure E-l and E-2 can be purchased from commercial sources or prepared according to methods familiar to one of ordinary skill in the art.
  • E-3 A mixture of E-l (in excess), E-2 and a base (e.g., potassium carbonate) is heated to obtain E-3 after any necessary work up.
  • a solution of E-3 and a base e.g. trimethylamine, DMAP
  • acyl chloride E-4 or carboxylic acid and DCC
  • General Reaction Scheme 6 provides an exemplary method (Method F) for preparation of Lipids of Formula (III).
  • G 1 , G 3 , R 1 and R 3 in General Reaction Scheme 6 are as defined herein for Formula (III), and GL refers to a one-carbon shorter homologue of Gl.
  • Compounds of structure F-l are purchased or prepared according to methods known in the art. Reaction of F-l with diol F-2 under appropriate condensation conditions (e.g., DCC) yields ester/alcohol F-3, which can then be oxidized (e.g., PCC) to aldehyde F-4. Reaction of F-4 with amine F-5 under reductive amination conditions yields a lipid of Formula (III).
  • lipids of Formula (III) are available to those of ordinary skill in the art.
  • other lipids of Formula (III) wherein L 1 and L 2 are other than ester can be prepared according to analogous methods using the appropriate starting material.
  • General Reaction Scheme 6 depicts preparation of a lipids of Formula (III), wherein G 1 and G 2 are the same; however, this is not a required aspect of the invention and modifications to the above reaction scheme are possible to yield compounds wherein G 1 and G 2 are different.
  • Suitable protecting groups include hydroxy, amino, mercapto and carboxylic acid.
  • Suitable protecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl (for example, t- butyldimethylsilyl , Lbutyldiphenylsilyl or trimethyl silyl), tetrahydropyranyl, benzyl, and the like.
  • Suitable protecting groups for amino, amidino and guanidino include /-butoxycarbonyl, benzyloxycarbonyl, and the like.
  • Suitable protecting groups for mercapto include -C(O)-R" (where R" is alkyl, aryl or arylalkyl), /;-methoxybenzyl, trityl and the like.
  • Suitable protecting groups for carboxylic acid include alkyl, aryl or arylalkyl esters.
  • Protecting groups may be added or removed in accordance with standard techniques, which are known to one skilled in the art and as described herein. The use of protecting groups is described in detail in Green, T.W. and P.G.M. Wutz, Protective Groups in Organic Synthesis (1999), 3rd Ed., Wiley.
  • the protecting group may also be a polymer resin such as a Wang resin, Rink resin or a 2-chlorotrityl-chloride resin.
  • the invention includes a nucleoside-modified nucleic acid molecule (e.g., a nucleoside-modified RNA) encoding a pro-growth factor.
  • a nucleoside-modified nucleic acid molecule e.g., a nucleoside-modified RNA
  • the progrowth factor is selected from CDK4, CCND1, SOX2, MYC, YAP, and any combination thereof.
  • the at least one pro-growth factor is selected from CDK4, CCND1, SOX2, MYC, YAP, NANOG, OCT4, SIRT1, an activating ligand of SIRT1, CTNND1, CTNNB1, ZONAB, E2F2, FGF1, FGF10, IGF1, IGF2, Col8al, Col8a2, Col4a3, Col4a4, SLC4al 1, Aqpl, WWTR1, and any combination thereof.
  • nucleotide sequences encoding a pro-growth factor can alternatively comprise sequence variations with respect to the original nucleotide sequences, 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. Therefore, the scope of the present invention includes nucleotide sequences that are substantially homologous to the known and/or reference nucleotide sequences which encode a pro-growth factor of interest.
  • nucleotide sequence is “substantially homologous” to any of the nucleotide sequences described herein when its nucleotide sequence has a degree of identity with respect to the nucleotide sequence of at least 60%, of at least 65%, of at least 70%, of at least 65%, of at least 80%, of at least 85%, of at least 90%, of at least 95%, of at least 96%, of at least 97%, of at least 98%, or of at least 99%.
  • a nucleotide sequence that is substantially homologous to a nucleotide sequence encoding a pro-growth factor can typically be isolated from a producer organism of the a pro-growth factor based on the information contained in the nucleotide sequence by means of introducing conservative or non-conservative substitutions, for example.
  • Other examples of possible modifications include the insertion of one or more nucleotides within the sequence, the addition of one or more nucleotides at the 3’ and/or 5’ end of the sequence, or the deletion of one or more nucleotides at the 3’ and/or 5’ end or from within the sequence.
  • the degree of identity between two polynucleotides is determined using computer algorithms and methods that are widely known for the persons skilled in the art.
  • nucleotide sequences that encode amino acid sequences of the pro-growth factor that preserve the cellular function of the pro-growth factor.
  • an amino acid sequence is “substantially homologous” to a known or reference amino acid sequence when its amino acid sequence has a degree of identity with respect to the amino acid sequence of at least 60%, of at least 65%, of at least 70%, of at least 65%, of at least 80%, of at least 85%, of at least 90%, of at least 95%, of at least 96%, of at least 97%, of at least 98%, or of at least 99%.
  • the identity between two amino acid sequences can be determined by using the BLASTP algorithm (BLAST Manual, Altschul, S., et ah, NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et ah, J. Mol. Biol. 215: 403-410 (1990)).
  • the LNP and/or composition of the invention comprises in vitro transcribed (IVT) nucleoside-modified RNA encoding a pro-growth factor as described herein.
  • the LNP and/or composition of the invention comprises one or more IVT nucleoside-modified RNAs encoding one or more pro-growth factors, wherein each pro-growth factor is encoded by a distinct nucleoside-modified RNA.
  • an IVT RNA can be introduced to a cell as a form of transient transfection.
  • the RNA is produced by in vitro transcription using a plasmid DNA template generated synthetically.
  • DNA of interest from any source can be directly converted by PCR into a template for in vitro mRNA synthesis using appropriate primers and RNA polymerase.
  • the source of the DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or any other appropriate source of DNA.
  • the desired template for in vitro transcription is a pro-growth factor capable of regenerating corneal endothelium.
  • the DNA to be used for PCR contains an open reading frame.
  • the DNA can be from a naturally occurring DNA sequence from the genome of an organism.
  • the DNA is a full length gene of interest of a portion of a gene.
  • the gene can include some or all of the 5' and/or 3' untranslated regions (UTRs).
  • the gene can include exons and introns.
  • the DNA to be used for PCR is a human gene.
  • the DNA to be used for PCR is a human gene including the 5' and 3' UTRs.
  • the DNA to be used for PCR is a gene from a pathogenic or commensal organism, including bacteria, viruses, parasites, and fungi.
  • the DNA to be used for PCR is from a pathogenic or commensal organism, including bacteria, viruses, parasites, and fungi, including the 5' and 3' UTRs.
  • the DNA can alternatively be an artificial DNA sequence that is not normally expressed in a naturally occurring organism.
  • An exemplary artificial DNA sequence is one that contains portions of genes that are ligated together to form an open reading frame that encodes a fusion protein.
  • the portions of DNA that are ligated together can be from a single organism or from more than one organism.
  • Genes that can be used as sources of DNA for PCR include genes that encode one or more pro-growth factors in an organism. In certain instances, the genes are useful for a short term treatment. In certain instances, the genes have limited safety concerns regarding dosage of the expressed gene.
  • a plasmid is used to generate a template for in vitro transcription of mRNA, which is used for transfection.
  • the RNA has 5' and 3' UTRs.
  • the 5' UTR is between zero and 3000 nucleotides in length.
  • the length of 5' and 3' UTR sequences to be added to the coding region can be altered by different methods, including, but not limited to, designing primers for PCR that anneal to different regions of the UTRs. Using this approach, one of ordinary skill in the art can modify the 5' and 3' UTR lengths required to achieve optimal translation efficiency following transfection of the transcribed RNA.
  • the 5' and 3' UTRs can be the naturally occurring, endogenous 5' and 3' UTRs for the gene of interest.
  • UTR sequences that are not endogenous to the gene of interest can be added by incorporating the UTR sequences into the forward and reverse primers or by any other modifications of the template.
  • the use of UTR sequences that are not endogenous to the gene of interest can be useful for modifying the stability and/or translation efficiency of the RNA. For example, it is known that AU-rich elements in 3' UTR sequences can decrease the stability of mRNA. Therefore, 3' UTRs can be selected or designed to increase the stability of the transcribed RNA based on properties of UTRs that are well known in the art.
  • the 5' UTR can contain the Kozak sequence of the endogenous gene.
  • a consensus Kozak sequence can be redesigned by adding the 5' UTR sequence.
  • Kozak sequences can increase the efficiency of translation of some RNA transcripts, but does not appear to be required for all RNAs to enable efficient translation. The requirement for Kozak sequences for many mRNAs is known in the art.
  • the 5' UTR can be derived from an RNA virus whose RNA genome is stable in cells.
  • various nucleotide analogues can be used in the 3' or 5' UTR to impede exonuclease degradation of the mRNA.
  • a promoter of transcription should be attached to the DNA template upstream of the sequence to be transcribed.
  • the RNA polymerase promoter becomes incorporated into the PCR product upstream of the open reading frame that is to be transcribed.
  • the promoter is a T7 RNA polymerase promoter, as described elsewhere herein.
  • Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for T7, T3 and SP6 promoters are known in the art.
  • the mRNA has both a cap on the 5' end and a 3' poly(A) tail which determine ribosome binding, initiation of translation and stability mRNA in the cell.
  • a circular DNA template for instance, plasmid DNA
  • RNA polymerase produces a long concatameric product, which is not suitable for expression in eukaryotic cells.
  • the transcription of plasmid DNA linearized at the end of the 3' UTR results in normal sized mRNA, which is effective in eukaryotic transfection when it is polyadenylated after transcription.
  • phage T7 RNA polymerase can extend the 3' end of the transcript beyond the last base of the template (Schenbom and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).
  • polyA/T sequence integrated into plasmid DNA can cause plasmid instability, which can be ameliorated through the use of recombination incompetent bacterial cells for plasmid propagation.
  • Poly(A) tails of RNAs can be further extended following in vitro transcription with the use of a poly(A) polymerase, such as E. coli polyA polymerase (E-PAP) or yeast polyA polymerase.
  • E-PAP E. coli polyA polymerase
  • yeast polyA polymerase E. coli polyA polymerase
  • increasing the length of a poly(A) tail from 100 nucleotides to between 300 and 400 nucleotides results in about a two-fold increase in the translation efficiency of the RNA.
  • the attachment of different chemical groups to the 3' end can increase mRNA stability. Such attachment can contain modified/artificial nucleotides, aptamers and other compounds.
  • ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. ATP analogs can further increase the stability of the RNA.
  • RNAs produced by the methods to include a 5' cap structure can be generated using Vaccinia capping enzyme and T -O-methyl transferase enzymes (CellScript, Madison, WI).
  • 5' cap is provided using techniques known in the art and described herein (Cougot, et ah, Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et ah, RNA, 7:1468-95 (2001); Elango, et ah, Biochim. Biophys. Res. Commun., 330:958-966 (2005)).
  • RNA can be introduced into target cells using any of a number of different methods, for instance, commercially available methods which include, but are not limited to, electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or the Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendort, Hamburg Germany), cationic liposome mediated transfection using lipofection, polymer encapsulation, peptide mediated transfection, or biolistic particle delivery systems such as “gene guns” (see, for example, Nishikawa, et al.
  • RNA of the invention is introduced to a cell with a method comprising the use of TransIT®-mRNA transfection Kit (Minis, Madison WI), which, in some instances, provides high efficiency, low toxicity, transfection.
  • RNA of the invention is introduced into a cell (e.g., a cell of a subject) via encapsulation within an LNP as described herein.
  • the LNP and/or composition of the present invention comprises a nucleoside-modified nucleic acid encoding a pro-growth factor as described herein.
  • the LNP and/or composition of the present invention comprises one or more nucleoside-modified nucleic acid(s) encoding a plurality of pro-growth factors.
  • each pro-growth factor is encoded by a distinct nucleoside-modified RNA.
  • the LNP and/or composition comprises a nucleoside-modified RNA.
  • the LNP and/or composition comprises a nucleoside-modified mRNA.
  • Nucleoside-modified mRNA have particular advantages over nonmodified mRNA, including for example, increased stability, low or absent innate immunogenicity, and enhanced translation. Nucleoside-modified mRNA useful in the present invention is further described in U.S. Patent Nos. 8,278,036, 8,691,966, and 8,835,108, each of 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.
  • nucleoside-modified mRNAs, each encoding a pro-growth factor has demonstrated the ability to regenerate corneal endothelium.
  • 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 ah, 2008, Mol Ther 16: 1833-1840; Anderson et ah, 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 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 nucleoside-modified nucleic acid molecule is a purified nucleoside-modified nucleic acid molecule.
  • the composition is purified to remove double-stranded contaminants.
  • a preparative high performance liquid chromatography (HPLC) purification procedure is used to obtain pseudouridine-containing RNA that has superior translational potential and no innate immunogenicity (Kariko et al., 2011, Nucleic Acids Research 39:el42).
  • the nucleoside-modified nucleic acid molecule is purified using non-HPLC methods. In certain instances, the nucleoside-modified nucleic acid molecule is purified using chromatography methods, including but not limited to HPLC and fast protein liquid chromatography (FPLC).
  • FPLC fast protein liquid chromatography
  • the present invention encompasses RNA, oligoribonucleotide, and polyribonucleotide molecules comprising pseudouridine or a modified nucleoside.
  • the composition comprises an isolated nucleic acid encoding a pro-growth factor, wherein the nucleic acid comprises a pseudouridine or a modified nucleoside.
  • the composition comprises a vector, comprising an isolated nucleic acid encoding a pro-growth factor, wherein the nucleic acid comprises a pseudouridine or a modified nucleoside.
  • the nucleoside-modified RNA of the invention is IVT RNA, as described elsewhere herein.
  • 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 x acp 3 (l-methyl-3-(3-amino-3- carboxypropyl) pseudouridine.
  • the modified nucleoside is m 1 'P (1- methylpseudouridine).
  • the modified nucleoside i methylpseudouridine).
  • the modified nucleoside i methyldihydrouridine).
  • the modified nucleoside is a pseudouridine moiety that is not further modified (T).
  • 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 present invention is a modified uridine (U).
  • the modified nucleoside is a modified cytidine (C).
  • the modified nucleoside is a modified adenosine (A).
  • the modified nucleoside is a modified guanosine (G).
  • the modified nucleoside of the present invention is m 5 C (5- methylcytidine). In another embodiment, the modified nucleoside is m 5 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 nriA (1 -methyladenosine); m 2 A (2- methyladenosine); Am (2'-0-methyladenosine); ms 2 m 6 A (2-methylthio-N 6 -methyladenosine); i 6 A (N 6 -isopentenyladenosine); ms 2 i 6 A (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 (2- methyl
  • a nucleoside-modified RNA of the present 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 0.1%. In another embodiment, 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 0.7%. In another embodiment, the fraction is 0.8%. In another embodiment, the fraction is 0.9%. 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%.
  • the fraction is 3%. In another embodiment, the fraction is 4%. In another embodiment, the fraction is 5%. In another embodiment, the fraction is 6%. In another embodiment, the fraction is 7%. In another embodiment, the fraction is 8%. In another embodiment, the fraction is 9%. 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%.
  • the fraction is 55%. In another embodiment, the fraction is 60%. In another embodiment, the fraction is 65%. In another embodiment, the fraction is 70%. In another embodiment, the fraction is 75%. In another embodiment, the fraction is 80%. In another embodiment, the fraction is 85%. In another embodiment, the fraction is 90%. Tn another embodiment, the fraction is 91%. In another embodiment, the fraction is 92%. In another embodiment, the fraction is 93%. In another embodiment, the fraction is 94%. In another embodiment, the fraction is 95%. In another embodiment, the fraction is 96%. In another embodiment, the fraction is 97%. In another embodiment, the fraction is 98%. In another embodiment, the fraction is 99%. 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 i.e., uridine, cytidine, guanosine, or adenosine
  • the fraction of modified residues is 0.2%.
  • the fraction is 0.3%.
  • the fraction is 0.4%.
  • the fraction is 0.5%.
  • the fraction is 0.6%. In another embodiment, the fraction is 0.7%. In another embodiment, the fraction is 0.8%. In another embodiment, the fraction is 0.9%. 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%. In another embodiment, the fraction is 5%. In another embodiment, the fraction is 6%. In another embodiment, the fraction is 7%. In another embodiment, the fraction is 8%. In another embodiment, the fraction is 9%. 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%.
  • 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%. Tn 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 55%. In another embodiment, the fraction is 60%. In another embodiment, the fraction is 65%. In another embodiment, the fraction is 70%. In another embodiment, the fraction is 75%. In another embodiment, the fraction is 80%. In another embodiment, the fraction is 85%. In another embodiment, the fraction is 90%. In another embodiment, the fraction is 91%. In another embodiment, the fraction is 92%. In another embodiment, the fraction is 93%.
  • the fraction is 94%. In another embodiment, the fraction is 95%. In another embodiment, the fraction is 96%. In another embodiment, the fraction is 97%. In another embodiment, the fraction is 98%. In another embodiment, the fraction is 99%. In another embodiment, the fraction is 100%. In another embodiment, 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%.
  • 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 present 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 4-fold factor.
  • translation is enhanced by a 5-fold factor.
  • translation is enhanced by a 6- fold factor.
  • translation is enhanced by a 7-fold factor.
  • translation is enhanced by a 8-fold factor.
  • translation is enhanced by a 9-fold factor.
  • translation is enhanced by a 10-fold factor. In another embodiment, translation is enhanced by a 15-fold factor. In another embodiment, translation is enhanced by a 20-fold factor. In another embodiment, 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.
  • 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 nucleoside-modified RNA of the present invention exhibits significantly less innate immunogenicity than an unmodified in vitro-synthesized RNA molecule of the same sequence.
  • the modified RNA molecule exhibits an innate immune response that is 2-fold less than its unmodified counterpart.
  • innate immunogenicity is reduced by a 3-fold factor.
  • innate immunogenicity is reduced by a 4-fold factor.
  • innate immunogenicity is reduced by a 5-fold factor.
  • innate immunogenicity is reduced by a 6-fold factor.
  • innate immunogenicity is reduced by a 7-fold factor.
  • innate immunogenicity is reduced by a 8-fold factor.
  • innate immunogenicity is reduced by a 9-fold factor. In another embodiment, innate immunogenicity is reduced by a 10-fold factor. In another embodiment, innate immunogenicity is reduced by a 15-fold factor. In another embodiment, innate immunogenicity is reduced by a 20-fold factor. In another embodiment, innate immunogenicity is reduced by a 50-fold factor. In another embodiment, innate immunogenicity is reduced by a 100-fold factor. In another embodiment, innate immunogenicity is reduced by a 200-fold factor. In another embodiment, innate immunogenicity is reduced by a 500-fold factor. In another embodiment, innate immunogenicity is reduced by a 1000-fold factor. In another embodiment, innate immunogenicity is reduced by a 2000-fold factor. In another embodiment, innate immunogenicity is reduced by another fold difference.
  • “exhibits significantly less innate immunogenicity” refers to a detectable decrease in innate immunogenicity.
  • the term refers to a fold decrease in innate immunogenicity (e.g., 1 of the fold decreases enumerated above).
  • the term refers to a decrease such that an effective amount of the nucleoside- modified RNA can be administered without triggering a detectable innate immune response.
  • the term refers to a decrease such that the nucleoside-modified RNA can be repeatedly administered without eliciting an innate immune response sufficient to detectably reduce production of the protein encoded by the modified RNA.
  • the decrease is such that the nucleoside-modified RNA can be repeatedly administered without eliciting an innate immune response sufficient to eliminate detectable production of the protein encoded by the modified RNA.
  • 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.
  • 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 nonhuman 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 intracameral, 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, intracameral injection, as well as intraocular, intravitreal, subcutaneous, intraperitoneal, intramuscular, intradermal, intrasternal 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.
  • a suitable vehicle e.g. sterile pyrogen-free water
  • 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 parent erally-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.
  • Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer systems.
  • 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.
  • 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.
  • a suitable vehicle e.g., sterile pyrogen-free water
  • 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.
  • Other parentally-administrable formulations that are useful include those that comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system.
  • 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 (1 85, Genaro, ed., Mack Publishing Co., Easton, PA), which is incorporated herein by reference.
  • the present invention provides a method of regenerating corneal endothelium in a subject in need thereof.
  • the method comprises administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a lipid nanoparticle (LNP) and at least one pharmaceutically acceptable carrier, diluent, and/or excipient, wherein the LNP comprises: (a) at least one nucleoside-modified ribonucleic acid (RNA) encoding at least one pro-growth factor, wherein each pro-growth factor is encoded by a distinct nucleoside- modified RNA; and (b) at least one ionizable lipid; wherein the LNP regenerates corneal endothelium in the subject.
  • the administering comprises intracameral injection.
  • the at least one nucleoside-modified RNA is messenger RNA (mRNA). In some embodiments, the at least one nucleoside-modified RNA comprises pseudouridine and/or 1-methyl-pseudouridine. In some embodiments, the at least one nucleoside- modified RNA is in vitro transcribed (IVT) RNA. In certain embodiments, the at least one nucleoside-modified RNA is IVT mRNA comprising pseudouridine and/or 1-methyl- pseudouridine.
  • mRNA messenger RNA
  • the at least one nucleoside-modified RNA comprises pseudouridine and/or 1-methyl-pseudouridine. In some embodiments, the at least one nucleoside- modified RNA is in vitro transcribed (IVT) RNA. In certain embodiments, the at least one nucleoside-modified RNA is IVT mRNA comprising pseudouridine and/or 1-methyl- pseudouridine.
  • the at least one ionizable lipid encapsulates the at least one nucleoside-modified RNA. In certain embodiments, the at least one ionizable lipid is a cationic lipid. Non-limiting examples of cationic lipids are described herein.
  • the LNP further comprises at least one surface-linked antibody, wherein the at least one surface-linked antibody targets a corneal endothelium cell surface marker.
  • the corneal endothelium cell surface marker is selected from N- cadherin, NCAM (CD56), connexin 43, integrin a3pi, integrin b5, VDAC3, aquaporin 1, chloride channel 3, SLC4A4, CD44, GPC4, and any combination thereof.
  • regenerating corneal endothelium in a subject in need thereof comprises inducing mitosis in at least 5%, at least 10%, at least 15%, or at least 20% of corneal endothelium cells in the subject.
  • the subject has vision loss due to a corneal endothelium disease or disorder (e.g., age-related corneal degeneration, Fuchs’ endothelial corneal dystrophy, and/or cataract surgical injury).
  • regenerating corneal endothelium in the subject treats the vision loss in the subject.
  • the subject is at risk of developing vision loss due to a corneal endothelium disease or disorder (e.g., age-related corneal degeneration, Fuchs’ endothelial corneal dystrophy, and/or cataract surgical injury).
  • a corneal endothelium disease or disorder e.g., age-related corneal degeneration, Fuchs’ endothelial corneal dystrophy, and/or cataract surgical injury.
  • regenerating corneal endothelium in the subject prevents the vision loss in the subject.
  • the subject is a human.
  • the at least one pro-growth factor comprises any one or more of cyclin-dependent kinase 4 (CDK4), cyclin DI (CCND1), sex determining region Y-box 2 (SOX2), c-myc transcription factor (MYC), and yes-associated protein 1 (YAP).
  • CDK4 cyclin-dependent kinase 4
  • CCND1 cyclin DI
  • SOX2 sex determining region Y-box 2
  • MYC c-myc transcription factor
  • YAP yes-associated protein 1
  • the at least one pro-growth factor comprises CDK4, CCND1, SOX2, MYC, and YAP.
  • the at least one pro-growth factor consists of CDK4, CCND1, SOX2, MYC, and YAP.
  • the at least one pro-growth factor comprises any one or more of CDK4, CCND1, SOX2, MYC, YAP, NANOG, OCT4, SIRT1, an activating ligand of SIRT1, CTNND1, CTNNB1, ZONAB, E2F2, FGF1, FGF10, IGF1, IGF2, Col8al, Col8a2, Col4a3, Col4a4, SLC4al l, Aqpl, and WWTR1.
  • the administering comprises administering a first dose. In certain embodiments, the administering further comprises administering one or more subsequent doses.
  • 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 encoding at least one pro-growth factor described herein to practice the methods of the invention.
  • the pharmaceutical compositions useful for practicing the invention may be administered to deliver a dose of from 1 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 present invention from 10 nM and 10 mM 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.
  • the invention further provides a kit for regenerating corneal endothelium in a subject in need thereof, the kit comprising (i) a pharmaceutical composition comprising a lipid nanoparticle (LNP) and at least one pharmaceutically acceptable carrier, diluent, and/or excipient, wherein the LNP comprises: (a) at least one nucleoside-modified ribonucleic acid (RNA) encoding at least one pro-growth factor, wherein each pro-growth factor is encoded by a distinct nucleoside-modified RNA; and (b) an ionizable lipid; wherein the LNP is capable of regenerating corneal endothelium in the subject; and (ii) instructional material(s) for intracam eral injection of a therapeutically effective amount of the pharmaceutical composition to the subject.
  • the instructional material(s) included in the kit further comprises instructions for carrying out the method of the invention for regenerating corneal endothelium in a subject in need thereof.
  • Example 1 mRNA-mediated induced regeneration of the corneal endothelium Materials and Methods
  • mice that were used in this study were bred for multiple generations into a Crl:CDl(ICR) mixed background.
  • Mice were housed in a temperature and light-controlled environment and received food and water ad libitum. Up to 5 mice of the same sex and similar age were housed in a cage. Mice were provided Bed-o’Cobs (The Andersons Lab Bedding), a porous cob material, as bedding and Shred-n’Rich nestlets (The Andersons Lab Bedding) for nesting and enrichment.
  • mice for intravital imaging of the eye was performed with the following amendments to the previously described protocol (Rompolas et al. Science 2016 Jun 17;352(6292): 1471-4).
  • Mice were initially anesthetized with IP injection of ketamine/xylazine cocktail (0.1 ml / 20 g body weight; 87.5 mg / kg Ketamine, 12.5 mg / kg Xylazine).
  • a deep plane of anesthesia was verified by checking pedal reflexes.
  • the mouse head was stabilized with a custom-made stereotaxic apparatus that includes palate bar and nose clamp but no ear bars. Precision, 3-axis micro-manipulators are used to adjust the head tilt so that the eye to be imaged is facing up.
  • a drop of eye gel (0.3 % Hypromellose) was used as an optically neutral interface between the eye and a glass coverslip, and to prevent dryness and irritation to the tissue during the anesthesia and imaging procedure.
  • the stage is placed on the microscope platform under the objective lens.
  • a heating pad is used to keep a stable body temperature and vaporized isoflurane is delivered through a nose cone to maintain anesthesia for the duration of the imaging process.
  • the eyes were rinsed with PBS and the mice were monitored and allowed to recover in a warm chamber before returned to the housing facility.
  • Image acquisition was performed with an upright Olympus FV1200MPE microscope, equipped with a Chameleon Vision II Ti: Sapphire laser.
  • the laser beam was focused through 10X, 20X or 25X objective lenses (Olympus UPLSAPO10X2, N.A. 0.40; UPLSAPO20X, N.A. 0.75; XLPLN25XWMP2, N.A. 1 .05).
  • An emitted fluorescence was collected by two multi-alkali and two gallium arsenide phosphide (GaAsP) non-descanned detectors (NDD).
  • GaAsP gallium arsenide phosphide
  • NDD1 419-458 nm NDD1 458-495 nm
  • GaAsP- NDDl 495-540 nm GaAsP -NDD2 575-630 nm.
  • GFP and Tomato reporters were excited at 930 nm and their signal was collected by GaAsP-NDDl and GaAsP -NDD2, respectively.
  • Second harmonic generation (SHG) signal was generated using 850 nm or 930 nm excitation wavelengths and detected by NDD1 or NDD2, respectively.
  • Serial optical sections were acquired in 2-5 pm steps, starting from the surface of the eye and capturing the entire thickness of the cornea (epithelium ⁇ 40 pm, stroma/endothelium ⁇ 80 pm). Expanded views of the cornea and limbus were obtained by acquiring a grid of sequential optical fields-of-view that were automatically stitched into one high-resolution tiled image using the microscope manufacturer software. Multi-day tracing experiments were done by re-imaging the same field-of-view or the entire eye at the indicated times after the initial acquisition. For each time point, inherent landmarks within the cornea, including the organization of the vasculature and collagen fibers (SHG), were used to consistently identify the limbus and navigate back to the original regions. Macroscopic images of the mouse eye were acquired under brightfield and fluorescence with an Olympus MVX10 Fluorescent Macro Zoom microscope fitted with Hamamatsu Orca CCD camera for digital imaging.
  • Photo-labeling experiments with the R26 PAGFP reporter mice were carried out with the same equipment and imaging setup as used for acquisition.
  • the pre-activated form of the fluorescent proteins was visualized by exciting with 850 nm wavelength and emission signal was collected in GaAsP-NDDl (495-540 nm). Excitation with 930 nm verified that no signal is emitted by the reporters before activation.
  • Photo-labeling was achieved by scanning a defined region-of-interest (ROI) at the plane of the basal layer of the epithelium, with the laser tuned to 750 nm wavelength, for 5-10 sec, using 5-10% laser power.
  • ROI region-of-interest
  • the z-plane was then moved down to the corneal endothelium, which served as a reference, and the same ROI was used for photolabeling cells in that layer.
  • a series of optical sections with a range that includes the entire thickness of the cornea, were acquired using the same acquisition settings as for GFP.
  • Visualizing the signal of the activated form of PAGFP only within the ROI confirmed the successful photo-labeling of basal epithelial or endothelial cells.
  • the same eyes were re-imaged at the indicated times to evaluate the changes of the labeled epithelial population and their movements compared to the endothelial reference cell group.
  • Corneal epithelial debridement wounds were generated as previously described (Chan and Werb 2015). Mice were initially anesthetized with IP injection of ketamine/xylazine cocktail and their eyes imaged under brightfield and fluorescence microscopy. Mice where then placed on a heating pad and observed under an Olympus SZ61 dissecting microscope. After topical application of Proparacaine the epithelium from the central part of the cornea was removed with an Algerbrush II ophthalmic brush to generate a scrape wound of 1.5 mm in diameter. The eyes were imaged again before the mice were allowed to recover.
  • mice ranging from 3-22 months of age mice were used for this procedure.
  • 1-2 drops of topical .5% proparacaine eye drops were instilled to each eye before the procedure.
  • Mice were initially anesthetized with IP injection of ketamine/xylazine cocktail and kept warm using a temperature-controlled heating pad throughout the whole procedure. A deep plane of anesthesia was verified by checking pedal reflexes.
  • the mouse head was stabilized by means of a palate bar and nose clamp and using a special sterotaxic apparatus that does not require the use of ear bars. Using precision micro-manipulators, the head angle was adjusted so that the treated eye was facing upwards.
  • the pupils were dilated with .5% tropicamide ophthalmic drops and the treated eye was examined under a stereomicroscope to ensure that the pupil is fully dilated and that the ocular muscles are relaxed so that there is no eye movement. The absence of eye movements ensures stability during the injection.
  • a diluted betadine solution was applied topically using a 1ml syringe to the ocular surface and fomices.
  • a custom-made microinjection apparatus was used that attaches directly to the same heated mounting platform that is used for intravital imaging.
  • the microinjector is composed of a hydraulic pump (Eppendorf CellTram Oil) connected to a 1mm inner diameter PTFE flexible tubing.
  • a fitting adaptor connects the tubing to a single-use, sterile 31 -gauge insulin pen needle (CareTouch).
  • the microneedle When attached to the tubing the microneedle is connected to a 3-axis manual micromanipulator to precisely control its movement during the injection.
  • a 5 pl solution is loaded into the syringe, ensuring that no air bubbles enter the system or are present at the tip of the needle.
  • the needle is guided to the mouse eye at a 45° angle, anteriorly relative to the limbus.
  • the cornea is gently punctured so that the tip of the microneedle enters the anterior chamber, ensuring that the loaded microneedle remains at a 45° angle relative to the limbus during the puncture. Contact with the iris or the lens should be avoided. During this process the eye may need to be supported with a pair of disposable plastic tweezers from the opposite side. Using the microsyringe pump, ⁇ 1.5 pl of solution is injected into the anterior chamber. This amount is injected gradually over 30 seconds before withdrawing the microneedle.
  • Antibiotic ointment (Bacitracin Zinc and Polymyxin B Sulfate Ophthalmic Ointment USP) was applied to the treated eyes as prophylactic.
  • constructs for the in vitro transcription (IVT) of cdk4, ccndl, myc, sox2 and yap- encoding mRNAs were custom designed and synthesized by VectorBuilder using a template plasmid carrying a T7 promoter.
  • a 5’ aptamer sequence for eIF4G binding was inserted between the T7 promoter and the start codon to optimize ribosome binding and translation initiation [Kariko, et al. Immunity.
  • IVT-mRNA was then cellulose-purified to remove dsRNA products from the reaction.
  • 293 T cells were cultured in 10% HI-FBS/1% PennStrep, then transfected with either luciferase IVT-mRNA or test IVT-mRNA. Cells were collected in cold DPBS and pelleted. Cell pellets were lysed with lx RIPA + protease inhibitor cocktail.
  • FIG. 14A shows a schematic of the mRNAs. Sequences for the 5’ UTR, 3’ UTR, and murine ORFs of the constructs encoding the mRNAs, and amino acid sequences of the translated murine ORFs, are provided in Table 1.
  • the filtered suspension was centrifuged at 400 g for 10 minutes at 4 °C. The supernatant was discarded. The cell pellet was washed twice with 0.04% bovine serum albumin (BSA) in IX PBS, centrifuging at 300 g for 5 minutes at 4 °C and the final pellet was resuspended on 40 pL of 2% BSA-PBS.
  • BSA bovine serum albumin
  • Objects were scaled, normalized, and the top 2500 variable genes were identified for principal component analysis (PC A).
  • PC A principal component analysis
  • UMAP Uniform Manifold Approximation and Projection
  • the three age objects were integrated according to the Seurat vignette [Stuart, et al., Cell. 2019 Jun 13;177(7): 1888- 1902. e21 ] .
  • Optimal clustering of cells was performed using the assistance of the clustree R package [Zappia, et al., Gigascience.
  • Volcano plots were created using the R package EnhancedVolcano using differentially expressed gene lists generated by Seurat’s FindMarker function (Wilcoxon Rank Sum tests with P ⁇ 0.05) [Blighe, et al., EnhancedVolcano: publication-ready volcano plots with enhanced colouring and labeling. 2020. R package version 1.8. 0],
  • Raw digital files from 2-photon imaging were acquired and saved in the OIB format using the microscope manufacturer’s software (FLUOVIEW, Olympus USA).
  • FLUOVIEW Olympus USA
  • a tiling method was used to reconstruct a single image from multiple full-thickness serial optical sections using the microscope acquisition software.
  • the microscope defines a square area consisting of 2 x 2 (XY) field-of-view with 10% overlap between them.
  • the microscope automatically acquires the four fields-of-view in a sequential pattern and uses information from the overlapping margins to stitch the individual field-of-view into a single image.
  • Raw image files were imported into ImageJ/Fiji (NIH) using Bio-Formats or to Imaris (Bitplane) for further analysis.
  • NIH ImageJ/Fiji
  • Bio-Formats or to Imaris Billplane
  • supervised image segmentation and blob detection was performed on individual optical sections. Identified blobs were manually validated and their number, size and signal intensity as mean grey values were measured.
  • LNP-mRNA lipid nanoparticle modified mRNA
  • corneal endothelial dystrophies include: 1) a gradual cell loss, and corresponding changes in the shape and size of individual cells with age, and 2) the appearance of guttae-like lesions; extracellular outgrowths that interrupt the lateral contacts between endothelial cells [Borboli and Colby. Ophthalmol Clin North Am. 2002 Mar;15(l): 17-25; Cibis, et al. Arch Ophthalmol. 1978 Mar;96(3):485-8], Live imaging of mouse corneas during different stages of adulthood, showed a consistent cell loss, accompanied by aberrant cellular morphology (polymegathism and pleomorphism), that correlated with age (FIGs. IB - IE).
  • FIG. IB, 1F-1 J Another unique feature of aged corneas revealed by this analysis, was a pronounced change in nuclear shape and size (FIGs. IB, 1F-1 J), which may indicate variations in chromatin organization and gene expression, as previously proposed for other cell types.
  • corneal endothelial cells Differential gene expression analysis of the identified corneal endothelial cells between young and aged mice revealed changes in key genes (FIGs. II, 4B). Specifically, several unique gene markers related to corneal endothelial cell function, such as Atpbl, Col8a2, Col4al, and Slc4al 1, were all downregulated in aged mice. Conversely, corneal endothelial cells from aged corneas upregulated keratin genes, including Krt5, and Krtl2, which are normally not associated with corneal endothelial cell identity. Taken together these experiments revealed a decline of the corneal endothelium during physiological aging, which is associated with changes in gene expression and results in progressive cell loss.
  • mice that express a cell-cycle reporter (CyclinBl-GFP) were used to capture proliferating cells during the tissue repair, by live imaging. The specificity and faithful kinetics of the reporter was validated by imaging the mitotically active corneal epithelium (FIGs. 8A-8B).
  • mRNAs When delivered systemically, using lipid nanoparticle vehicles, mRNAs are endocytosed primarily by hepatocytes, but localized induced expression has been previously demonstrated when mRNAs are injected in situ. It was contemplated herein that local delivery of modified mRNA would be especially advantageous for the corneal endothelium, due to the protected environment of the anterior eye chamber and the relatively slow turnover of the aqueous humor. In principle, these conditions would aid the retention and endocytosis of the mRNA cargo by corneal endothelial cells. To test this, GFP-encoding mRNA was directly (intracamerally) injected in the eyes of wild type mice and expression was validated by live imaging (FIGs. 9A, 10A-10C).
  • in vivo lineage tracing was performed by longitudinal live imaging. For this, mice harboring a fluorescent Cre reporter were used. Recombination and clonal labeling were induced by local injection of modified Cre- encoding mRNA (FIG. 9C). Imaging of the corneas at 24 hours after injection confirmed Cre- induced recombination and labeling of corneal endothelial cells (FIG. 9D). Furthermore, no new recombination events were observed after 24 hours, indicating that mRNA uptake is limited within this time window.
  • corneas After injection of the Cre-encoding mRNA, the corneas were imaged to document the location of labeled cells, and then re-imaged after two months, which confirmed that the tissue was fully quiescent, with no evidence of proliferation or other changes to the corneal endothelial cells during this time (FIG. 12B).
  • definitive evidence is provided herein that corneal endothelial cells are activated and can undergo one or more cycles of cell division before returning into a quiescent state (FIGs. 9E, 12B).
  • activation of corneal endothelial cells occurs exclusively at the wound margins, while the rest of the tissue is maintained in a quiescent state throughout the repair process and thereafter (FIGs.
  • a promising therapeutic strategy to revert the disease progression and restore vision is to modulate intrinsic molecular pathways to promote corneal endothelial cell proliferation in the absence of any injury.
  • Modified mRNA technology has made it possible to transiently modulate the protein expression program of cells in vivo, offering an appealing vehicle for the implementation of this therapeutic strategy.
  • a therapeutic strategy to reprogram corneal endothelial cells into a pro-regenerative state without injury was developed herein, and the live imaging system was used to validate its efficacy. It is contemplated herein that by forcing exogenous expression of key enzymes and transcription factors, one can activate the relevant signaling pathways to induce the corneal endothelial cells to undergo mitosis.
  • Cdk4 and Ccndl(cyclin DI) typically form a complex that promotes progression of the cell cycle.
  • Myc and Sox2 - two of the Yamanaka factors for somatic cell re-programming - are known to promote proliferation in various cell types, including in human corneal endothelial cells in vitro and in a rat cryoinjury model in vivo.
  • Yap is a downstream effector of the Hippo pathway and critical for mediating contact inhibition growth, which is hypothesized to be a major driver of cellular quiescence in the corneal endothelium.
  • FIG. 15A To test the efficacy of the CCMSY factors to induce proliferation of corneal endothelial cells in vivo, and to document any potential adverse effects, concurrent lineage tracing of the cells that uptake and express the CCMSY mRNAs after eye injection was performed (FIG. 15A). To validate this assay, injection of a mix of different mRNAs that encode traceable reporters (GFP + mCherry or GFP + Cre-recombinase) was first performed. It was confirmed that cells which expressed one reporter always expressed the other. This indicates that a mix of encapsulated mRNA molecules is equally taken up and expressed by the same corneal endothelial cells in vivo (FIG. IOC).
  • GFP + mCherry or GFP + Cre-recombinase traceable reporters
  • a Cre-encoding mRNA injected in a Cre reporter mouse line was used as a surrogate to label and track the corneal endothelial cells that express the CCMSY factors.
  • cells that received the Cre + CCMSY mRNAs underwent mitosis (FIGs. 15B-15D, 16A-16C).
  • This spur of proliferation led to a measurable increase in corneal endothelial cell density effectively reversing the corneas of one-year-old mice to that of a three-month-old (FIGs. 15E-15H).
  • Control corneas that only received the Cre mRNA remained virtually unchanged with no evidence of proliferation, consistent with the previous lineage tracing experiments (FIGs.
  • EEU 5-ethynyl-2’-deoxyuridine
  • FIG. 18B Only sparse cells were observed to retain EDU in the mCherry only control mice (FIG. 18B).
  • modified mRNA-based therapies for corneal endothelial dysfunction is advantageous, due to the lack of genomic integration and inherent instability and short half-life of mRNAs in the cytoplasm. This is in line with the present data showing a transient uptick in cell proliferation during the first 24 hours after injection, and a quick return to quiescence.
  • stability and performance of therapeutic mRNAs can be improved, by 5’ and 3’ sequence optimizations, synthetic capping, and incorporation of modified nucleosides as is well-known in the art.
  • Embodiment 1 A lipid nanoparticle (LNP), wherein the LNP comprises:
  • RNA nucleoside-modified ribonucleic acid
  • Embodiment 2 The LNP of embodiment 1, wherein the at least one pro-growth factor comprises any one or more of cyclin-dependent kinase 4 (CDK4), cyclin DI (CCND1), sex determining region Y-box 2 (SOX2), c-myc transcription factor (MYC), and yes-associated protein 1 (YAP).
  • CDK4 cyclin-dependent kinase 4
  • CCND1 cyclin DI
  • SOX2 sex determining region Y-box 2
  • MYC c-myc transcription factor
  • YAP yes-associated protein 1
  • Embodiment 3 The LNP of embodiment 1 or 2, wherein the LNP further comprises at least one surface-linked antibody, wherein the at least one surface-linked antibody targets a corneal endothelium cell surface marker.
  • Embodiment 4 The LNP of any previous embodiment, wherein the at least one nucleoside-modified RNA is messenger RNA (mRNA).
  • mRNA messenger RNA
  • Embodiment 5 The LNP of any previous embodiment, wherein the at least one nucleoside-modified RNA comprises pseudouridine and/or 1-methyl-pseudouridine.
  • Embodiment 6 The LNP of any previous embodiment, wherein the at least one nucleoside-modified RNA is in vitro transcribed (IVT) RNA.
  • IVTT in vitro transcribed
  • Embodiment 7 The LNP of any previous embodiment, wherein the at least one progrowth factor comprises CDK4, CCND1, SOX2, MYC, and YAP.
  • Embodiment 8 The LNP of any previous embodiment, wherein the at least one progrowth factor consists of CDK4, CCND1, SOX2, MYC, and YAP.
  • Embodiment 9 The LNP of any previous embodiment, wherein the corneal endothelium cell surface marker is selected from N-cadherin, NCAM (CD56), connexin 43, integrin a3pi, integrin b5, VDAC3, aquaporin 1, chloride channel 3, SLC4A4, CD44, GPC4, and any combination thereof.
  • the corneal endothelium cell surface marker is selected from N-cadherin, NCAM (CD56), connexin 43, integrin a3pi, integrin b5, VDAC3, aquaporin 1, chloride channel 3, SLC4A4, CD44, GPC4, and any combination thereof.
  • Embodiment 10 The LNP of any previous embodiment, wherein the at least one ionizable lipid encapsulates the at least one nucleoside-modified RNA.
  • Embodiment 11 The LNP of any previous embodiment, wherein the at least one ionizable lipid is a cationic lipid.
  • Embodiment 12 The LNP of any previous embodiment, wherein one or more of the following applies:
  • the at least one pro-growth factor comprises or consists of CDK4, and further wherein the CDK4 comprises SEQ ID NO: 2 or SEQ ID NO: 12;
  • the at least one pro-growth factor comprises or consists of CCND1, and further wherein the CCND1 comprises SEQ ID NO: 4 or SEQ ID NO: 14;
  • the at least one pro-growth factor comprises or consists of SOX2, and further wherein the SOX2 comprises SEQ ID NO: 6 or SEQ ID NO: 16;
  • the at least one pro-growth factor comprises or consists of MYC, and further wherein the MYC comprises SEQ ID NO: 8 or SEQ ID NO: 18; and (v) the at least one pro-growth factor comprises or consists of YAP, further wherein the YAP comprises SEQ ID NO: 10 or SEQ ID NO: 20.
  • Embodiment 13 The LNP of any previous embodiment, wherein regenerating corneal endothelium in the subject comprises inducing mitosis in at least 5%, at least 10%, at least 15%, or at least 20% of corneal endothelium cells in the subject.
  • Embodiment 14 The LNP of any previous embodiment, wherein the subject has vision loss due to a corneal endothelium disease or disorder (e.g., age-related corneal degeneration, Fuchs’ endothelial corneal dystrophy, and/or cataract surgical injury).
  • a corneal endothelium disease or disorder e.g., age-related corneal degeneration, Fuchs’ endothelial corneal dystrophy, and/or cataract surgical injury.
  • Embodiment 15 The LNP of any previous embodiment, wherein regenerating corneal endothelium in the subject treats the vision loss in the subject.
  • Embodiment 16 The LNP of any previous embodiment, wherein the subject is a human.
  • Embodiment 17 A pharmaceutical composition comprising the LNP of any previous embodiment and at least one pharmaceutically acceptable carrier, diluent, and/or excipient.
  • Embodiment 18 The LNP of any previous embodiment, for use in a method of regenerating corneal endothelium in the subject.
  • Embodiment 19 A method of regenerating corneal endothelium in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a lipid nanoparticle (LNP) and at least one pharmaceutically acceptable carrier, diluent, and/or excipient, wherein the LNP comprises:
  • RNA nucleoside-modified ribonucleic acid
  • Embodiment 20 The method of any previous embodiment, wherein the administering comprises intracameral injection.
  • Embodiment 21 The method of any previous embodiment, wherein the at least one progrowth factor comprises any one or more of cyclin-dependent kinase 4 (CDK4), cyclin DI (CCND1), sex determining region Y-box 2 (SOX2), c-myc transcription factor (MYC), and yes- associated protein 1 (YAP).
  • CDK4 cyclin-dependent kinase 4
  • CCND1 cyclin DI
  • SOX2 sex determining region Y-box 2
  • MYC c-myc transcription factor
  • YAP yes- associated protein 1
  • Embodiment 22 The method of any previous embodiment, wherein the LNP further comprises at least one surface-linked antibody, wherein the at least one surface-linked antibody targets a corneal endothelium cell surface marker.
  • Embodiment 23 The method of any previous embodiment, wherein the at least one nucleoside-modified RNA is messenger RNA (mRNA).
  • mRNA messenger RNA
  • Embodiment 24 The method of any previous embodiment, wherein the at least one nucleoside-modified RNA comprises pseudouridine and/or 1-methyl-pseudouridine.
  • Embodiment 25 The method of any previous embodiment, wherein the at least one nucleoside-modified RNA is in vitro transcribed (IVT) RNA.
  • IVT in vitro transcribed
  • Embodiment 26 The method of any previous embodiment, wherein the at least one progrowth factor comprises CDK4, CCND1, SOX2, MYC, and YAP.
  • Embodiment 27 The method of any previous embodiment, wherein the at least one progrowth factor consists of CDK4, CCND1, SOX2, MYC, and YAP.
  • Embodiment 28 The method of any previous embodiment, wherein the corneal endothelium cell surface marker is selected from N-cadherin, NCAM (CD56), connexin 43, integrin a3pi, integrin b5, VDAC3, aquaporin 1, chloride channel 3, SLC4A4, CD44, GPC4, and any combination thereof.
  • the corneal endothelium cell surface marker is selected from N-cadherin, NCAM (CD56), connexin 43, integrin a3pi, integrin b5, VDAC3, aquaporin 1, chloride channel 3, SLC4A4, CD44, GPC4, and any combination thereof.
  • Embodiment 29 The method of any previous embodiment, wherein the at least one ionizable lipid encapsulates the at least one nucleoside-modified RNA.
  • Embodiment 30 The method of any previous embodiment, wherein the at least one ionizable lipid is a cationic lipid.
  • Embodiment 31 The method of any previous embodiment, wherein one or more of the following applies:
  • the at least one pro-growth factor comprises or consists of CDK4, and further wherein the CDK4 comprises SEQ ID NO: 2 or SEQ ID NO: 12;
  • the at least one pro-growth factor comprises or consists of CCND1, and further wherein the CCND1 comprises SEQ ID NO: 4 or SEQ ID NO: 14;
  • the at least one pro-growth factor comprises or consists of SOX2, and further wherein the SOX2 comprises SEQ ID NO: 6 or SEQ ID NO: 16;
  • the at least one pro-growth factor comprises or consists of MYC, and further wherein the MYC comprises SEQ ID NO: 8 or SEQ ID NO: 18;
  • the at least one pro-growth factor comprises or consists of YAP, further wherein the YAP comprises SEQ ID NO: 10 or SEQ ID NO: 20.
  • Embodiment 32 The method of any previous embodiment, wherein regenerating corneal endothelium in a subject in need thereof comprises inducing mitosis in at least 5%, at least 10%, at least 15%, or at least 20% of corneal endothelium cells in the subject.
  • Embodiment 33 The method of any previous embodiment, wherein the administering comprises administering a first dose.
  • Embodiment 34 The method of any previous embodiment, wherein the administering further comprises administering one or more subsequent doses.
  • Embodiment 35 The LNP of any previous embodiment, wherein the subject has vision loss due to a corneal endothelium disease or disorder (e.g., age-related corneal degeneration, Fuchs’ endothelial corneal dystrophy, and/or cataract surgical injury).
  • a corneal endothelium disease or disorder e.g., age-related corneal degeneration, Fuchs’ endothelial corneal dystrophy, and/or cataract surgical injury.
  • Embodiment 36 The LNP of any previous embodiment, wherein regenerating corneal endothelium in the subject treats the vision loss in the subject.
  • Embodiment 37 The method of any previous embodiment, wherein the subject is a human.
  • Embodiment 38 A kit for regenerating corneal endothelium in a subject in need thereof, the kit comprising:
  • a pharmaceutical composition comprising a lipid nanoparticle (LNP) and at least one pharmaceutically acceptable carrier, diluent, and/or excipient, wherein the LNP comprises: (a) at least one nucleoside-modified ribonucleic acid (RNA) encoding at least one pro-growth factor, wherein each pro-growth factor is encoded by a distinct nucleoside-modified RNA; and (b) an ionizable lipid; wherein the LNP is capable of regenerating corneal endothelium in the subject; and
  • instructional material(s) for intracameral injection of a therapeutically effective amount of the pharmaceutical composition to the subject (ii) instructional material(s) for intracameral injection of a therapeutically effective amount of the pharmaceutical composition to the subject.
  • Embodiment 39 The kit of any previous embodiment, wherein the at least one progrowth factor comprises any one or more of cyclin-dependent kinase 4 (CDK4), cyclin DI (CCND1), sex determining region Y-box 2 (SOX2), c-myc transcription factor (MYC), and yes- associated protein 1 (YAP).
  • CDK4 cyclin-dependent kinase 4
  • CCND1 cyclin DI
  • SOX2 sex determining region Y-box 2
  • MYC c-myc transcription factor
  • YAP yes- associated protein 1
  • Embodiment 40 The kit of any previous embodiment, wherein the LNP further comprises at least one surface-linked antibody, wherein the at least one surface-linked antibody targets a corneal endothelium cell surface marker.
  • Embodiment 41 The kit of any previous embodiment, wherein the at least one nucleoside-modified RNA is messenger RNA (mRNA).
  • mRNA messenger RNA
  • Embodiment 42 The kit of any previous embodiment, wherein the at least one nucleoside-modified RNA comprises pseudouridine and/or 1-methyl-pseudouridine.
  • Embodiment 43 The kit of any previous embodiment, wherein the at least one nucleoside-modified RNA is in vitro transcribed (IVT) RNA.
  • IVT in vitro transcribed
  • Embodiment 44 The kit of any previous embodiment, wherein the at least one progrowth factor comprises CDK4, CCND1, SOX2, MYC, and YAP.
  • Embodiment 45 The kit of any previous embodiment, wherein the at least one progrowth factor consists of CDK4, CCND1, SOX2, MYC, and YAP.
  • Embodiment 46 The kit of any previous embodiment, wherein the corneal endothelium cell surface marker is selected from N-cadherin, NCAM (CD56), connexin 43, integrin a3pi, integrin b5, VDAC3, aquaporin 1, chloride channel 3, SLC4A4, CD44, GPC4, and any combination thereof.
  • the corneal endothelium cell surface marker is selected from N-cadherin, NCAM (CD56), connexin 43, integrin a3pi, integrin b5, VDAC3, aquaporin 1, chloride channel 3, SLC4A4, CD44, GPC4, and any combination thereof.
  • Embodiment 47 The kit of any previous embodiment, wherein the at least one ionizable lipid encapsulates the at least one nucleoside-modified RNA.
  • Embodiment 48 The kit of any previous embodiment, wherein the at least one ionizable lipid is a cationic lipid.
  • Embodiment 49 The kit of any previous embodiment, wherein one or more of the following applies:
  • the at least one pro-growth factor comprises or consists of CDK4, and further wherein the CDK4 comprises SEQ ID NO: 2 or SEQ ID NO: 12;
  • the at least one pro-growth factor comprises or consists of CCND1, and further wherein the CCND1 comprises SEQ ID NO: 4 or SEQ ID NO: 14;
  • the at least one pro-growth factor comprises or consists of SOX2, and further wherein the SOX2 comprises SEQ ID NO: 6 or SEQ ID NO: 16;
  • the at least one pro-growth factor comprises or consists of MYC, and further wherein the MYC comprises SEQ ID NO: 8 or SEQ ID NO: 18;
  • the at least one pro-growth factor comprises or consists of YAP, further wherein the YAP comprises SEQ ID NO: 10 or SEQ ID NO: 20.
  • Embodiment 50 The kit of any previous embodiment, wherein regenerating corneal endothelium in a subject in need thereof comprises inducing mitosis in at least 5%, at least 10%, at least 15%, or at least 20% of corneal endothelium cells in the subject.
  • Embodiment 51 The LNP of any previous embodiment, wherein the subject has vision loss due to a corneal endothelium disease or disorder (e.g., age-related corneal degeneration, Fuchs’ endothelial corneal dystrophy, and/or cataract surgical injury).
  • a corneal endothelium disease or disorder e.g., age-related corneal degeneration, Fuchs’ endothelial corneal dystrophy, and/or cataract surgical injury.
  • Embodiment 52 The LNP of any previous embodiment, wherein regenerating corneal endothelium in the subject treats the vision loss in the subject.
  • Embodiment 53 The kit of any previous embodiment, wherein the subject is a human.

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Abstract

L'invention concerne la régénération de l'endothélium cornéen pour traiter une perte de vision chez un sujet en ayant besoin. L'invention comprend une nanoparticule lipidique (LNP) capable de régénérer l'endothélium cornéen chez un sujet en ayant besoin. La LNP comprend : (a) au moins un acide ribonucléique (ARN) à modification nucléosidique codant pour au moins un facteur de pro-croissance, chaque facteur de pro-croissance étant codé par un ARN à modification nucléosidique distinct; et (b) au moins un lipide ionisable. L'invention concerne également des compositions pharmaceutiques comprenant la LNP de l'invention, un kit comprenant la composition pharmaceutique, et un procédé de régénération de l'endothélium cornéen chez un sujet en ayant besoin.
PCT/US2023/077845 2022-10-26 2023-10-26 Compositions et procédés pour régénérer l'endothélium cornéen WO2024092086A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20200255859A1 (en) * 2017-07-31 2020-08-13 Reflection Biotechnologies Limited Cellular models of and therapies for ocular diseases
WO2021150891A1 (fr) * 2020-01-24 2021-07-29 The Trustees Of The University Of Pennsylvania Utilisation d'arnm modifié par un nucléoside codant pour un facteur de croissance pour la régénération d'un tissu parodontal et d'un os
WO2022235586A1 (fr) * 2021-05-03 2022-11-10 Astellas Institute For Regenerative Medicine Procédés de génération de cellules endothéliales cornéennes matures

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20200255859A1 (en) * 2017-07-31 2020-08-13 Reflection Biotechnologies Limited Cellular models of and therapies for ocular diseases
WO2021150891A1 (fr) * 2020-01-24 2021-07-29 The Trustees Of The University Of Pennsylvania Utilisation d'arnm modifié par un nucléoside codant pour un facteur de croissance pour la régénération d'un tissu parodontal et d'un os
WO2022235586A1 (fr) * 2021-05-03 2022-11-10 Astellas Institute For Regenerative Medicine Procédés de génération de cellules endothéliales cornéennes matures

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
YUHONG WANG, RAJALA AMMAJI, RAJALA RAJU: "Lipid Nanoparticles for Ocular Gene Delivery", JOURNAL OF FUNCTIONAL BIOMATERIALS, vol. 6, no. 2, 8 June 2015 (2015-06-08), pages 379 - 394, XP055404997, DOI: 10.3390/jfb6020379 *

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