[go: up one dir, main page]

WO2024073042A1 - Ocular delivery of therapeutic agents - Google Patents

Ocular delivery of therapeutic agents Download PDF

Info

Publication number
WO2024073042A1
WO2024073042A1 PCT/US2023/034113 US2023034113W WO2024073042A1 WO 2024073042 A1 WO2024073042 A1 WO 2024073042A1 US 2023034113 W US2023034113 W US 2023034113W WO 2024073042 A1 WO2024073042 A1 WO 2024073042A1
Authority
WO
WIPO (PCT)
Prior art keywords
cyclo
side chain
cargo
pkkkrkv
amino acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2023/034113
Other languages
French (fr)
Inventor
Ziqing QIAN
Haoming Liu
Xiang Li
Patrick Dougherty
Pingjuan LI
Silvia PASINI
Xiulong SHEN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Entrada Therapeutics Inc
Original Assignee
Entrada Therapeutics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Entrada Therapeutics Inc filed Critical Entrada Therapeutics Inc
Publication of WO2024073042A1 publication Critical patent/WO2024073042A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • 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

Definitions

  • ocular therapeutic agents for treating ocular diseases exist or have been proposed.
  • Some ocular therapeutic agents such as oligonucleotides, polypeptides, or intracellularly acting small molecule therapeutics, may have limited effectiveness unless they are delivered to the correct eye location, cell-type, and/or to an intracellular compartment where their effects may be realized.
  • the anatomy and physiology of the eye presents challenges to delivering therapeutic agents to a desired eye location and/or specific cells of the eye.
  • the eye includes static and dynamic barriers that tend to exclude entry of xenobiotics and discourage active absorption of therapeutic agents (see, for example, Gote, et al., J Pharm Exp Ther, September 2019, 370(3): 602-624).
  • Static barriers include tissue that provides a passive physical barrier to therapeutic agent penetration (see Rowe-Rendleman, et al., Invest Opthamol Vis Sci, 2014 Apr, 55(4):2714-2730).
  • Dynamic barriers include physiological clearance mechanisms such as aqueous turnover, vitreous flow, ocular drug metabolism, and eye metabolizing enzymes (see, id).
  • barriers for intraocular administration such as intravitreal injection
  • barriers for intraocular administration includes static barriers such as the vitreous humor and dynamic barriers such as anterior aqueous humor flow, posterior trans-retinal flow, and efflux transporters (see, id).
  • Barriers of suprachoroidal administration such as via suprachoroidal injection, includes static barriers, such as the choriod, Bruch’s membrane, PRE tight junctions, and the retina, as well as dynamic barriers such as choroidal circulation and retinal circulation (see, id).
  • Barriers for periocular administration includes static barriers, such as the sclera, the choriod, Bruch’s membrane, RPE tight junctions, the retina, as well as dynamic barriers such as subconjunctival-episcleral lymph and blood vessels, choriodal circulation, and retinal circulation (see, id).
  • Barriers for topical administration includes static barriers, such as the cornea, the conjunctiva, the sclera, the choriod, Bruch’s membrane, RPE tight junctions, and the retina, as well as dynamic barriers, such as tears and lacrimal drainage, subconjunctival-espisleral lymph and blood vessels, choroidal circulation, and retinal circulation (see, id).
  • a therapeutic agent not only needs to be delivered to an appropriate location of the eye, but also needs to be delivered to the appropriate cells and cellular compartments of the eye.
  • Carrier systems such as polymers, cationic liposomes, or chemical modifications, for example, by the covalent attachment of cholesterol molecules, have been used to facilitate intracellular delivery. Still, intracellular delivery efficiency by these approaches is often low and improved delivery systems to increase efficacy of intracellular delivery have remained elusive.
  • CPPs cell penetrating peptides
  • the present disclosure relates to, among other things, cargo conjugates and compositions containing the same, for delivering a cargo to the eye.
  • the cargo conjugate may include a cyclic cell penetrating peptide and a therapeutic agent.
  • the therapeutic agent may be designed to have a biological effect, for example, to treat a disease of the eye.
  • the cargo conjugates and compositions containing the same may be used to treat ocular diseases.
  • the present di sclosure is related to, among other things, methods of admi ni storing the cargo conjugates and compositions containing the same to a subject in need thereof.
  • the method may include identifying a subject that may benefit from the delivery of a therapeutic agent to the eye.
  • the subject may have, or be at risk of having, an ocular disease.
  • the method may further include delivering the cargo conjugate or composition containing the same directly to the eye.
  • cargo conjugate and/or compositions containing the same are delivered to the surface of the eye.
  • cargo conjugate and/or compositions containing the same are intraocularly injected.
  • cargo conjugate and/or compositions containing the same are injected into the retina.
  • cargo conjugate and/or compositions containing the same are delivered intravitreally.
  • a method of delivering a therapeutic agent to the eye of a subject comprises administering a therapeutically effective amount of a cargo conjugate to the eye of the subject.
  • the cargo conjugate comprises (i) a cargo comprising the therapeutic agent that selectively binds to a target molecule associated with a disease of the eye; and (ii) an ocular delivery' construct comprising an exocyclic peptide (EP), a first cyclic cell penetrating peptide (cCPP) and one or more linkers.
  • EP exocyclic peptide
  • cCPP first cyclic cell penetrating peptide
  • R1, R2, and R3 are each independently H or an aryl or heteroaryl side chain of an amino acid; at least two of R1, R2, and R3 are an aryl or heteroaryl side chain of an amino acid; R4 and Re are independently H or an amino acid side chain; peptide is an exocyclic peptide (EP) comprising from 2 to 10 amino acids ;
  • M is a bonding group; each m is independently an integer from 0-3; n is an integer from 0-2; x' is an integer from 0-20; y is an integer from 1-5; q is 1-4; z' is an integer from 1-23; and
  • Cargo is a therapeutic moiety.
  • the amino acid residue comprising an aryl or heteroaryl group is phenylalanine or 3-(2-naphthyl)-alanine.
  • Ri and Re are, independently, H or a side chain of an amino acid selected from arginine, citrulline, serine or histidine.
  • at least one of R4 and Re are H.
  • at least one of R» and Re are an amino acid side chain of arginine.
  • at least one of R4 and Re are an amino acid side chain of serine.
  • at least one of Rt and Re are an amino acid side chain of histidine.
  • the cCPP of the ocular delivery construct has a sequence selected from: FfORrRrQ, FGFGRGRQ; GfFGrGrQ, FfOGRGRQ; FfFGRGRQ; FfrDGrGrQ; FGFGRRRQ; and FGFRRRRQ.
  • a method of delivering a therapeutic agent to an eye of a subject comprising administering a therapeutically effective amount of a cargo conjugate to the eye of the subject.
  • the cargo conjugate comprises (i) a cargo comprising the therapeutic agent that selectively binds to a target molecule associated with a disease of the eye; and (ii) an ocular delivery construct comprising an exocyclic peptide (EP), a first cyclic cell penetrating peptide (cCPP) and one or more linkers, wherein the ocular delivery construct has the structure:
  • Ri, Rz, and R3 can each independently be H or an amino acid residue having a side chain comprising an aryl or heteroaryl group; at least two of Ri, Rz, and R? is an aryl or heteroaryl side chain of an amino acid;
  • R4, and R6 are independently H or an amino acid side chain
  • M is a bonding group; each m is independently an integer from 0-3; n is an integer from 0-2; x' is an integer from 0-20; y is an integer from 1-5; z' is an integer from 1-23; and
  • Cargo is a therapeutic moiety.
  • the amino acid residue comprising an aryl or heteroaryl group is phenylalanine, beta homophenylalanine, or 3-(2-naphthyl)-alanine.
  • R.4 and Rs are, independently, H or a side chain of an amino acid selected from arginine, citrulline, serine or histidine.
  • at least one of R4 and Rr> are H.
  • at least one of R4 and Re are an amino acid side chain of arginine.
  • at least one of R.4 and Rs are an amino acid side chain of serine.
  • at least one of R» and Rs are an amino acid side chain of histidine.
  • the cCPP of the ocular delivery construct has a sequence selected from: ⁇ hF-FOSRSRQ and ⁇ hF-F ⁇ DGRGRQ.
  • M comprises ,or , wherein t' is 0 to 10.
  • M comprises . In embodiments, M comprises wherein t' is 0 to 10. In embodiments, M comprises w
  • the EP comprises 1 or 2 amino acids comprising a side chain comprising a guanidine group, or a protonated form thereof.
  • the EP comprises 1, 2, 3, or 4 lysine residues.
  • the EP comprises one of the following sequences: PKKKRKV; KR, RR, KKK; KGK; KBK; KBR; KKK; KRR; RKK; RRR; KKKK; KKRK; KRKK; KRRK; RKKR; RRRR; KGKK; KKGK; KKKKK; KKKRK; KBKBK; KKKRKV; PGKKRKV; PKGKRKV; PKKGRKV; PKKKGKV; PKKKRGV; or PKKKRKG.
  • EP comprises PKGKRKV.
  • the cargo comprises a therapeutic oligonucleotide.
  • the therapeutic oligonucleotide is an antisense oligonucleotide.
  • the antisense oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO).
  • the cargo comprises a therapeutic peptide.
  • the disease of the eye is selected from one or more of diabetic retinopathy; glaucoma; retinitis pigmentosa; Usher syndrome; retinal tears or holes; retinal detachment; retinal ischemia; damage associated with laser therapy including photodynamic therapy; surgical light induced iatrogenic retinopathy; drug-induced retinopathies; autosomal dominant optic atrophy; toxic and/or nutritional amblyopias; Leber's hereditary optic neuropathy; atypical retinitis pigmentosa; Bardet-Biedl syndrome; blue-cone monochromacy; cataracts; central areolar choroidal dystrophy; choroideremia; cone dystrophy; rod dystrophy; rod-cone dystrophy; congenital stationary nightblindness; cytomegalovirus retinitis, diabetic macular edema; dominant drusen; giant cell arteritis; Goldmann Favre dystrophy; graves' ophthalm
  • administering a cargo conjugate to directly to an eye of the subject comprises local administration.
  • local administration is selected from topical administration, intravitreal administration, periocular administration, subretinal administration, suprachoroidal administration, and intracameral administration.
  • the method further comprises identifying a subject having an ocular disease.
  • the subject is suffering from an ocular disease or disorder, or is at risk of developing, an ocular disease or disorder.
  • the subject has a genetic disease or disorder of the eye.
  • the subject is a mammal In embodiments, the mammal is a human.
  • administration of the compound modulates expression or activity of a target molecule.
  • administration of the compound downregulates expression or activity of the target molecule.
  • administration of the compound upregulates expression or activity of the target molecule.
  • the target molecule is a protein.
  • the target molecule is an oligonucleotide.
  • the cargo conjugate is administered in a pharmaceutical composition.
  • FIG. 1A is a schematic drawing of the eye with a section of the retina shown as schematically enlarged. Adapted from Simple Anatomy of the Retina by Helga Kolb, Webvision, the Organization of the Retina and Visual System (webvision.med.utah.edu/book/part-i- foundations/simple-anatomy-of-the-retina/).
  • FIG. IB is a schematic drawing of layers and cell components of the retina. Adapted from Koeppen & Stanton: Berne and Levy Physiology, 6 th Edition, 2008 by Mosby, an imprint of Elsevier, Inc
  • FIG. 2 shows examples of modified nucleotides used in antisense oligonucleotides described herein.
  • FIGS. 3A-3D illustrate examples of conjugation chemistries for connecting an antisense compound (AC) , such as an antisense oligonucleotide (ASO), to a cyclic cell penetrating peptide.
  • AC antisense compound
  • ASO antisense oligonucleotide
  • FIG. 3A shows amide bond formation between peptides with a carboxylic acid group or with TFP activated ester and primary amine residues at the 5' end of an AC.
  • FIG. 3B shows conjugation of secondary amine or primary amine modified AC at 3' and peptide-TFP ester through amide bond formation.
  • FIG. 3C shows conjugation of a peptide-azide to the 5' cyclooctyne modified AC via copper-free azide-alkyne cycloaddition.
  • FIG. 3D demonstrates another example of conjugation between a 3' modified cyclooctyne ACs or 3' modified azide ACs and CPP containing linker-azide or linker-alkyne/cyclooctyne moiety, via a copper-free azide-alkyne cycloaddition or cupper catalyzed azide-alkyne cycloaddition, respectively (click reaction).
  • FIG. 4 shows an example of conjugation chemistry for connecting an ASO and cCPP with an additional linker modality containing a polyethylene glycol (PEG) moiety.
  • FIG. 5 shows images of sectioned mouse eye tissue treated with a vehicle or a PMO-EEV conjugate. Tissue was stained for nuclear material (DAPI, blue) and for PMOs (anti-PMO antibody, green).
  • FIG. 6 shows images of sectioned mouse retinal tissue treated with a vehicle or a PMO- EEV conjugate.
  • the tissue was stained for nuclear material (DAPI, blue) and for PMOs (anti-PMO antibody, green).
  • the top images show both the DAPI and anti-PMO stains and the bottom images show only the anti-PMO stains.
  • RPE retinal pigment epithelium
  • ONL outer nuclear layer
  • INL inner nuclear layer
  • GCL ganglion cell layer
  • FIG. 7 shows images of sectioned mouse retinal tissue treated with a vehicle or a PMO- EEV conjugate.
  • the tissue was stained for nuclear material (DAPI, blue) and for glial fibrillary acidic protein (GFAP; pink).
  • DAPI nuclear material
  • GFAP glial fibrillary acidic protein
  • FIG. 8 is a plot showing the percent exon skipping after mice were treated with various cargo conjugates.
  • Methods and compositions are provided herein that can be used to transport a therapeutic moiety or therapeutic agent to the cells of the eye of a subject. Methods and compositions are provided herein may be used to treat ocular diseases.
  • the cargo may be a therapeutic moiety for which delivery to a cell of the eye is desired.
  • the therapeutic moiety may be a molecule useful for treating a disease of the eye.
  • the therapeutic moiety selectively binds to a target molecule associated with an ocular disease.
  • the target molecule is a macromolecule implicated in a disease or pathology of the eye.
  • the target molecule is a polypeptide or protein.
  • the target molecule is an oligonucleotide.
  • the oligonucleotide target comprises DNA.
  • the oligonucleotide target comprises genomic DNA. In embodiments, the oligonucleotide target comprises RNA. In embodiments, the oligonucleotide target comprises mRNA. In embodiments, the target is associated with an ocular disease. In embodiments, selective binding of the therapeutic moiety with the target molecule is useful for the treatment of a disease, pathology or other abnormal state or condition of the eye. In embodiments, selective binding of the therapeutic moiety to the target molecule upregulates expression or activity of the target molecule. In embodiments, selective binding of the therapeutic moiety to the target molecule downregulates expression or activity of the target molecule.
  • the term “ocular delivery construct” refers to a compound that, when conjugated to cargo and ophthalmically administered, enhances uptake of the cargo in a cell of the eye relative to uptake of the cargo alone (when not conjugated to the ocular delivery construct).
  • the ocular delivery construct may preferentially targets a tissue of the eye.
  • the ocular delivery construct targets one or more structures, tissues, layers, and/or cells of eye.
  • the ocular delivery construct comprises a cyclic cell penetrating peptide (cCPP); a compound comprising a cCPP and a linker; a compound comprising a cCPP and an exocyclic peptide; or a compound comprising an endosomal escape vehicle (EEV) which comprises a cCPP, an exocyclic peptide, and a linker.
  • cCPP cyclic cell penetrating peptide
  • EEV endosomal escape vehicle
  • an ocular delivery construct provided herein can be used to transport a cargo, such as a therapeutic agent, to a cell of the eye of subject.
  • the ocular delivery construct transports a therapeutic moiety to an intracellular compartment of a cell.
  • an ocular delivery construct transports a therapeutic moiety to the retina of a subject.
  • the ocular delivery construct transports a therapeutic moiety intracellularly to cells of one or more cell types within the retina.
  • the ocular delivery construct preferentially transports a therapeutic agent intracellularly to cells of one or more cell type within the retina.
  • a cargo conjugate or a composition comprising a cargo conjugate may be ophthalmically administered.
  • the term “ophthalmically administered,” “ophthalmic administration,” “ocularly administered,” and “ocular administration” refer to local delivery of a compound or composition directly to one or more components of the eye (described in detail later herein). Such terms exclude systematic administration of the cargo conjugate.
  • ocular administration of a therapeutic may allow for a more effective treatment than systematic administration.
  • Systemic administration in some cases, may not achieve effective concentrations of therapeutics in the eye due in part to systemic clearance mechanisms and blood-ocular barriers.
  • Ocular administration may avoid, mitigate, or circumvent one or more systemic clearance mechanisms, such as first-pass metabolism, and/or blood-ocular barriers, such as the blood-aqueous barrier and the blood-retinal barrier to achieve therapeutically effective concentrations in cells of the eye.
  • the blood-ocular barriers may hinder a therapeutic from penetrating the eye and/or reaching portions of the eye at a therapeutically relevant concentration when systemically administered.
  • ocular administration may result in fewer side effects than systemic administration of a therapeutic because ocular administration provides local delivery of the therapeutic to the tissue to be treated.
  • a cargo conjugate may be targeted to and/or localize to one or more structures, layers, and/or cell types of the eye.
  • an ocularly administered cargo conjugate may be targeted to and/or localize to one or more structures, layers, and/or cell types of the eye.
  • targeted to x refers to the cargo conjugate or delivery construct preferentially interacting with x.
  • a cargo conjugate or delivery construct target to a particular system, organ, structure, layer, tissue type, or cell type may interact with other one or more additional systems, on or more additional organs, one or more additional tissue types, one or more additional cell types, or any combination thereof.
  • Reference to a cargo conjugate or delivery construct targeted to x includes all the components comprising x.
  • a cargo conjugate targeted to the eye includes the cargo conjugate being targeted to one or more structures, tissues, layers, and/or cells that make up the eye.
  • a cargo conjugate or delivery construct As used herein in the context of biodistribution of a delivery construct or a cargo conjugate, the terms “localize” and “localized” refer to the physical position of the cargo conjugate within a body, an organ, a tissue, or a cell. Reference to a cargo conjugate or delivery construct being localized within a particular locale includes all the sublocales comprising the locale.
  • a cargo conjugate or delivery construct localized to the eye includes the cargo conjugate or delivery construct being localized to one or more structures, tissues, layers, and/or cells that make up the eye and can be referred to herein as an “ocular delivery construct.”
  • An ocular delivery construct localized to the retina may be further localized to one or more of the layers and/or cells that comprise the retina.
  • the anatomy, physiology, and chemical composition of the eye provide barriers to delivery of therapeutic agents. Many therapeutics struggle to penetrate target tissues in the eye, such as the cornea or retina, which can result in poor bioavailability.
  • the eye has static and dynamic barriers that inhibit therapeutic agents from reaching target tissues at therapeutically effective concentrations.
  • Static barriers of the eye include tissues that provide a passive physical barrier to therapeutic agent penetration.
  • Dynamic barriers include physiological clearance mechanisms and barriers that include aqueous turnover, vitreous flow, ocular drug metabolism, and eye metabolizing enzymes.
  • main structures in a mammalian eye include, the cornea, iris, pupil, aqueous humor, lens, vitreous humor, retina, and optic nerve.
  • the retina is a layered structure containing distinct layers of neurons interconnected by synapses, some of which are shown in FIG. IB.
  • Retina layers from the anterior to posterior surface include: inner limiting membrane, nerve fiber layer (NFL), ganglion cell layer (GCL), inner plexiform layer, inner nuclear layer (INL), middle limiting membrane, outer plexiform layer, outer nuclear layer (ONL), outer (external) limiting membrane, photoreceptor layer, and the retinal pigment epithelium (RPE).
  • the cells in the retina can be subdivided into several cell types shown in FIG. IB: ganglion cells, Muller cells (Muller glia), bipolar cells, amacrine cells, horizontal cells, photoreceptor cells (rods and cones), and pigment cells.
  • the majority of cell types in the retina span one, two, or three retinal layers (see FIG. IB).
  • Some cell types are elongated such that a first part of the cells of a cell type is in a first retinal layer, a second part of the cells of the cell type is in a second retinal layer, and a third part of the cells in the cell type is in a third retinal layer.
  • the nuclei of the photoreceptor cells are generally in the outer nuclear layer
  • the rods/cones of the photoreceptor cells are generally in the photoreceptor layer
  • the synapses of the photoreceptor cells are generally in the outer plexiform layer.
  • an ocular delivery construct can be used to transport a therapeutic moiety (as a conjugate construct) to a structure of the eye.
  • an ocular delivery construct may be used to transport a therapeutic moiety to the retina.
  • the ocular delivery construct may be used to transport a therapeutic moiety to one or more retinal layer.
  • the ocular delivery construct may be used to transport a therapeutic moiety to one or more of the inner limiting membrane, nerve fiber layer (NFL), ganglion cell layer (GCL), inner plexiform layer, inner nuclear layer (INL), middle limiting membrane, outer plexiform layer, outer nuclear layer (ONL), outer (external) limiting membrane, photoreceptor layer, and the retinal pigment epithelium (RPE).
  • NNL nerve fiber layer
  • GCL ganglion cell layer
  • inner plexiform layer inner nuclear layer
  • INNL inner nuclear layer
  • ONL middle limiting membrane
  • outer plexiform layer outer nuclear layer
  • outer (external) limiting membrane outer (external) limiting membrane
  • photoreceptor layer and the retinal pigment epithelium (RPE).
  • RPE retinal pigment epithelium
  • the ocular delivery construct may be used to transport a therapeutic moiety to and within one or more of a ganglion cell, a Muller cell (Muller glia), a bipolar cell, an amacrine cell, a horizontal cell, a photoreceptor cell (rods and cones), and a pigment cell.
  • a cargo conjugate when ophthalmically administered, may be targeted to and/or localize in the retina.
  • the cargo conjugate may be targeted to and/or localize in one or more of the layers of the retina such as the inner limiting membrane, nerve fiber layer (NFL), ganglion cell layer, inner plexiform layer, inner nuclear layer, middle limiting membrane, outer plexiform layer, outer nuclear layer, outer (external) limiting membrane, photoreceptor layer, and retinal pigment epithelium, and/or one or more of the cell types of the retina such as a ganglion cell, a Muller cell, a bipolar cell, an amacrine cell, a horizontal cell, a photoreceptor cell (rods and cones), and a pigment cell.
  • the layers of the retina such as the inner limiting membrane, nerve fiber layer (NFL), ganglion cell layer, inner plexiform layer, inner nuclear layer, middle limiting membrane, outer plexiform layer, outer nuclear layer, outer (external) limiting membrane
  • the ocular delivery constructs provided herein may be conjugated to a therapeutic moiety for which delivery to the eye is desired.
  • the cargo conjugate may include a therapeutic moiety designed to provide a therapeutic effect to a subject inflicted with an ocular disease.
  • the cargo conjugate includes a therapeutic moiety useful for treating a disease of the eye (i.e., an ocular disease).
  • the therapeutic moiety targets a protein or gene associated with a disease of the eye.
  • the cargo conjugates and compositions may be used to treat an ocular disease.
  • the therapeutic moiety modulates expression of a protein or gene associated with a disease of the eye.
  • the therapeutic moiety upregulates expression of a protein or gene associated with a disease of the eye. In embodiments, the therapeutic moiety downregulates expression of a protein or gene associated with a disease of the eye. In embodiments, the therapeutic moiety modulates the composition of mRNA. In embodiments, the therapeutic moiety modulates processing of the mRNA. In embodiments, the therapeutic moiety modulates splicing of a pre-mRNA target transcript. In embodiments, the therapeutic moiety modulates alternative splicing of a pre-mRNA target transcript. In embodiments, modulation of splicing or alternative splicing induces exon skipping and/or exon inclusion.
  • Non-limiting examples of ocular diseases that a cargo conjugate may be used to treat include, diabetic retinopathy; glaucoma; retinitis pigmentosa (RP), such as autosomal dominant RP, autosomal recessive RP, sector RP, and RP associated with Usher syndrome; retinal tears or holes; retinal detachment; retinal ischemia; damage associated with laser therapy including photodynamic therapy; surgical light induced iatrogenic retinopathy; drug-induced retinopathies; autosomal dominant optic atrophy; toxic and/or nutritional amblyopias; Leber’s hereditary optic neuropathy; atypical retinitis pigmentosa; Bardet-Biedl syndrome; blue-cone monochromacy; cataracts; central areolar choroidal dystrophy; choroideremia; cone dystrophy; rod dystrophy; rodcone dystrophy; congenital stationary’ night blindness; cytomegalovirus retinitis; diabetic
  • a cargo conjugate having a therapeutic moiety designed to treat an ocular disease is provided.
  • a composition comprising such a cargo conjugate is ocularly delivered to a subject in need thereof.
  • the composition is administered directly the retina.
  • the composition is administered intravitreally.
  • the cargo conjugate may include a therapeutic moiety (TM) for which delivery to the eye is desired, for example, to treat an ocular disease TMs can include antisense oligonucleotides (ASO), polypeptides, and small molecules.
  • TM therapeutic moiety
  • ASO antisense oligonucleotides
  • a TM is an effector that modulates target gene expression and/or target protein activity.
  • a target gene is the gene of which modulation of gene expression and/or protein activity is desired.
  • a target transcript is the pre-mRNA or mRNA transcript that is transcribed from the target gene.
  • a target protein is the polypeptide or protein encoded by the target transcript.
  • the target gene, target transcript, or target protein may be associated with a disease state.
  • the target gene, target transcript, or target protein may be associated with an ocular disease.
  • a target gene may be a mutated gene associated with the disease state; a target transcript may have a coding sequence associated with the disease state, may be aberrantly spliced in the disease state, may be present in reduced or increased levels in the disease state, or the like; a target protein may be present in reduced or increased levels in the disease state, may have aberrant function, reduced function, or no function in the disease state, or the like; or any combination thereof.
  • an “ocular target” or “target molecule” can be a target gene, a target transcript, or a target protein associated with an ocular disease.
  • the target gene, target transcript, and target protein are a gene, transcript, and protein associated with a disease of the retina.
  • a TM may exert a therapeutic effect through any suitable mechanism.
  • a cargo conjugate includes a TM that modulates one or more aspects of target gene expression, such as transcription and translation, and/or target protein activity.
  • a TM may function, relative to the disease phenotype, to downregulate target gene expression; upregulate target gene expression; inhibit target protein function; increase target protein function; or any combination thereof.
  • the cargo conjugate includes a TM that modulates one or more aspects of pre-mRNA processing of a target transcript.
  • Pre-mRNA molecules are made in the nucleus and are processed before or during transport to the cytoplasm for translation. Processing of the pre- mRNAs includes addition of a 5' methylated cap and an approximately 200-250 base poly(A) tail to the 3' end of the transcript.
  • mRNA processing also includes splicing of the pre-mRNA, which occurs in the maturation of 90-95% of mammalian mRNAs. Introns (or intervening sequences) are regions of a primary transcript (or the DNA encoding it) that are not included in the coding sequence of the mature mRNA.
  • Exons are regions of a primary transcript that remain in the mature mRNA.
  • the exons are spliced together to form the mature mRNA sequence.
  • Splice junctions are also referred to as splice sites with the 5' side of the junction often called the “5* splice site,” or “splice donor site” and the 3' side called the “3' splice site” or “splice acceptor site.”
  • the 3 ' end of an upstream exon is j oined to the 5 ' end of the downstream exon.
  • the unspliced RNA (or pre-mRNA) has an exon/intron junction at the 5' end of an intron and an intron/exon junction at the 3' end of an intron.
  • the exons are contiguous at what is sometimes referred to as the exon/exon junction or boundary in the mature mRNA.
  • Cryptic splice sites are those which are less often used but may be used when the usual splice site is blocked or unavailable. Cryptic splice sites may result from one or more mutations in an intron. Such mutations may result in a high occurrence of splicing at the cryptic splice site.
  • a TM may modulate alternative splicing of a pre-mRNA transcript by, for example, inducing exon skipping and/or inducing exon inclusion. Induction of exon skipping and/or exon inclusion may result in a mRNA that has a different combination of exons than is observed in a mRNA transcript of a diseased state.
  • Some genetic mutations alter splicing which can result in the inclusion of exons not normally present in the processed mRNA and/or exclusion of exons normally present in the processed mRNA. Such inclusions and exclusions may result in a truncated protein, a protein with decreased function, a protein with no function, an unstable protein, or any combination thereof. Similarly, mutations in exons that do not necessarily affect splicing, may result in a truncated protein, a protein with decreased function, a protein with no function, an unstable protein, or any combination thereof.
  • the TM is an antisense oligonucleotide (ASO).
  • ASO is an oligonucleotide that binds (e.g., hybridizes) to a portion of an RNA transcript.
  • the ASO may comprise natural or modified nucleotides.
  • the ASO may comprise one or more modified nucleosides, one or more modified intemucleoside linkages, one or more conjugate groups, or combinations thereof.
  • the ASO may be used to modulate pre-mRNA processing by modulating splicing of the pre-mRNA target transcript.
  • modulation of splicing refers to altering the processing of a pre-mRNA target transcript such that the spliced mRNA transcript contains either a different combination of exons as a result of exon skipping or exon inclusion, a deletion in one or more exons, or the deletion or addition of a sequence not normally found in the spliced mRNA of the disease state (e.g., an intron sequence). Modulation of splicing may result in alternative splicing. As such modulation of splicing may result in modulation of alternative splicing.
  • ASO hybridization to a target sequence in a pre-mRNA target transcript modifies splicing to produce a mature mRNA encoding a fully functional protein, a partially functional protein, or a non-functional protein.
  • ASO hybridization to a target sequence in a pre-mRNA target transcript modifies splicing of a pre-mRNA in a diseased cell to produce a mature mRNA encoding a protein that is present in a non-diseased cell (e g., a wild-type protein).
  • ASO hybridization results in alternative splicing of the target pre- mRNA.
  • ASO hybridization results in skipping of one or more exons.
  • exon skipping refers to modulation of splicing in a cell to produce a mature mRNA lacking one or more exons relative to a mature mRNA in the cell for which exon skipping has not occurred.
  • an ASO induces exon skipping to produce a mature mRNA that lacks one or more exons associated with a disease state.
  • an ASO induces exon skipping to produce a mature mRNA that lacks one or more pseudo-exons associated with a disease state.
  • an ASO induces exon skipping to produce a mature mRNA that lacks one or more exons associated with the disease state but, as a result of the exon skipping includes one or more exons that is not present in a mature mRNA of the disease state.
  • the one or more exons that are included may be exons present in wild-type mature mRNA that are missing in a mature mRNA in the disease state.
  • the skipped exon comprises a frameshift mutation, a nonsense mutation, or a missense mutation.
  • the skipped exon sequence comprises a nucleic acid deletion, substitution, or insertion.
  • the skipped exon itself does not comprise a sequence mutation, but a neighboring intron or exon comprises a mutation.
  • ASO hybridization to a target sequence within a target pre-mRNA prevents inclusion of an exon sequence in the mature mRNA molecule.
  • antisense oligonucleotides hybridization to a target sequence within a target pre-mRNA results in preferential expression of a wild type target protein isomer.
  • antisense oligonucleotides hybridization to a target sequence within a target pre-mRNA results in expression of a re-spliced target protein comprising an active fragment of a wild type target protein.
  • ASOs can be designed to promote exon skipping in a target transcript to result in, for example, a protein of the healthy phenotype (full length and fully functional), a full-length protein having at least some function, a nonfunctional full-length protein, a fully functional truncated protein, a truncated protein having at least some function, or a non-functional truncated protein.
  • Exon skipping may also be used to introduce a premature stop codon in a target transcript.
  • mRNA comprising a premature stop codon may undergo nonsense mediated decay.
  • Inducing nonsense mediate decay of a target transcript may be beneficial to treat diseases characterized by expression of a deleterious target protein or high concentrations of a target protein and/or target transcript.
  • An ASO can induce exon skipping by binding to and sterically inhibiting splicing of a target transcript (see, e.g., International Patent Application No. International Patent Application No. PCT/US22/28357, filed on 9 May 2022, and entitled COMPOSITIONS AND METHODS FOR MODULATING mRNA SPLICING, which application is hereby incorporated herein by reference in its entirety).
  • ASO hybridization results in exon inclusion.
  • exon inclusion refers to modulation of splicing in a cell to produce a mature mRNA that includes one or more exons relative to a mature mRNA in the cell for which exon inclusion has not occurred.
  • an ASO induces exon inclusion to produce a mature mRNA that includes one or more exons that are deleted in a disease state.
  • an ASO induces exon inclusion to produce a mature mRNA that includes one or more exons whose deletion is associated with the disease state, but as a result of the exon inclusion, the mature mRNA includes the exons not present in the mature mRNA of the disease state.
  • the one or more exons that are included may be exons present in wild-type mature mRNA that are missing in a mature mRNA in the disease state.
  • ASO hybridization to a target sequence within a pre-mRNA target transcript induces inclusion of one or more exon sequences in the mature mRNA molecule.
  • ASO hybridization to a target sequence within a pre-mRNA target transcript results in preferential expression of a wild type target protein isomer.
  • antisense oligonucleotides hybridization to a target sequence within a target pre-mRNA results in correction of alternative splicing observed in a disease state.
  • ASOs can be designed to promote intron inclusion in a target transcript to result in, for example, a protein of the healthy phenotype (full length and fully functional), a full-length protein having at least some function, a nonfunctional full-length protein, a fully functional truncated protein, a truncated protein having at least some function, or a non-functional truncated protein.
  • the TM may be an ASO that modulates pre-mRNA processing by inhibiting the poly adenylation of a target transcript. Inhibition of addition of a poly(A) tail to target transcript may decrease the levels of the target transcript and/or target protein levels.
  • ASOs conjugated to cyclic peptides for modulating polyadenylation of mRNA is disclosed in International Patent Application No. PCT/US22/28354, filed on 9 May 2022, and entitled COMPOSITIONS AND METHODS FOR MODULATING GENE EXPRESSION, which application is hereby incorporated herein by reference in its entirety.
  • the TM may modulate one or more aspects of mRNA translation.
  • the TM may be an ASO that prevents degradation of a mRNA target transcript. Since a single mRNA transcript is used as a translation template to produce multiple protein copies, stabilizing a target mRNA transcript may increase the number of times the transcript is used as a template, thereby increasing the levels of the protein encoded by the target transcript. Stabilization of a target mRNA transcript may increase the resistance of the target transcript to degradation (e.g., 5 "-3' and/or 3 '-5' exonuclease degradation), thereby increasing the half-life of the target transcript.
  • ASO that prevents degradation of a mRNA target transcript. Since a single mRNA transcript is used as a translation template to produce multiple protein copies, stabilizing a target mRNA transcript may increase the number of times the transcript is used as a template, thereby increasing the levels of the protein encoded by the target transcript. Stabilization of a target mRNA transcript may increase the resistance of the target
  • Therapeutic moieties may stabilize an mRNA target transcript by binding to, for example, the 3' untranslated region, the 5' untranslated region, or both. Increasing the stability of an mRNA target transcript may be beneficial to treat diseases characterized by low concentrations of a protein
  • the TM may modulate target gene expression by decreasing the stability of a mRNA target transcript and/or preventing translation of the mRNA target transcript.
  • a TM may be an ASO that sterically prevents translation of an mRNA target transcript and/or increases the susceptibility of the mRNA target transcript to decay.
  • the TM may induce nonsense mediated decay of the target transcript.
  • the ASO comprises DNA, and binding of the ASO to the target transcript creates a DNA/RNA hybrid that can be degraded by the enzyme RNaseH. Decreasing the stability and/or preventing translation of an mRNA target transcript may be beneficial to treat diseases where reducing the amount of a target protein is desired.
  • the ASO modulates one or more aspects of target gene transcription or translation through steric blocking.
  • the following review article describes the mechanisms of steric blocking and applications thereof and is incorporated by reference herein in its entirety: Roberts et al. Nature Reviews Drug Discoveiy (2020) 19: 673-694.
  • the ASO may modulate one or more aspects of gene expression and/or protein function through various mechanisms some of which are disclosed herein.
  • the TM may modulate the activity of a target protein.
  • the TM may be a polypeptide or a small molecule that functions to inhibit or increase the function of a target protein.
  • a cargo conjugate having a TM designed to treat Usher syndrome such as Usher syndrome type II or Usher syndrome type Ila is provided.
  • a composition comprising such a cargo conjugate is ocularly delivered to a subject in need thereof, for example to treat Usher syndrome.
  • the composition is administered directly the retina.
  • the composition is administered intravitreally.
  • Usher syndrome type HA (OMIM 276901) is the most common type of Usher syndrome and is characterized by progressive vision loss and congenital moderate hearing loss.
  • Usherin (USH2A; NCBI Gene ID:7399), a protein implicated in Usher syndrome type ILA, regulates the long-term maintenance of retinal photoreceptors.
  • Pathogenic mutations in the gene that encodes usherin that disrupt usherin production for example, by introducing a premature stop codon that results in a truncated protein and/or non-functional protein, can lead to degeneration of photoreceptors.
  • Mutations in exon 13 of the USH2A gene are the most recurring mutations in Usher syndrome type IIA and include, for example, c.2299delG and C.2276G > T (See Dulla, et al., Molecular Therapy, 4 August 2021, 29(8):2441-2455). Skipping of exon 13 in USH2A pre-mRNA represents a potential therapeutic strategy for treating Usher syndrome type IIA.
  • Dulla, et al. illustrate that a functional truncated usherin results from exon skipping in the USH2A transcripts of zebrafish using antisense oligonucleotides.
  • the following proteins and genes, which are associated with Usher syndrome, may be targeted to treat Usher syndrome: myosin VILA (MY07A), harmonin (USH1C), cadherin 23 (CDH23), protocadherin 15 (PDCH15), USH1G, SANS, usherin (USH2A), VLGR1, GRP98, whirlin (DBFB3), and clarin-1 (USH3A).
  • myosin VILA MY07A
  • harmonin USH1C
  • CDH23 cadherin 23
  • PDCH15 protocadherin 15
  • USH1G SANS
  • usherin USH2A
  • VLGR1, GRP98 whirlin
  • DBFB3A clarin-1
  • a cargo conjugate includes a TM that modulates target gene expression and/or target protein activity related to one or more target genes and/or target proteins associated with Usher syndrome type I I A.
  • the target gene is USH2A and the target protein is usherin.
  • the TM is an ASO that modulates one or more aspects of transcription and/or translation of one or more target genes associated with Usher syndrome type IIA.
  • the ASO is directed to a target nucleotide sequence within USH2A pre-mRNA and modulates one or more aspects of USH2A pre-mRNA splicing.
  • ASO induces exon skipping of one or more exons of a USH2A target transcript.
  • the cargo conjugate comprising the ASO induces exon skipping of exon 13 of a human USH2A target transcript.
  • skipping of an exon in the USH2A pre-mRNA results in an in-frame deletion to produce a truncated usherin protein that has at least partial activity.
  • skipping of exon 13 of the USH2A target transcript results in an in-frame deletion to produce a truncated usherin protein that has at least partial activity.
  • the ASO is a phosphorodiamidate morpholino oligomer (PMO).
  • the cargo conjugate comprising the ocular delivery construct and the ASO that results in skipping of one or more exons of a human USH2A target transcript is ocularly delivered to a subject in need thereof.
  • the composition is administered directly the retina. In embodiments, the composition is administered intravitreally.
  • a cargo conjugate has a TM designed to treat diabetic retinopathy (DR).
  • a composition comprising such a cargo conjugate is ocularly delivered to a subject in need thereof, for example, to treat diabetic retinopathy
  • the composition is administered directly the retina.
  • the composition is administered intravitreally.
  • Diabetic retinopathy is the leading cause of blindness in Americans between 20 and 74 years of age.
  • Clinical and epidemiological studies have identified a genetic component to DR, but data from studies aimed at identifying genes or genome regions associated with DR have been inconsistent (see, e.g., Cho and Sobrin, Curr Diab Rep, 2014 Aug, 14(8):515).
  • EPO erythropoietin
  • TCF7L2 transcription factor 7-like 2
  • a cargo conjugate includes a TM that modulates target gene expression and/or target protein activity related to one or more target genes and/or target proteins associated with DR.
  • a cargo conjugate may include a TM that modulates one or more aspects of gene expression and/or target protein activity of one or both of EPO and TCF7L2.
  • the TM is an ASO.
  • a cargo conjugate has a TM designed to treat glaucoma.
  • a composition comprising such a cargo conjugate is ocularly delivered to a subject in need thereof, for example, to treat glaucoma.
  • the composition is administered directly the retina.
  • the composition is administered intravitreally.
  • Glaucoma is the leading cause of blindness in the United States. Glaucoma is generally caused by damage to the optic nerve by increases in eye pressure. A number of proteins and genes have been associated with glaucoma including myocilin (MYOC), optineurin (OPTN); tankbinding kinase 1 (TBK1); ATP-binding cassette transporter Al (ABCA1); actin filament associated protein 1 (AFAP1), GDP-Mannose 4,6-Dehydratase (GMDS); phosphomannomutase 2 (PMM2); transforming growth factor beta receptor 3 (TGFBR3); fibronectin type III domain containing 3B (FNDC3B); rho guanine nucleotide exchange factor 12 (ARHGEF 12); growth arrest specific 7 (GAS7); forkhead Box Cl (FOXCI); ataxin 2 (ATXN2); thioredoxin reductase 2 (TXNRD2); ependymin related 1 (EPDRl);
  • a cargo conjugate has a TM designed to treat retinitis pigmentosa (RP).
  • a composition comprising such a cargo conjugate is ocularly delivered to a subject in need thereof, for example to treat RP.
  • the composition is administered directly to the retina.
  • the composition is administered intravitreally.
  • RP is characterized by the loss of photoreceptor cells over time, leading to progressive vision loss. A number of genes have been associated with RP, with the majority of them expressed in either the photoreceptors or the retinal pigment epithelium of the eye (See, e.g., Ferari et al., Curr Genomics, 2011 Jun, 12(4):238-249).
  • bestrophin-1 BEST1
  • carbonic anhydrase IV CA4
  • cone-rod homeobox protein CRX
  • fascin homolog 2 actin-bundling protein
  • FSCN2 actin-bundling protein
  • GUI1B guanylate cyclase activator IB
  • IMPDH1 inosine 5 '-monophosphate dehydrogenase 1
  • KLHL7 nuclear receptor subfamily 2, group E, member 3
  • NRL nuclear receptor subfamily 2, group E, member 3
  • NRL neural retina leucine zipper protein
  • PRPF3 pre-mRNA processing factor 3 homolog
  • PRPF8 pre-mRNA processing factor 8 homolog PRPF8
  • PRP31 pre-mRNA processing factor 31 homolog
  • PRPH2 peripherin 2
  • a cargo conjugate includes a TM that modulates target gene expression and/or target protein activity related to one or more target genes and/or target proteins associated with RP.
  • Retinitis pigmentosa may be autosomal recessive.
  • the following proteins and genes are associated with autosomal recessive RP: RP22; RP29; RP32, ATP-binding cassette; subfamily A (ABC1), member 4 (ABCA4); bestrophin-1 (BEST1); chromosome 2 open reading frame 71 (C2ORF71); ceramide kinase-like protein (CERKL); cyclic nucleotide gated channel alphal (CNGA1); cyclic nucleotide gated channel betal (CNGB1); crumbs homolog 1 (CRB1); eyes shut homolog (EYS, RP25); family with sequence similarity 161 member A (FAM161A); NAD(+)- specific isocitrate dehydrogenase 3 beta (IDH3B); interphotoreceptor matrix proteoglycan 2 (IMPG2); lecithin retinol acyltransferase (IMP
  • Retinitis pigmentosa may be X-linked.
  • the following proteins and genes are associated with X-linked RP: retinitis pigmentosa 2 protein (RP2); RP6; RP23; RP24; RP34; and retinitis pigmentosa GTPase regulator (RPGR).
  • RP2 retinitis pigmentosa 2 protein
  • RP6 retinitis pigmentosa 2 protein
  • RP23 retinitis pigmentosa 2 protein
  • RPGR retinitis pigmentosa GTPase regulator
  • a cargo conjugate includes a TM that modulates target gene expression and/or target protein activity related to one or more target genes and/or target proteins associated with X-linked RP.
  • Retinitis pigmentosa may be autosomal dominant Autosomal dominate RP (adRP) is caused by heterozygous mutations in the PRFPF31 gene.
  • the PRPF31 gene encodes human pre- mRNA processing factor 3 (PRPF31).
  • PRPF31 is a ubiquitous pre-mRNA splicing factor that is a part of the small nuclear ribonucleoprotein complex of the spliceosome. More than 130 genetic PRPF31 mutations are known, some of which are loss of function variants. Penetrance of PRPF31 - assoacited adRP is incomplete; that is, carriers may be asymptomatic but still pass the disease to offspring.
  • the major determinant of PRPF31 mutation penetrance is thought to be the expression level of the nonmutant PRPF31 allele (See Ali-Nasser Et. al., Mol Vis, 2022; 28, pg359-368, PM1D: 36338669, PMCID: PMC9603903).
  • Carriers that have a high expression level of the nonmutant PRPF31 protein may not be symptomatic. Wild-type PRPF31 gene expression is highly variable and impacted by several factors such as CCR4-NOT.
  • CCR4-NOT transcription complex subunit 3
  • CNOT3 transcription complex subunit 3
  • CCR4-NOT binds to the promoter of PRPF31 inhibiting transcription of PRPF31.
  • CCR4-NOT is encoded by the CNOT3 gene. Modulating the activity and/or level of the CCR4-NOT t protein and/or the level of CNOT3 transcripts may be used to modulate PRPF31 gene expression.
  • a cargo conjugate that includes a TM that modulates target gene expression and/or target protein activity related to one or more target genes and/or target proteins associated with adRP.
  • the target gene or target protein may be PRPF31 (PRPF31), CNOT3 (CCR4-NOT), or both.
  • the TM increases PRPF31 target transcript and/or protein levels.
  • the TM decreases CCR4-NOT target protein levels and/or CNOT3 target transcript levels which may result in the increase of PRPF31 transcript levels and/or PRPF31 protein levels.
  • the TM induces nonsense mediate decay of CNOT3 target transcripts thereby reducing the level of CNOT3 target transcripts and/or CCR4- NOT target protein levels.
  • the TM is an ASO that hybridizes to at least a portion of a CNOT3 target transcript to induce nonsense mediated decay of the CNOT3 target transcript.
  • a cargo conjugate has a TM designed to treat Bardet-Biedl syndrome.
  • a composition comprising such a cargo conjugate is ocularly delivered to a subject in need thereof, for example to treat Bardet-Biedl syndrome.
  • the composition is administered directly the retina.
  • the composition is administered intravitreally
  • Bardet-Biedl syndrome is characterized rode and/or cone dystrophy.
  • a number of proteins and genes have been associated with Bardet-Biedl syndrome including BBS1, BBS2, ARL6 (BBS3), BBS4, BBSS, MKKS (BBS7), TTCB (BBSS), PTHB1 (BBS9), BBS10, TRIM32 (BBS11), BBS12, MKS1 (BBS13), CEP290/NPNP6/LCA10 (BBS14), WDPCP/FRITZ (BBS15), SDCCAG8 (BBS16), LZTFL1 (BBS17), BBIP1/10 (BBS18), IFT27 (BBS19), and AZI1/CEP131 (BBS20) (See, Priya, et al., Indian J Opthamol, 2016 Sept, 64(9):620-627).
  • a cargo conjugate includes a TM that modulates target gene expression and/or target protein activity related to one or more target genes
  • a cargo conjugate has a TM designed to treat autosomal dominant optic atrophy (ADOA).
  • a composition comprising such a cargo conjugate is ocularly delivered to a subject in need thereof, for example, to treat ADOA.
  • the composition is administered directly the retina.
  • the composition is administered intravitreally.
  • ADOA Autosomal dominant optic atrophy
  • ADOA is associated with mutations in the OPA1 gene.
  • the OPA1 (3q38-q29) gene encodes the OPA1 protein, a ubiquitously expressed mitochondrial GTPase that is involved in regulating mitochondrial function such as, for example, oxidative phosphorylation, mitochondrial DNA maintenance, and apoptosis.
  • the OP Al gene includes 30 exons which encode OP Al precursor proteins.
  • the OPA1 pre-cursor proteins are targeted to the mitochondria and cleaved into OPA1 long-form proteins which become anchored to the inner mitochondrial membrane, or OPA1 short-form proteins which are soluble within the cell.
  • OPA1 haploinsufficiency see Kushnareva, et al., Cell Death Dis. 2016 Jul; 7(7): e2309, dor 10.1038/cddis.2016.160).
  • Increasing the global expression of OPA1 may allow for an increase in the levels of full-length functional OPAl.
  • a cargo conjugate includes a TM that modulates target gene expression and/or target protein activity related to one or more target genes and/or target proteins associated with ADOA.
  • the target gene is OPAl.
  • the I'M acts to increase OPAl target transcript levels which may increase in functional OPAl protein levels.
  • the TM is an ASO.
  • the ASO is a PMO.
  • the TM may be used to target the 5' UTR and/or the 3' UTR of a OPAl target transcript to stabilize the transcript.
  • One of skill in the art may design suitable antisense oligonucleotides targeting the gene and/or the transcript of human OPAl based on the sequence of the gene, which is available at ncbi.nlm.nih.gov/gene/4976, “OPAl mitochondrial dynamin like GTPase [ Homo sapiens (human) ],” Gene ID: 4976.
  • a cargo conjugate has a TM designed to treat Leber congenital amaurosis (LCA).
  • a composition comprising such a cargo conjugate is ocularly delivered to a subject in need thereof, for example to treat LCA.
  • the composition is administered directly the retina.
  • the composition is administered intravitreally.
  • Leber congenital amaurosis (LCA) is a form of inherited retinal degradation causing blindness or visual impairment, often before the age of one.
  • LCA is a heterogenous disease in which mutants of at least 15 genes are associated with LCA.
  • CEP290 (15%), GUCY2D (12%) and CRB1 (10%) are the most frequently mutated LCA genes (See Prog Retin Eye Res. 2008 Jul;27(4):391-419. Doi. 10.1016/j.preteyeres.2008.05.003). At least 35 different mutations have been identified in The CEP290 gene that are associated with LCA.
  • the gene product of CEP290 is the centrosomal protein 290 kDA protein (CEP290; also known as nephrovystin-6 (NPHP6), MKS3, and BBS14).
  • CEP290 plays a role in the centrosome.
  • CEP290 also plays a role in cilia development, such as the primary cilia of the photoreceptor cells of the retina.
  • a cargo conjugate includes a TM that modulates target gene expression and/or target protein activity related to one or more target genes and/or target proteins associated with LCA.
  • the target gene is CEP290.
  • a common hypomorphic is located within intron 26 of a CEP290 transcript (c.2991+1655A>G) (see Collin, et al., Molecular Therapy-Nucleic Acids (2012) 1, el4; doi:10.1038/mtna.2012.3).
  • This mutation creates a cryptic slice donor site within intron 26.
  • the cryptic splice site results in the inclusion of an aberrant 128 base pair exon into the transcript and introduces a pre-mature stop codon (p.C998X) into the transcript Ultimately, patients with this mutation have low levels for none) of the full-length functional CEP290 protein.
  • the TM acts to increase full-length CEP290 transcript levels.
  • the full- length CEP290 transcripts lack the aberrant 128 base pair exon addition and the pre-mature stop codon.
  • the TM induces exon skipping of the aberrant 128 base pair exon in a CEP290 target transcript to restore normal splicing and result in an increase of full-length CEP290 transcript levels and/or protein levels.
  • the TM is an ASO that induces exon skipping by targeting the cryptic splice donor site to sterically block splicing factors from binding to the CEP290 target transcript to complete the cryptic splicing.
  • the ASO is a PMO.
  • Suitable antisense oligonucleotides targeting the transcript or gene of human CEP290 based on the sequence of the gene, which is available at ncbi.nlm.nih.gov/gene/80184, “entrosomal protein 290 [ Homo sapiens (human) ],” Gene ID: 80184. Macular dystrovhy/Stargardt disease
  • a cargo conjugate has a TM designed to treat macular dystrophy.
  • a composition comprising such a cargo conjugate is ocularly delivered to a subject in need thereof, for example to treat glaucoma.
  • the composition is administered directly the retina.
  • the composition is administered intravitreally.
  • Macular dystrophy is a group of diseases that cause deterioration of the retina. Examples of macular dystrophy diseases include Stargardt disease, Vitelliform macular dystrophy, and North Carolina macular dystrophy. Stargardt disease (STGD) is the most common form of macular dystrophy. STGD is characterized by the buildup of lipofuscin (lipid containing residues of lysosomal digestion) on the macula. The accumulation of lipofuscin damages the cells responsible for sharp central vision.
  • STGD Stargardt disease
  • STGD There are three known subtypes of STGD; STGD 1 , STGD2, and STDG3.
  • STDG1 is caused by mutations in the ABCA4 gene.
  • the ABCA4 gene encodes an ATP-binding cassette transporter subfamily A member 4 protein (ABCA4; See ABCA4 ATP binding cassette subfamily A member 4 [ Homo sapiens (human)], Gene ID NO; 24, ncbi.nlm.nih.gov/gene/24). ABCA4 is involved in the visual cycle.
  • ABC4A is involved in the clearance of trans-retinal and excess 11 -ci s retinal/11-cis-retinal Schiff-base conjugates from photoreceptor cells to prevent the buildup of toxic bisretinoid compounds (See Quazi and Molday, PNAS, vol 111, no 13, pg. 5024-5029, doi: 10.1073/pnas.1400780111).
  • Dysfunctional ABCA4 may result in accumulation of bisretinoid compounds which can damage cells.
  • ABC4 gene Common mutations in the ABC4 gene include c.5461- 101T>C and c.4539_2001G>A (See, e g., Dulla, et al., Investigative Opthalmology & Visual Science, July 2018, Vol., 59, 5315; and Imo, Et. AL, Genes (Basel), 2019 Jun 14; 10(6):452, doi: 10.3390/genesl0060452).
  • a cargo conjugate includes a TM that modulates target gene expression and/or target protein activity related to one or more target genes and/or target proteins associated with STGD, such as STGD1, STGD2, and STDG3.
  • the target gene is ABCA4.
  • the TM is an ASO.
  • the ASO is a PMO.
  • the cargo conjugates may include a TM that targets such genes or gene products.
  • the cargo conjugates provided herein comprises an ocular delivery construct and a therapeutic moiety or therapeutic agent.
  • the therapeutic moiety may be any suitable therapeutic moiety for treating a disease of the eye.
  • the terms “therapeutic moiety” or “therapeutic agent” refer to any molecule (e.g., polypeptide, small molecule, oligonucleotide, gene editing machinery) that is designed to have and/or has prophylactic or other biological activity.
  • the therapeutic moiety selectively binds to a target molecule associated with an ocular disease.
  • the target molecule is a macromolecule implicated in a disease or pathology of the eye.
  • the target molecule is a polypeptide or protein.
  • the target molecule is an oligonucleotide.
  • the oligonucleotide target comprises DNA.
  • the oligonucleotide target comprises genomic DNA.
  • the oligonucleotide target comprises RNA.
  • the oligonucleotide target comprises mRNA.
  • the target molecule is associated with an ocular disease.
  • selective binding of the therapeutic moiety with the target molecule is useful for the treatment of a disease, pathology or other abnormal state or condition of the eye.
  • selective binding of the therapeutic moiety to the target molecule upregulates expression or activity of the target molecule.
  • selective binding of the therapeutic moiety to the target molecule downregulates expression or activity of the target molecule.
  • the therapeutic moiety comprises a therapeutic oligonucleotide.
  • the therapeutic moiety comprises a polypeptide.
  • the therapeutic moiety comprises a small molecule.
  • the therapeutic moiety includes one or more components of gene editing machinery.
  • the therapeutic moiety comprises a therapeutic oligonucleotide.
  • the therapeutic oligonucleotide comprises an antisense oligonucleotide (ASO).
  • ASO antisense oligonucleotide
  • the therapeutic oligonucleotide comprises siRNA, RNAi, microRNA, antagomir, an aptamer, a ribozyme, an immunostimulatory oligonucleotide, a decoy oligonucleotide, a supermir, a miRNA mimic, a miRNA inhibitor, or a combination thereof (See, for example, Chery, J., “RNA therapeutics: RNAi and antisense mechanisms and clinical applications,” Postdoc J, July 2016, 4(7):35-50, and Zhu, et al., “RNA-based therapeutics: an overview and prospectus,: Cell Death & Disease, 23 July 2022, 12(644) (doi: 10.1038/s41419-022-05075-2).
  • therapeutic oligonucleotides are provided that include from about 5 to about 100 nucleic acids in length. In embodiments, the therapeutic oligonucleotide is from about 5 to about 50, about 8 to about 40, about 10 to about 30, about 15 to about 30, or about 20 to about 30 nucleotides in length. In embodiments, the therapeutic oligonucleotide includes one or more modified nucleosides, one or more modified internucleoside linkages, one or more conjugate groups, or combinations thereof.
  • the therapeutic oligonucleotide is an antisense oligonucleotide (ASO) directed to a target gene or a target transcript associated with a disease of the eye.
  • ASO antisense oligonucleotide
  • the ASO may be directed to and bind to a target nucleotide sequence located within a target gene or a target transcript.
  • the target nucleotide sequence is within a target gene and/or target transcript associated with an eye disease.
  • antisense oligonucleotide refers to an oligonucleotide that is at least partially complementary to a target sequence within a target polynucleotide.
  • An antisense oligonucleotide (ASO) is a single stranded molecule that contains DNA, RNA, or combinations or modifications thereof that are at least partially complementary to a chosen sequence, e.g., a target nucleotide sequence within a target gene or target transcript.
  • An ASO may modulate one or more aspects of gene expression and/or protein function via hybridization of the ASO with a target nucleotide sequence.
  • the ASO may modulate one or more aspects of gene expression and/or protein junction through various mechanisms.
  • hybridization of an ASO to a target nucleotide sequence of a target transcript may modulate splicing of a target transcript such as, for example, via exon skipping; exon inclusion; alternative splicing; prevent polyadenylation of the target transcript; increase the target transcript stability; induce target transcript degradation; prevent translation of the target transcript; or any combination thereof.
  • ASOs have been demonstrated to be effective and targeted inhibitors of protein synthesis, and, consequently, can be used to modulate gene expression of a targeted gene.
  • the ASO hybridizes with a target sequence of a target gene or target transcript having sequence from about 5 to about 50 nucleotides in length, which can also be referred to as the length of the ASO.
  • the ASO is from about 5 to about 50, about 8 to about 40, about 10 to about 30, about 15 to about 30, or about 20 to about 30 nucleotides in length.
  • the ASO is at least about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15, and up to about 16, about 17, about 18, about 19, about 20, about 21 , about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31 , about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, or about 50 nucleotides in length.
  • the ASO is about 15 nucleotides in length.
  • the ASO is about 16 nucleotides in length.
  • the ASO is about 17 nucleotides in length.
  • the ASO is about 18 nucleotides in length. In embodiments, the ASO is about 19 nucleotides in length. In embodiments, the ASO is about 20 nucleotides in length. In embodiments, the ASO is about 21 nucleotides in length. In embodiments, the ASO is about 22 nucleotides in length. In embodiments, the ASO is about 23 nucleotides in length. In embodiments, the ASO is about 24 nucleotides in length. In embodiments, the ASO is about 25 nucleotides in length. In embodiments, the ASO is about 26 nucleotides in length. In embodiments, the ASO is about 27 nucleotides in length. In embodiments, the ASO is about 28 nucleotides in length. In embodiments, the ASO is about 29 nucleotides in length. In embodiments, the ASO is about 30 nucleotides in length.
  • the ASO may be less than about 100 percent complementary to a target nucleotide sequence.
  • percent complementarity refers to the number of nucleobases of an ASO that have nucleobase complementarity with a corresponding nucleobase of target nucleotide sequence by the total length (number of nucleobases) of the ASO.
  • the ASOs contain no more than about 15%, no more than about 10%, no more than 5%, or no mismatches.
  • the ASOs are at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, or 100% complementary to a target nucleic acid.
  • Percent complementarity of an oligonucleotide is calculated by dividing the number of complementary nucleobases by the total number of nucleobases of the oligonucleotide.
  • Percent complementarity of a region of an oligonucleotide is calculated by dividing the number of complementary nucleobases in the region by the total number of nucleobases region.
  • incorporation of nucleotide affinity modifications allows for a greater number of mismatches compared to an unmodified compound.
  • certain oligonucleotide sequences may be more tolerant to mismatches than other oligonucleotide sequences.
  • One of ordinary skill in the art is capable of determining an appropriate number of mismatches between a an ASO and a target nucleotide sequence, such as by determining melting temperature (Tm). Tm or change in Tm (ATm) can be calculated by techniques that are familiar to one of ordinary skill in the art. For example, techniques described in Freier et al. (Nucleic Acids Research, 1997, 25, 22: 4429-4443) allow one of ordinary skill in the art to evaluate nucleotide modifications for their ability to increase the melting temperature of an RNA:DNA duplex.
  • Design of a therapeutic oligonucleotides will depend upon the target gene.
  • Targeting a therapeutic oligonucleotide to a particular target nucleotide sequence can be a multistep process. The process usually begins with the identification of gene of interest. The transcript of the gene of interest is analyzed and a target nucleotide sequence is identified. In embodiments, the target gene is a gene associated with a disease of the eye.
  • a therapeutic oligonucleotide can be designed that inhibits expression of a target gene.
  • Methods for designing, synthesizing, and screening therapeutic nucleotides for antisense activity against a preselected target nucleic acid and/or target gene can be found, for example in "Antisense Drug Technology, Principles, Strategies, and Applications” Edited by Stanley T. Crooke, CRC Press, Boca Raton, Florida, which is incorporated by reference in its entirety for any purpose.
  • the efficacy of a therapeutic oligonucleotide may be assessed by evaluating the antisense activity effected by their administration.
  • the term "antisense activity” refers to any detectable and/or measurable activity attributable to the hybridization of a therapeutic oligonucleotide to its target nucleotide sequence. Such detection and/or measuring may be direct or indirect.
  • antisense activity is assessed by detecting and or measuring the amount of target protein in a cell or population of cells before and after administration of the therapeutic oligonucleotide to the cell or population of cells.
  • antisense activity is assessed by detecting and/or measuring the amount of target transcript in a cell or population of cells.
  • Therapeutic oligonucleotides comprise nucleosides linked through intemucleoside linkages.
  • Nucleosides include a pentose sugar (e.g., ribose or deoxyribose) and a nitrogenous base (nucleobase or simply base) covalently attached to sugar.
  • the naturally occurring (traditional) bases found in DNA and/or RNA are adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U).
  • a naturally occurring (traditional) nucleoside linkage is a phosphodiester bond.
  • the therapeutic oligonucleotides may have all natural sugars, natural bases, and natural intemucleoside linkages.
  • nucleosides are routinely used for incorporation into therapeutic oligonucleotides to enhance one or more properties, such as nuclease resistance, pharmacokinetics, or affinity for a target gene or target transcript.
  • Non-limiting examples of nucleosides are provided in FIG. 2 and in Khvorova et al. Nature Biotechnology (2017) 35: 238-248, which is incorporated by reference herein in its entirety.
  • a therapeutic oligonucleotide has one or more modified nucleosides.
  • a therapeutic oligonucleotide has one or more modified sugars.
  • a therapeutic oligonucleotide has one or more modified bases.
  • a therapeutic oligonucleotide has one or more modified intemucleoside linkages.
  • nucleobase is any group that contains one or more atoms or groups of atoms capable of hydrogen bonding to a base of another nucleic acid.
  • nucleobases many modified nucleobases or nucleobase mimetics known to those skilled in the art are amenable with the compounds described herein.
  • modified nucleobase and nucleobase mimetic can overlap, but generally a modified nucleobase refers to a nucleobase that is fairly similar in structure to the parent nucleobase, such as for example a 7-deaza purine, a 5-methyl cytosine, or a G-clamp, whereas a nucleobase mimetic generally includes more complicated structures, such as for example a tricyclic phenoxazine nucleobase mimetic. Methods for preparation of the above noted modified nucleobases are well known to those skilled in the art.
  • a therapeutic oligonucleotide includes one or more nucleosides having a modified sugar moiety.
  • the furanosyl sugar ring of a natural nucleoside can be modified.
  • a furanosyl sugar ring may be modified in any suitable manner, including, but not limited to, addition of a substituent group, bridging of two non-geminal ring atoms to form a bicyclic nucleic acid (BNA) and substitution of an atom or group such as -S-, -N(R)- or -C(R1 )(R2) for the ring oxygen at the 4'-position.
  • BNA bicyclic nucleic acid
  • Modified sugar moieties are well known and can be used to alter, typically increase, the affinity of the antisense compound for its target and/or increase nuclease resistance.
  • a representative list of modified sugars includes, but is not limited to, non- bicyclic substituted sugars, especially non-bicyclic 2'-substituted sugars having a 2'-F, 2 -OCH3 or a 2'-O(CH2)2-OCH3 substituent group, and 4-thio modified sugars.
  • Sugars can also be replaced with a sugar mimetic group, for example, a morpholino ring, a methylenemorpholine ring, among others.
  • a therapeutic oligonucleotide may include one or more bicyclic modified sugars (SNA's), such as, for example, LNA (4*-(CH2)-O-2' bridge), 2'-thio-LNA (4'-(CH2)-S-2' bridge), 2'-amino-LNA (4'-(CH2)-NR-2' bridge), ENA (4'-(CH2)2-O-2' bridge), 4'-(CH2)3-2' bridged BNA, ⁇ -(CIECHfCIB))- ⁇ bridged BNA” cEt (4'-(CH(CH3>0-2' bridge), and cMOE BNAs (4'-(CH(CH2OCH3)-O-2' bridge).
  • SNA's bicyclic modified sugars
  • a therapeutic oligonucleotide may include one or more locked nucleic acids" (LN As) in which the 2'-hydroxyl group of the ribosyl sugar ring is linked to the 4' carbon atom of the sugar ring thereby forming a 2'-C,4'-C-oxymethylene linkage to form the bicyclic sugar moiety.
  • LNA monomers adenine, cytosine, guanine, 5- methyl-cytosine, thymine and uracil, along with their oligomerization, and nucleic acid recognition properties have been described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). LNAs and preparation thereof are also described in WO 98/39352 and WO 99/14226.
  • Intemucleoside linking groups link the nucleosides or otherwise modified monomer units of an oligonucleotide together.
  • the two main classes of intemucleoside linking groups are defined by the presence or absence of a phosphorus atom.
  • Representative phosphorus containing intemucleoside linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidate, phosphorodiamidate, and phosphorothioates.
  • non-phosphorus containing intemucleoside linking groups include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-O-C(O)-S-), thionocarbamate (-O-C(OXNH)-S-); siloxane (-O-Si(H)2-O-); and N.N'-dimethylhydrazine (- CH2-N(CH3)-N(CH3)-).
  • Therapeutic oligonucleotides having non-phosphorus intemucleoside linking groups are referred to as oligonucleosides.
  • Modified intemucleoside linkages can be used to alter, typically increase, nuclease resistance of the therapeutic oligonucleotides.
  • Intemucleoside linkages having a chiral atom can be prepared racemic, chiral, or as a mixture.
  • Representative chiral intemucleoside linkages include, but are not limited to, alkylphosphonates and phosphorothi oates. Methods of preparation of phosphorous- containing and non-phosphorous-containing linkages are well known to those skilled in the art.
  • a phosphate group can be linked to the 2', 3' or 5' (or 6', for a 6 membered ring, such as a methylenemorpholine ring) hydroxyl moiety of the sugar (or sugar mimetic).
  • the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound.
  • the phosphate groups are commonly referred to as forming the intemucleoside backbone of the oligonucleotide.
  • the normal linkage or backbone of RNA and DNA is a 3' to 5* phosphodiester linkage.
  • the oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO) comprising a backbone of methylenemorpholine rings linked through phosphorodiamidate intemucleoside linkages.
  • PMO phosphorodiamidate morpholino oligomer
  • PMOs are uncharged nucleic acid analogs bind to target nucleic acid through base paring. PMOs that bind to mRNA may block interaction of proteins to the mRNA through steric blockade (See, e.g., Nan and Zhang, Front. Microbiol. 20 April 2019 (doi.org/10.3389/fmicb .2018.00750)). As uncharged, or net neutral charged, oligonucleotides, PMOs are particularly effective for intracellular delivery with ocular delivery construct.
  • the therapeutic oligonucleotides may contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations that may be defined, in terms of absolute stereochemistry, as (R) or (S); a or 0; or as (D) or (L). Included in the antisense compounds provided herein are all such possible isomers, as well as their racemic and optically pure forms.
  • therapeutic oligonucleotides are modified by covalent attachment of one or more conjugate groups.
  • conjugate groups modify one or more properties of the attached therapeutic oligonucleotides including but not limited to pharmacodynamic, pharmacokinetic, binding, absorption, cellular distribution, cellular uptake, chaige and clearance.
  • Conjugate groups are routinely used in the chemical arts and are linked directly or via an optional linking moiety or linking group to a parent compound such as a therapeutic oligonucleotides.
  • Conjugate groups include without limitation, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins and dyes.
  • the conjugate group is a polyethylene glycol (PEG), and the PEG is conjugated to either the therapeutic oligonucleotide, a linker, an EP, or the cyclic peptide.
  • the therapeutic moiety comprises one or more component of gene-editing machineiy.
  • gene-editing machinery refers to protein, nucleic acids, or combinations thereof, which may be used to edit a genome.
  • Non-limiting examples of gene-editing machinery include guide RNAs (gRNAs), nucleases, nuclease inhibitors, and combinations and complexes thereof.
  • the gene editing machineiy may be used to repair a mutated gene or to introduce a mutation into a gene.
  • the gene may be a gene associated with a disease of the eye.
  • a linker conjugates the ocular delivery construct to the one or more components of gene-editing machinery. Any linker described in this disclosure or that is known to a person of skill in the art may be utilized.
  • the therapeutic moiety includes a guide RNA (gRNA).
  • gRNA guide RNA targets a genomic loci in a prokaryotic or eukaryotic cell.
  • the gRNA is a single-molecule guide RNA (sgRNA).
  • a sgRNA includes a spacer sequence and a scaffold sequence.
  • a spacer sequence is a short nucleic acid sequence used to target a nuclease (e.g., a Cas9 nuclease) to a specific nucleotide region of interest (e.g., a genomic DNA sequence to be cleaved).
  • the spacer may be about 17-24 bases in length, such as about 20 bases in length.
  • the spacer targets a site that immediately precedes a 5' protospacer adjacent motif (PAM).
  • the PAM sequence may be selected based on the desired nuclease.
  • the PAM sequence may be any one of the PAM sequences shown in Table 1 below, wherein N refers to any nucleic acid, R refers to A or G, Y refers to C or T, W refers to A or T, and V refers to A or C or G. Table 1. Nucleases and PAM sequences
  • a spacer may target a sequence of a mammalian gene, such as a human gene. In embodiments, the spacer may target a mutant gene. In embodiments, the spacer may target a coding sequence. In embodiments, the spacer may target an exonic sequence. In embodiments, the spacer may target a polyadenylation site (PS). In embodiments, the spacer may target a sequence element of a PS. In embodiments, the spacer may target a polyadenylation signal (PAS), an intervening sequence (IS), a cleavage site (CS), a downstream element (DES), or a portion or combination thereof. In embodiments, a spacer may target a splicing element (SE) or a cis-splicing regulatory element (SRE).
  • PS polyadenylation site
  • PAS polyadenylation signal
  • IS intervening sequence
  • CS cleavage site
  • DES downstream element
  • a spacer may target a splicing element (SE) or
  • the scaffold sequence is the sequence within the sgRNA that is responsible for nuclease (e.g., Cas9) binding.
  • the scaffold sequence does not include the spacer/targeting sequence.
  • the scaffold may be about 10 to about 150 nucleotides in length, or about 50 to about 100 nucleotides in length.
  • the gRNA is single guide RNA molecule comprising the spacer and the scaffold.
  • the gRNA comprises two molecules that hybridize to form the gRNA.
  • An example of a gRNA that includes two molecules is a gRNA comprising a crRNA and tracrRNA.
  • the gRNA or one or more components thereof may further include a poly(A) tail.
  • a compound that includes a CPP is conjugated to a nucleic acid that includes a gRNA or a component thereof.
  • the nucleic acid includes about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11 , about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 gRNAs or components thereof.
  • the gRNAs recognize the same target. In embodiments, the gRNAs recognize different targets.
  • a compound that includes a CPP is conjugated to a nucleic acid designed to express the gRNA within a cell.
  • the nucleic acid may include a promoter sequence to drive expression of the gRNA.
  • the therapeutic moiety includes a nuclease.
  • the nuclease is a Type II, Type V-A, Type V-B, Type VC, Type V-U, Type VI-B nuclease.
  • the nuclease is a transcription, activator-like effector nuclease (TALEN), a meganuclease, or a zinc-finger nuclease or a modified form or variant thereof.
  • TALEN transcription, activator-like effector nuclease
  • TALEN transcription, activator-like effector nuclease
  • meganuclease or a zinc-finger nuclease or a modified form or variant thereof.
  • the nuclease is a Cas9, Casl2a (Cpfl), Casl2b, Casl2c, Tnp-B like, Casl3a (C2c2), Casl3b, or Casl4 nuclease or a modified form or variant thereof.
  • the nuclease is a Cas9 nuclease or a Cpfl nuclease
  • the ocular delivery construct is conjugated to a nucleic acid encoding a nuclease.
  • the nucleic acid encoding a nuclease includes a sequence encoding a promoter, wherein the promoter drives expression of the nuclease.
  • the therapeutic moiety includes a ribonucleoprotein (RNP) that includes a gRNA and a nuclease.
  • RNP ribonucleoprotein
  • a RNP is a complex of a gRNA bound to a nuclease.
  • the gRNA, the nuclease, or both may be covalently attached to an ocular delivery construct to form a cargo conjugate having an RNP therapeutic moiety.
  • a composition that includes: (a) a cargo conjugate comprising an ocular delivery' construct conjugated to a gRNA and (b) a nuclease is delivered to a cell.
  • a composition that includes: (a) a cargo conjugate comprising an ocular delivery construct conjugated to a nuclease and (b) an gRNA is delivered to a cell.
  • a composition that includes: (a) a first cargo conjugate comprising a first delivery construct conjugated to a gRNA and (b) a second cargo conjugate comprising a second delivery construct conjugated to a nuclease is delivered to a cell.
  • a cargo conjugate comprises an ocular delivery construct conjugated to a nucleic acid encoding a gRNA and/or a nuclease.
  • the nucleic acid encoding a nuclease and a gRNA includes a sequence encoding a promoter, wherein the promoter drives expression of the nuclease and the gRNA.
  • the nucleic acid encoding a nuclease and a gRNA includes two promoters, wherein a first promoter controls expression of the nuclease and a second promoter controls expression of the gRNA.
  • the nucleic acid encoding a gRNA and a nuclease encodes from about 1 to about 20 gRNAs, or from about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, or about 19, and up to about 20 gRNAs.
  • the gRNAs recognize different targets. In embodiments, the gRNAs recognize the same target.
  • the therapeutic moiety includes a nuclease inhibitor.
  • a limitation of gene editing is potential off-target editing.
  • the delivery of a nuclease inhibitor may limit off-target editing.
  • the nuclease inhibitor is a polypeptide, polynucleotide, or small molecule.
  • the therapeutic moiety includes a therapeutic polypeptide.
  • the therapeutic polypeptide includes a peptide inhibitor.
  • the peptide inhibitor inhibits a protein associated with a disease of the eye.
  • the therapeutic polypeptide includes a peptide replacement therapy to functionally replace an aberrantly expressed protein associated with a disease of the eye.
  • the therapeutic moiety includes a protein or a fragment thereof. In embodiments, the therapeutic moiety includes an RNA binding protein or an RNA binding fragment thereof. In embodiments, the therapeutic moiety includes an enzyme. In embodiments, the therapeutic moiety includes an RNA-cleaving enzyme or an active fragment thereof.
  • the therapeutic moiety includes an antibody or an antigen-binding fragment.
  • Antibodies and antigen-binding fragments can be derived from any suitable source, including human, mouse, camelid (e.g., camel, alpaca, llama), rat, ungulates, or non-human primates (e.g., monkey, rhesus macaque).
  • antibody includes intact polyclonal or monoclonal antibodies and antigenbinding fragments thereof.
  • a native immunoglobulin molecule includes two heavy chain polypeptides and two light chain polypeptides.
  • Each of the heavy chain polypeptides associate with a light chain polypeptide by virtue of interchain disulfide bonds between the heavy and light chain polypeptides to form two heterodimeric proteins or polypeptides (i.e., a protein that includes two heterologous polypeptide chains).
  • the two heterodimeric proteins then associate by virtue of additional interchain disulfide bonds between the heavy chain polypeptides to form an immunoglobulin protein or polypeptide.
  • the therapeutic moiety is an antigen-binding fragment that binds to a target protein associated with a disease of the eye.
  • An antibody may modulate the activity of the target protein to which it binds.
  • the therapeutic moiety is an antigen-binding fragment that binds to a target transcript of a protein (Ye et al., PNAS (2008), 105(l):82-87; and Jung et al., (RNA (2014), 20(6): 805-814).
  • an antigen-binding fragment that binds to a target protein includes 1, 2, 3, 4, 5, or all 6 CDRs of a variable heavy chain (VH) and/or a variable light chain (VL) sequence from an antibody that specifically binds to the target protein.
  • the antigen binding fragment includes 1, 2, or 3 of the CDRs of a camelid single domain antibody such as the VHH region.
  • the antigen-binding fragment that binds to a target protein is a portion of a full-length antibody, such as Fab, F(ab’)2, Fab’, Fv fragments, minibodies, diabodies, single domain antibody (dAb), single-chain variable fragments (scFv), multispecific antibodies formed from antibody fragments, or any other modified configuration of the immunoglobulin molecule that includes an antigen-binding site or fragment of the required specificity.
  • the therapeutic moiety includes a bispecific antibody.
  • Bispecific antibodies are antibodies that can simultaneously bind two separate and unique antigens (or different epitopes of the same antigen).
  • the therapeutic moiety includes a bispecific antibody that can simultaneously bind to a target protein associated with an eye disease and another target protein.
  • Non-limiting examples include scFv (single-chain variable fragment), BsDb (bispecific diabody), scBsDb (single-chain bispecific diabody), scBsTaFv (single-chain bispecific tandem variable domain), DNL-(Fab)3 (dock-and-lock trivalent Fab), sdAb (singledomain antibody), and BssdAb (bispecific single-domain antibody).
  • BsAbs with an Fc region are useful for carrying out Fc mediated effector functions such as antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. They have the half-life of normal IgG.
  • BsAbs without the Fc region rely solely on their antigen-binding capacity for carrying out therapeutic activity. Due to their smaller size, these fragments have better solid-tumor penetration rates. BsAb fragments do not require glycosylation, and they may be produced in bacterial cells. The size, valency, flexibility and half-life of BsAbs to suit the application.
  • the therapeutic moiety includes a “diabody.”
  • diabody refers to a bispecific antigen-binding antibody fragment in which VH and VL domains are expressed in a single polypeptide chain using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen-binding sites (see, e g., Holliger et al.. Proc. Natl. Acad. Sci. USA 90:6444-48 (1993) and Poljak et al., Structure 2:1121- 23 (1994)).
  • Diabodies may be designed to bind to two distinct antigens and are bi-specific antigen binding constructs.
  • the therapeutic moiety includes a “nanobody” or a “single domain antibody” (which can also be referred to herein as sdAbs or VHH).
  • Single domain antibody refers to an antigen-binding fragment that includes a single monomeric variable antibody domain comprising one variable domain (VH) of a heavy-chain antibody.
  • VH variable domain
  • the variable chain is the VHH of a cam elid single chain antibody.
  • the therapeutic moiety includes a minibody.
  • the therapeutic moiety is an antibody mimetic.
  • Antibody mimetics are compounds that, like antibodies, can specifically bind antigens, but that are not structurally related to antibodies. They are usually artificial peptides or proteins with a molar mass of about 3 to 20 kD (compared to the molar mass of antibodies at -150 kDa.). Examples of antibody mimetics include affibody molecules affilins, affimers, affitins, alphabodies anticalins, avimers, DARPins, fynomers Kunitz domain peptides and monobodies.
  • the therapeutic moiety includes a peptide.
  • the peptide acts as an agonist, increasing the activity of a target protein.
  • the peptide acts as an antagonist, decreasing the activity of a target protein.
  • the peptide is configured to inhibit protein-protein interaction (PPI). Protein-protein interactions (PPIs) are important in many biochemical processes, including transcription of nucleic acid and various post-translational modifications of translated proteins.
  • PPIs can be experimentally determined by biophysical techniques such as X-ray crystallography, NMR spectroscopy, surface plasma resonance (SPR), bio-layer interferometry (BLI), isothermal titration calorimetry (ITC), radio-ligand binding, spectrophotometric assays and fluorescence spectroscopy.
  • biophysical techniques such as X-ray crystallography, NMR spectroscopy, surface plasma resonance (SPR), bio-layer interferometry (BLI), isothermal titration calorimetry (ITC), radio-ligand binding, spectrophotometric assays and fluorescence spectroscopy.
  • Peptides that inhibit protein-protein interaction can be referred to as peptide inhibitors.
  • the therapeutic moiety includes a peptide inhibitor.
  • the peptide inhibitor includes from about 5 to about 100 amino acids, from about 5 to about 50 amino acids; from about 15 to about 30 amino acids; or from about 20 to about 40 amino acids.
  • the peptide inhibitor includes one or more chemical modifications, for example, to reduce proteolytic degradation and/or to improve in vivo half-life.
  • the peptide inhibitor includes one or more synthetic amino acids and/or a backbone modification.
  • the peptide inhibitor has an a-helical structure.
  • the peptide inhibitor is configured to disrupt one or more function of a protein associated with a disease of the eye. In embodiments, the peptide inhibitor is configured to disrupt formation of protein complexes. In embodiments, binding of the peptide inhibitor to the protein blocks dimer formation.
  • the therapeutic moiety includes a small molecule for treating a di sease of the eye.
  • the small molecule does not readily gain access to an intracellular compartment of a cell of the eye when delivered by itself (not conjugated to an ocular delivery construct).
  • a cargo conjugate includes an ocular delivery construct.
  • An ocular delivery construct may be used to transport a cargo (e.g., a therapeutic moiety) to a structure of the eye, an eye tissue, and/or a cell-type of the eye.
  • a cargo e.g., a therapeutic moiety
  • an ocular delivery construct may be used to transport a cargo across a cell membrane, for example, to deliver the cargo to the cytosol or nucleus of a cell in the eye.
  • An ocular delivery construct can comprise a cyclic cell penetrating peptide (cCPP); a cCPP and a linker; a cCPP and an exocyclic peptide (EP); or an endosomal escape vehicle (EEV) which comprises a cCPP, an EP, and a linker.
  • cCPP cyclic cell penetrating peptide
  • EP exocyclic peptide
  • EEV endosomal escape vehicle
  • the configuration of the ocular delivery construct depends at least in part on the components of the ocular delivery construct. Two or more components that are coupled, conjugated, or linked are a part of the same compound.
  • the ocular delivery construct comprises a cCPP, and the cCPP is conjugated to the cargo to from a cargo conjugate.
  • the ocular delivery construct comprises a cCPP and a linker, and the cCCP is coupled to the linker and the linker is conjugated to the cargo to form a cargo conjugate.
  • the ocular delivery construct comprises a cCPP and an EP, and the cCPP is coupled to the EP and the EP is conjugated to the cargo to from the cargo conjugate; the cCPP is coupled to the EP and the cCPP is conjugated to the cargo to form the cargo conjugate; or both the cCPP and the EP are conjugated to the cargo to form the cargo conjugate.
  • the ocular delivery construct comprises an EEV
  • the EP is coupled to the cCPP
  • the cCPP is coupled to the linker, and the linker is conjugated to the cargo
  • the cCPP is coupled to the EP
  • the EP is coupled to the linker, and the linker is conjugated to the cargo
  • the cCPP is coupled to the linker, the EP is coupled to the linker, and the EP is conjugated to the cargo
  • the EP is coupled to the linker, the cCPP is coupled to the linker, and the cCPP is conjugated to the cargo
  • the EP is coupled to the linker, the cCPP is coupled to the linker, and the linker is conjugated to the cargo to from a cargo conjugate.
  • the ocular delivery constructs comprise at least one cell penetrating peptide (CPP).
  • the cell penetrating peptide can be a cyclic cell penetrating peptide (cCPP)
  • the ocular delivery constructs comprise one cell penetrating peptide (cCPP).
  • the ocular delivery constructs comprise two cell penetrating peptides (cCPP).
  • the cCPP is capable of penetrating a cell membrane.
  • the cCPP can deliver the cargo to the cytosol of the cell.
  • the cCPP can deliver the cargo to a cellular location where a target gene, target transcript, and/or target protein is located.
  • an EP, and/or a linker at least one bond or lone pair of electrons on the cCPP can be replaced.
  • the total number of amino acid residues in the cCPP is in the range of from 6 to 20 amino acid residues, e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues, inclusive of all ranges and subranges therebetween.
  • the cCPP can comprise 6 to 13 amino acid residues.
  • the cCPP can comprise 6 to 10 amino acids.
  • cCPP comprising 6-10 amino acid residues can have a structure according to any of Formula I-A to I-E:
  • AAi, AA2, AA3, AA4, AAs, AAe, AA?, AAg, AAg, and AA10 are amino acid residues.
  • the cCPP can comprise 6 to 8 amino acids.
  • the cCPP can comprise 8 amino acids.
  • Each amino acid in the cCPP may be a natural or non-natural amino acid.
  • Abbreviations used herein for some natural and non-natural amino acids are shown in Table 2.
  • amino acid refers to compounds having an amino group and a carboxylic acid group. Most amino acids (except for glycine) also have a side chain. As used herein, “amino acid side chain” or “side chain” refers to the characterizing substituent bound to the a-carbon of the amino acid.
  • an “a-amino acid” i s an amino acid in which the amino group is attached to the first (alpha) carbon adjacent to the carboxylic acid group, such that the carbon atom of the carbonyl is separated from the nitrogen atom of the amino group by one carbon atom.
  • a “b-amino acid” (also called “beta-amino acid,” and “P-amino acid”) is an analog of an a -amino acid in which the amino group is attached to the second (beta) carbon, rather than the alpha-carbon, such that the carbon atom of the carbonyl is separated from the nitrogen atom of the amino group by two carbon atoms.
  • b-amino acids include but are not limited to b-alanine and b-homophenylalanine.
  • An “uncharged” amino acid is an amino acid that does not have a charge at a physiological pH (between 5.0 and 8.0). It is noted that histidine can exist in neutral or positively charged forms at physiological pH.
  • a side chain that does not comprise an aryl or heteroaryl group can be referred to herein as a “non-aiyl” side chain.
  • the side chain that does not comprise an aryl or heteroaryl group can be uncharged and is referred to herein as an uncharged, non-aryl side chain.
  • Amino acids with uncharged non-aryl amino side chains include, but are not limited to, histidine, threonine; serine; leucine; isoleucine, valine; neopentylglycine, alanine; homoalanine, homoserine; 3-(4-thiazolyl)-alanine; 3-(4-furanyl)-alanine; 3-(4-thienyl)-alanine; and b-amino acid derivatives thereof.
  • non-natural amino acid refers to an organic compound that is a congener of a natural amino acid in that it has a structure similar to a natural amino acid so that it mimics the structure and reactivity of a natural amino acid.
  • the non-natural amino acid can be a modified amino acid, and/or amino acid analog, that is not one of the 20 common naturally occurring amino acids or the rare natural amino acids selenocysteine or pyrrolysine.
  • Non-natural amino acids can also be a D-isomer of a natural amino acid.
  • suitable amino acids include, but are not limited to, alanine, allosoleucine, arginine, citrulline, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, napthylalanine, phenylalanine, proline, pyroglutamic acid, serine, threonine, tryptophan, tyrosine, valine, a derivative thereof, or combinations thereof.
  • the cCPP can comprise 2 contiguous amino acids with hydrophobic side chains. In embodiments, the cCPP can comprise 3 contiguous amino acids with hydrophobic side chains.
  • the hydrophobicity of amino acid residues can be measured and/or calculated using a variety of techniques. In embodiments, the hydrophobicity of an amino acid residue can be determined by calculating its consensus value on the consensus scale of D. Eisenberg et al., using the method described in D. Eisenberg et al., “Hydrophobic Moments and Protein Structure,” Faraday Symp. Chem. Soc. 1982, 17, 109-120 (e.g., D. Eisenberg et al.).
  • a hydrophobic amino acid is an amino acid that has a hydrophobic side chain.
  • one or two amino acids in the cCPP can have no side chain. In embodiments, all amino acids in the cCPP have a side chain. As used herein, when no side chain is present, the amino acid has two hydrogen atoms on the carbon atom(s) (e.g., -CH?) linking the amine and carboxylic acid of the amino acid residue.
  • the amino acid having no side chain can be glycine or beta-alanine.
  • the cCPP can comprise from 6 to 20, from 6 to 10, or from 6 to 8 amino acid residues, wherein: (i) at least two amino acids can, independently, be glycine, b-alanine, serine, histidine or 4-aminobutyric acid; (ii) at least two amino acids can have a side chain comprising an aryl or heteroaryl group; and (iii) at least two amino acid has a side chain comprising a guanidine group, or a protonated form thereof.
  • two amino acids can, independently, be glycine, b-alanine, serine, histidine or 4-aminobutyric acid;
  • two or three amino acids can have a side chain comprising an aryl or heteroaryl group; and
  • two amino acid has a side chain comprising a guanidine group, or a protonated form thereof.
  • one amino acid of the cCPP can be glycine, b-alanine, serine, histidine, or 4-aminobutyric acid.
  • two amino acids can be, independently, glycine, b- alanine, serine, histidine, or 4-aminobutyric acid.
  • three amino acids can be glycine, b-alanine, serine, histidine, or 4-aminobutyric acid.
  • one amino acid of the cCPP can have a side chain comprising an aryl or heteroaryl group.
  • two amino acids of the cCPP can have a side chain comprising an aryl or heteroaryl group.
  • three amino acids of the cCPP can have a side chain comprising an aryl or heteroaryl group.
  • one amino acid of the cCPP can have a side chain that does not comprise an aryl or heteroaryl group, referred to herein as a “non-aryl” side chain.
  • the side chain that does not comprise an aryl or heteroaryl group can be uncharged and is referred to herein as an uncharged, non-aryl side chain.
  • two amino acids of the CPP e g., cCPP
  • three amino acids of the CPP e.g., cCPP
  • Amino acids with uncharged non-aryl amino side chains include, but are not limited to, histidine; threonine; serine; leucine; isoleucine; valine; neopentylglycine; alanine; homoalanine; homoserine; 3-(4-thiazolyl)-alanine, 3-(4-furanyl)- alanine; and 3-(4-thienyl)-alanine.
  • one amino acid of the cCPP has a side chain comprising a guanidine group, or a protonated form thereof.
  • two amino acids of the cCPP can have a side chain comprising a guanidine group, or a protonated form thereof.
  • three amino acids of the cCPP can have a side chain comprising a guanidine group, or a protonated form thereof.
  • four amino acids of the cCPP can have a side chain comprising a guanidine group, or a protonated form thereof.
  • the cCPP can comprise 4 to 20 amino acids, such as 6 to 20 amino acids, wherein: (i) at least one amino acid has a side chain comprising a guanidine group, or a protonated form thereof; (ii) at least one amino acid has no side chain or a side chain comprising , or a protonated form thereof; and (iii) at least two amino acids independently have a side chain comprising an aryl or heteroaryl group.
  • At least two amino acids can have no side chain or a side chain comprising , or a protonated form thereof.
  • the amino acid when no side chain is present, the amino acid has two hydrogen atoms on the carbon atom(s) (e g., -CH2-) linking the amine and carboxylic acid.
  • the amino acid having no side chain can be glycine or P-alanine.
  • the cCPP can comprise from 6 to 20 amino acid residues, wherein: (i) at least two amino acids can be glycine, p-alanine, or 4-aminobutyric acid residues; (ii) at least two amino acids can have a side chain comprising an aryl or heteroaryl group; and (iii) at least two amino acids can have a side chain comprising a guanidine group, , or a protonated form thereof.
  • the cCPP can comprise from 6 to 20 amino acid residues, wherein: (i) 2 amino acids can independently be glycine, P-alanine, or 4-aminobutyric acid residues; (ii) 2 or 3 amino acids can have a side chain comprising an aryl or heteroaryl group; and (iii) 2 amino acids have a side chain comprising a guanidine group, , or a protonated form thereof.
  • the cCPP can comprise from 6 to 20 amino acid residues, wherein: (i) at least three amino acids can independently be glycine, P-alanine, or 4-aminobutyric acid residues; (ii) at least one amino acid can have a side chain comprising an aryl or heteroaryl group; and (iii) at least one amino acid can have a side chain comprising a guanidine group, , or a protonated form thereof.
  • the cCPP can comprise (i) 1, 2, 3, 4, 5, or 6 glycine, P-alanine, 4- aminobutyric acid residues, or combinations thereof.
  • the cCPP can comprise (i) 2 glycine, P-alanine, 4-aminobutyric acid residues, or combinations thereof.
  • the cCPP can comprise (i) 3 glycine, P-alanine, 4-aminobutyric acid residues, or combinations thereof.
  • the cCPP can comprise (i) 4 glycine, p-alanine, 4-aminobutyric acid residues, or combinations thereof.
  • the cCPP can comprise (i) 5 glycine, P-alanine, 4- aminobutyric acid residues, or combinations thereof. In embodiments, the cCPP can comprise (i) 6 glycine, P-alanine, 4-aminobutyric acid residues, or combinations thereof. In embodiments, the cCPP can comprise (i) 3, 4, or 5 glycine, P-alanine, 4-aminobutyric acid residues, or combinations thereof. In embodiments, the cCPP can comprise (i) 3 or 4 glycine, P-alanine, 4-aminobutyric acid residues, or combinations thereof.
  • the cCPP can comprise (i) 1, 2, 3, 4, 5, or 6 glycine residues.
  • the cCPP can comprise (i) 2 glycine residues.
  • the cCPP can comprise (i) 3 glycine residues.
  • the cCPP can comprise (i) 4 glycine residues.
  • the cCPP can comprise (i) 5 glycine residues.
  • the cCPP can comprise (i) 6 glycine residues.
  • the cCPP can comprise (i) 3, 4, or 5 glycine residues.
  • the cCPP can comprise (i) 3 or 4 glycine residues.
  • the cCPP can comprise (i) 2 or 3 glycine residues.
  • the cCPP can comprise (i) 1 or 2 glycine residues.
  • the cCPP can comprise (i) 3, 4, 5, or 6 glycine, p-alanine, 4-aminobutyric acid residues, or combinations thereof. In embodiments, the cCPP can comprise (i) 3 glycine, p- alanine, 4-aminobutyric acid residues, or combinations thereof. In embodiments, the cCPP can comprise (i) 4 glycine, P-alanine, 4-aminobutyric acid residues, or combinations thereof. In embodiments, the cCPP can comprise (i) 5 glycine, P-alanine, 4-aminobutyric acid residues, or combinations thereof.
  • the cCPP can comprise (i) 6 glycine, P-alanine, 4- aminobutyric acid residues, or combinations thereof. In embodiments, the cCPP can comprise (i) 3, 4, or 5 glycine, P-alanine, 4-aminobutyric acid residues, or combinations thereof. In embodiments, the cCPP can comprise (i) 3 or 4 glycine, P-alanine, 4-aminobutyric acid residues, or combinations thereof.
  • the cCPP can comprise at least three glycine residues. In embodiments, the cCPP can comprise (i) 3, 4, 5, or 6 glycine residues. In embodiments, the cCPP can comprise (i) 3 glycine residues. In embodiments, the cCPP can comprise (i) 4 glycine residues. In embodiments, the cCPP can comprise (i) 5 glycine residues. In embodiments, the cCPP can comprise (i) 6 glycine residues. In embodiments, the cCPP can comprise (i) 3, 4, or 5 glycine residues. The cCPP can comprise (i) 3 or 4 glycine residues.
  • none of the glycine, P-alanine, or 4-aminobutyric acid residues in the cCPP are contiguous.
  • two or three glycine, P-alanine, 4-or aminobutyric acid residues can be contiguous.
  • two glycine, P-alanine, or 4-aminobutyric acid residues can be contiguous.
  • none of the glycine residues in the cCPP are contiguous.
  • each glycine residues in the cCPP can be separated by an amino acid residue that is not glycine.
  • two or more of the glycine residues in the cCPP are contiguous. In embodiments, two or three glycine residues are contiguous. In embodiments, two glycine residues are contiguous
  • the cCPP can comprise (ii) 2, 3, 4, 5 or 6 amino acid residues independently having a side chain comprising an aryl or heteroaryl group. In embodiments, the cCPP can comprise (ii) 2 amino acid residues independently having a side chain comprising an aryl or heteroaryl group. In embodiments, the cCPP can comprise (ii) 3 amino acid residues independently having a side chain comprising an aryl or heteroaryl group. In embodiments, the cCPP can comprise (ii) 2, 3, or 4 amino acid residues independently having a side chain comprising an aryl or heteroaryl group. In embodiments, the cCPP can comprise (ii) 2 or 3 amino acid residues independently having a side chain comprising an aryl or heteroaiyl group.
  • the cCPP can comprise (ii) 2, or 3amino acid residues independently having a side chain comprising an aryl group. In embodiments, the cCPP can comprise (ii) 2 amino acid residues independently having a side chain comprising an aryl group. In embodiments, the cCPP can comprise (ii) 3 amino acid residues independently having a side chain comprising an aryl group. In embodiments, the cCPP can comprise (ii) 2 or 3 amino acid residues independently having a side chain comprising an aryl group.
  • the aryl group can be a 6- to 14-membered aryl.
  • Aryl can be phenyl, naphthyl or anthracenyl, each of which is optionally substituted.
  • Aryl can be phenyl or naphthyl, each of which is optionally substituted.
  • the heteroaryl group can be a 6- to 14-membered heteroaryl having 1, 2, or 3 heteroatoms selected from N, O, and S. Heteroaryl can be pyridyl, quinolyl, or isoquinolyl.
  • the amino acid residue having a side chain comprising an aryl or heteroaryl group can each independently be bis(homonaphthylalanine); homonaphthylalanine; naphthylalanine; phenylglycine; bis(homophenylalanine); homophenylalanine, phenylalanine, tryptophan, or tyrosine, each of which is optionally substituted with one or more substituents.
  • the amino acid having a side chain comprising an aryl or heteroaryl group can each independently be selected from: naphthylalanine; homophenylalanine and phenylalanine.
  • the amino acid residue having a side chain comprising an aryl or heteroaryl group can each be independently a residue of phenylalanine, naphthylalanine, phenyl glycine, homophenylalanine, homonaphthylalanine, bis(homophenylalanine), bis-(homonaphthylalanine), tryptophan, or tyrosine, each of which is optionally substituted with one or more substituents.
  • the amino acid residue having a side chain comprising an aryl group can each independently be a residue of tyrosine, phenylalanine; 1 -naphthylalanine, 2-naphthylalanine; tryptophan; phenylglycine; homophenylalanine; or P-homophenylalanine;.
  • the amino acid residue having a side chain comprising an aryl group can each independently be a residue of phenylalanine, 2- naphthylalanine, homophenylalanine, P-homophenylalanine or homonaphthylalanine, each of which is optionally substituted with one or more substituents.
  • the amino acid residue having a side chain comprising an aryl group can each be independently a residue of phenylalanine, 2- naphthylalanine, homophenylalanine, or P-homophenylalanine, each of which is optionally substituted with one or more substituents.
  • the amino acid residue having a side chain comprising an aryl group can each be independently a residue of phenylalanine or 2-naphthylalanine, each of which is optionally substituted with one or more substituents.
  • At least one amino acid residue having a side chain comprising an aryl group can be a residue of phenylalanine.
  • Two amino acid residues having a side chain comprising an aryl group can be residues of phenylalanine.
  • Each amino acid residue having a side chain comprising an aryl group can be a residue of phenylalanine.
  • none of the amino acids having the side chain comprising the aryl or heteroaryl group are contiguous.
  • two amino acids having the side chain comprising the aryl or heteroaryl group can be contiguous.
  • two contiguous amino acids can have opposite stereochemistry.
  • the two contiguous amino acids can have the same stereochemistry.
  • three amino acids having the side chain comprising the aryl or heteroaryl group can be contiguous.
  • three contiguous amino acids can have the same stereochemistry.
  • three contiguous amino acids can have alternating stereochemistry.
  • the amino acid residues comprising aryl or heteroaryl groups can be L-amino acids.
  • the amino acid residues comprising aryl or heteroaryl groups can be D-amino acids.
  • the amino acid residues comprising aryl or heteroaryl groups can be a mixture of D- and L-amino acids.
  • the optional substituent can be any atom or group which does not significantly reduce (e.g., by more than 50%) the cytosolic delivery efficiency of the cCPP, e.g., compared to an otherwise identical sequence which does not have the substituent.
  • the optional substituent can be a hydrophobic substituent or a hydrophilic substituent.
  • the optional substituent can be a hydrophobic substituent.
  • the substituent can increase the solvent-accessible surface area (as defined herein) of the hydrophobic amino acid.
  • the substituent can be halogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, acyl, alkylcarbamoyl, alkylcarboxamidyl, alkoxycarbonyl, alkylthio, or arylthio.
  • the substituent can be halogen.
  • guanidine refers to the structure:
  • guanidine As used herein, a protonated form of guanidine refers to the structure:
  • Guanidine replacement groups refer to functional groups on the side chain of amino acids that will be positively charged at or above physiological pH or those that can recapitulate the hydrogen bond donating and accepting activity of guanidinium groups.
  • the guanidine replacement groups may facilitate cell penetration and delivery of a therapeutic agent while reducing toxicity associated with guanidine groups or protonated forms thereof.
  • the cCPP can comprise at least one amino acid having a side chain comprising a guanidine or guanidinium replacement group.
  • the cCPP can comprise two amino acids having a side chain comprising a guanidine or guanidinium replacement group.
  • the cCPP can comprise three amino acids having a side chain comprising a guanidine or guanidinium replacement group.
  • the cCPP can comprise four amino acids having a side chain comprising a guanidine or guanidinium replacement group.
  • the cCPP can comprise five amino acids having a side chain comprising a guanidine or guanidinium replacement group.
  • the cCPP can comprise six amino acids having a side chain comprising a guanidine or guanidinium replacement group.
  • the guanidine or guanidinium group can be an isostere of guanidine or guanidinium.
  • the guanidine or guanidinium replacement group can be less basic than guanidine.
  • guanidine replacement group refers to , or a protonated form thereof.
  • a cCPP comprising from 6 to 20 amino acids residues, wherein: (i) at least two amino acids have a side chain comprising a guanidine group, or a protonated form thereof; (ii) at least two amino acids have no side chain or a side chain comprising , or a protonated form thereof; and (iii) at least two amino acids residues independently have a side chain comprising an aryl or heteroaryl group.
  • At least two amino acids residues can have no side chain or a side chain comprising protonated form thereof.
  • the amino acid residue when no side chain is present, the amino acid residue have two hydrogen atoms on the carbon atom(s) (e g., -CH2-) linking the amine and carboxylic acid.
  • Glycine or b-alanine are examples of amino acids with no side chain.
  • the cCPP can comprise at least one amino acid having a side chain comprising one of the following moieties: , or a protonated form thereof. [0203] In embodiments, the cCPP can comprise at least two amino acids each independently having one of the following moieties
  • At least two amino acids can have a side chain comprising the same moiety selected from: , or a protonated form thereof.
  • at least one amino acid can have a side chain comprising or a protonated form thereof
  • At least two amino acids can have a side chain comprising , or a protonated form thereof.
  • one, two, three, or four amino acids can have a side chain comprising , or a protonated form thereof.
  • one amino acid can have a side chain comprising , or a protonated form thereof.
  • two amino acids can have a side chain comprising , or a protonated form thereof. , or a protonated form thereof, can be attached to the terminus of the amino acid side chain, can be attached to the terminus of the amino acid side chain.
  • the cCPP can comprise (iii) 2, 3, 4, 5 or 6 amino acid residues independently having a side chain comprising a guanidine group, guanidine replacement group, or a protonated form thereof.
  • the cCPP can comprise (iii) 2 amino acid residues independently having a side chain comprising a guanidine group, guanidine replacement group, or a protonated form thereof.
  • the cCPP can comprise (iii) 3 amino acid residues independently having a side chain comprising a guanidine group, guanidine replacement group, or a protonated form thereof.
  • the cCPP can comprise (iii) 4 amino acid residues independently having a side chain comprising a guanidine group, guanidine replacement group, or a protonated form thereof. In embodiments, the cCPP can comprise (iii) 5 amino acid residues independently having a side chain comprising a guanidine group, guanidine replacement group, or a protonated form thereof. In embodiments, the cCPP can comprise (iii) 6 amino acid residues independently having a side chain comprising a guanidine group, guanidine replacement group, or a protonated form thereof.
  • the cCPP can comprise (iii) 2, 3, 4, or 5 amino acid residues independently having a side chain comprising a guanidine group, guanidine replacement group, or a protonated form thereof. In embodiments, the cCPP can comprise (iii) 2, 3, or 4 amino acid residues independently having a side chain comprising a guanidine group, guanidine replacement group, or a protonated form thereof. In embodiments, the cCPP can comprise (iii) 2 or 3 amino acid residues independently having a side chain comprising a guanidine group, guanidine replacement group, or a protonated form thereof.
  • the amino acid residues can independently have a side chain comprising the guanidine group, guanidine replacement group, or the protonated form thereof that are not contiguous.
  • two amino acid residues can independently have the side chain comprising the guanidine group, guanidine replacement group, or the protonated form thereof can be contiguous.
  • three amino acid residues can independently have the side chain comprising the guanidine group, guanidine replacement group, or the protonated form thereof can be contiguous.
  • four amino acid residues can independently have the side chain comprising the guanidine group, guanidine replacement group, or the protonated form thereof can be contiguous.
  • the contiguous amino acid residues can have the same stereochemistry. In embodiments, the contiguous amino acids can have alternating stereochemistry. [0206] In embodiments, the amino acid residues independently having the side chain comprising the guanidine group, guanidine replacement group, or the protonated form thereof, can be L-amino acids. In embodiments, the amino acid residues independently having the side chain comprising the guanidine group, guanidine replacement group, or the protonated form thereof, can be D-amino acids. In embodiments, the amino acid residues independently having the side chain comprising the guanidine group, guanidine replacement group, or the protonated form thereof, can be a mixture of L- or D-amino acids.
  • each amino acid residue having the side chain comprising the guanidine group, or the protonated form thereof can independently be a residue of arginine, homoarginine, 2-amino-3 -propionic acid, 2-amino-4-guanidinobutyric acid or a protonated form thereof.
  • each amino acid residue having the side chain comprising the guanidine group, or the protonated form thereof can independently be a residue of arginine or a protonated form thereof.
  • each amino acid having the side chain comprising a guanidine replacement group, or protonated form thereof can independently be or a protonated form thereof.
  • the cCPP can comprise a first amino acid having a side chain comprising an aryl or heteroaryl group and a second amino acid having a side chain comprising an aryl or heteroaryl group, wherein an N-terminus of a first glycine forms a peptide bond with the first amino acid having the side chain comprising the aryl or heteroaryl group, and a C-terminus of the first glycine forms a peptide bond with the second amino acid having the side chain comprising the aryl or heteroaryl group.
  • first amino acid often refers to the N-terminal amino acid of a peptide sequence
  • first amino acid is used to distinguish the referent amino acid from another amino acid (e.g., a “second amino acid”) in the cCPP such that the term “first amino acid” may or may not refer to an amino acid located at the N-terminus of the peptide sequence.
  • the cCPP can comprise an N-terminus of a second glycine forms a peptide bond with an amino acid having a side chain comprising an aryl or heteroaryl group, and a C- terminus of the second glycine forms a peptide bond with an amino acid having a side chain comprising a guanidine group, or a protonated form thereof.
  • the cCPP can comprise a first amino acid having a side chain comprising a guanidine group, or a protonated form thereof, and a second amino acid having a side chain comprising a guanidine group, or a protonated form thereof, wherein an N-terminus of a third glycine forms a peptide bond with a first amino acid having a side chain comprising a guanidine group, or a protonated form thereof, and a C-terminus of the third glycine forms a peptide bond with a second amino acid having a side chain comprising a guanidine group, or a protonated form thereof.
  • the cCPP can comprise a residue of asparagine, aspartic acid, glutamine, glutamic acid, or homoglutamine. In embodiments, the cCPP can comprise a residue of asparagine. In embodiments, the cCPP can comprise a residue of glutamine.
  • the cCPP can comprise a residue of tyrosine; phenylalanine; 1- naphthylalanine, 2-naphthylalanine; tryptophan; homophenylalanine; or p-homophenylalanine.
  • the cCPP can comprise at least one D amino acid. In embodiments, the cCPP can comprise one to fifteen D amino acids. In embodiments, the cCPP can comprise one to ten D amino acids. In embodiments, the cCPP can comprise 1, 2, 3, 4, 5, 6, 7 or 8 D amino acids. In embodiments, the cCPP can comprise at least one L amino acid. In embodiments, the cCPP can comprise one to fifteen L amino acids. In embodiments, the cCPP can comprise one to ten L amino acids.
  • the cCPP can comprise 1, 2, 3, 4, 5, 6, 7 or 8 L amino acids. In embodiments, the cCPP can comprise 2, 3, 4, 5, 6, 7, or 8 contiguous amino acids having alternating D and L chirality. In embodiments, the cCPP can comprise three contiguous amino acids having the same chirality. In embodiments, the cCPP can comprise two contiguous amino acids having the same chirality. In embodiments, at least two of the amino acids can have the opposite chirality. In embodiments, the at least two amino acids having the opposite chirality can be adjacent to each other. In embodiments, at least three amino acids can have alternating stereochemistry relative to each other.
  • the at least three amino acids having the alternating chirality relative to each other can be adjacent to each other. In embodiments, at least four amino acids have alternating stereochemistry relative to each other. In embodiments, the at least four amino acids having the alternating chirality relative to each other can be adjacent to each other. In embodiments, at least two of the amino acids can have the same chirality. In embodiments, at least two amino acids having the same chirality can be adjacent to each other. In embodiments, at least two amino acids have the same chirality and at least two amino acids have the opposite chirality. In embodiments, the at least two amino acids having the opposite chirality can be adjacent to the at least two amino acids having the same chirality.
  • adjacent amino acids in the cCPP can have any of the following sequences: D-L; L- D; D-L-L-D; L-D-D-L; L-D-L-L-D; D-L-D-D-L; D-L-L-D-L; or L-D-D-L-D.
  • the amino acid residues that form the cCPP can all be L-amino acids.
  • the amino acid residues that form the cCPP can all be D-amino acids.
  • At least two of the amino acids can have a different chirality.
  • at least two amino acids having a different chirality can be adjacent to each other.
  • at least three amino acids can have different chirality relative to an adjacent amino acid.
  • at least four amino acids can have different chirality relative to an adjacent amino acid.
  • at least two amino acids have the same chirality and at least two amino acids have a different chirality.
  • one or more amino acid residues that form the cCPP can be achiral.
  • the cCPP can comprise a motif of 3, 4, or 5 amino acids, wherein two amino acids having the same chirality can be separated by an achiral amino acid.
  • the cCPPs can comprise the following sequences: D/L-X-D/L; D/L-X-D/L-X; D/L-X-D/L-X-D/L; D- X-D; D-X-D-X; D-X-D-X-D; L-X-L; L-X-L-X; or L-X-L-X-L, wherein DZL indicates that the amino acid can be a D or an L amino acid and X is an achiral amino acid.
  • the achiral amino acid can be glycine.
  • an amino acid having a side chain comprising: protonated form thereof can be adjacent to an amino acid having a side chain comprising an aryl or
  • an amino acid having a side chain comprising: , or a protonated form thereof can be adjacent to at least one amino acid having a side chain comprising a guanidine or protonated form thereof.
  • an amino acid having a side chain comprising a guanidine or protonated form thereof can be adjacent to an amino acid having a side chain comprising an aryl or heteroaryl group.
  • two amino acids having a side chain comprising: protonated forms there can be adjacent to each other.
  • two amino acids having a side chain comprising a guanidine or protonated form thereof are adjacent to each other.
  • a cCPP can comprise at least two contiguous amino acids having a side chain can comprise an aryl or heteroaryl group and at least two non-adjacent amino acids having a side chain comprising: or a protonated form thereof.
  • a cCPP can comprise at least two contiguous amino acids having a side chain comprising an aryl or heteroaryl group and at least two non-adjacent amino acids having a side chain comprising , or a protonated form thereof.
  • the adjacent amino acids can have the same chirality.
  • the adjacent amino acids can have the opposite chirality.
  • Other combinations of amino acids can have any arrangement of D and L amino acids, e.g., any of the sequences described in the preceding paragraph.
  • At least two amino acids having a side chain comprising: protonated form thereof are alternating with at least two amino acids having a side chain comprising a guanidine group or protonated form thereof.
  • the cCPP can comprise the structure of Formula (A):
  • Ri, Ra, Rs, R», RS, Re, and R 7 are independently H or an amino acid side chain;
  • AAsc is an amino acid side chain; and q is 1, 2, 3 or 4.
  • the cCPP of the general Formula (A) can have any configuration and/or amino acid side chain as described in the published PCT application NO. US2020/066459 (WO2021127650A1) or US Patent No. 11,225,506.
  • AAsc can be , wherein t can be an integer from
  • AAsc can be , wherein t can be 0 or an integer from 1 to 5. t can be 1 to 5. T is
  • t can be 2. t can be 3.
  • AAsc can be conjugated to a linker.
  • the cCPP are of the general Formula (A) or a protonated form thereof, wherein:
  • Ri, R2, and Rs are each independently H or an aryl or heteroaiyl side chain of an amino acid; at least two of Ri, Ri, and Rs is an aryl or heteroaryl side chain of an amino acid;
  • R4, RS, Re, R 7 are independently H or an amino acid side chain; at least two of Ri, Rs, Re, R 7 is H or a side chain of 3-guanidino-2-aminopropionic acid, 4-guanidino-2-aminobutanoic acid, arginine, homoarginine, N-methylarginine, N,N- dimethylarginine, 2,3 -diaminopropionic acid, 2,4-diaminobutanoic acid, lysine, N-methyllysine, N,N-dimethyllysine, N-ethyllysine, N,N,N-trimethyllysine, 4-guanidinophenylalanine, citrulline, serine, histidine, N,N-dimethyllysine, 0-homoarginine, 3-(l-piperidinyl)alanine;
  • AAsc is an amino acid side chain; and q is 1, 2, 3 or 4.
  • the cCPP is of Formula (A), where at least one of R*, Rs, Re, R 7 are independently an uncharged, non-aromatic side chain of an amino acid.
  • at least two of Ri, Rs, Re, R 7 are, independently, H or a side chain of serine, histidine, or citrulline.
  • compounds that include a cyclic peptide having from 6 to 12 amino acids, wherein at least two amino acids of the cyclic peptide are charged amino acids; at least two amino acids of the cyclic peptide are aromatic hydrophobic amino acids; and at least two amino acids of the cyclic peptide are uncharged, non-aromatic amino acids.
  • at least two charged amino acids of the cyclic peptide are arginine.
  • at least two aromatic, hydrophobic amino acids of the cyclic peptide are phenylalanine, naphtha alanine (3- Naphth-2-yl-alanine), or a combination thereof.
  • At least two uncharged, non- aromatic amino acids of the cyclic peptide are citrulline, glycine or a combination thereof.
  • the compound is a cyclic peptide having from 6 to 12 amino acids wherein two amino acids of the cyclic peptide are arginine; at least two amino acids are aromatic, hydrophobic amino acids selected from phenylalanine, naphtha alanine, homophenylalanine and combinations thereof; and at least two amino acids are uncharged, non-aromatic amino acids selected from citrulline, serine, histidine, glycine, and combinations thereof.
  • the compound is a cyclic peptide having from 6 to 12 amino acids wherein two amino acids of the cyclic peptide are arginine; two or three amino acids are, independently, aromatic, hydrophobic amino acids selected from phenylalanine, 2 -naphthylalanine, 0-homophenylalanine; and two amino acids are independently, uncharged, non-aromatic amino acids selected from citrulline, serine, histidine, and glycine.
  • the cCPP can comprise the structure of Formula (I):
  • Ri. Rz, and Rs can each independently be H or an amino acid residue having a side chain comprising an aryl or heteroaryl group; at least two of Ri, Rz, and Rs is an aryl or heteroaiyl side chain of an amino acid;
  • R* and R6 are independently H or an amino acid side chain
  • AAsc is an amino acid side chain; q is 1, 2, 3 or 4; and each m is independently an integer 0, 1, 2, or 3.
  • a cCPP of Formula (A) may be of Formula (I)
  • the cCPP are of Formula (I) or (A), where Ri, Rz, and R3 can each independently be H; -alkylene-ary 1; or -alkylene -heteroaryl. Ri, Rz, and Rs can each independently be H, -Ci- 3 alkylene-aryl or -Cisalkylene-heteroaryl. Ri, Rz, and Rs can each independently be H or -alkylene-ary] . Ri, Rz, and Rs can each independently be H or -Ci-salkylene-aryl. Ci-salkylene can be methylene. Aryl can be a 6- to 14-membered aryl.
  • Heteroaryl can be a 6- to 14-membered heteroaryl having one or more heteroatoms selected from N, O, and S.
  • Aryl can be selected from phenyl, naphthyl, or anthracenyl.
  • Aryl can be phenyl or naphthyl.
  • Aryl can be phenyl.
  • Heteroaiyl can be pyridyl, quinolyl, and isoquinolyl.
  • Ri, Rz, and Rs can each independently be H, -Ci- salkylene-Ph or -C1-a3lkylene-Naphthyl.
  • Ri, Rz, and Rs can each independently be H, -CHzPh, or -CHzNaphthyl.
  • Ri, R2, and Rs can each independently be H or -CHzPh.
  • the cCPP are of Formula (I) or (A), where Ri, R2, and R3 can each independently be the side chain of tyrosine, phenylalanine; 1 -naphthylalanine; 2-naphthylalanine; tryptophan; 3-benzothienylalanine, 4-phenylphenylalanine; 3,4-difluorophenylalanine; 4- trifluoromethylphenylalanine; 2,3,4,5,6-pentafluorophenylalanine; homophenylalanine; P- homophenylalanine; 4-tert-butyl-phenylalanine; 4-pyridinylalanine; 3-pyridinylalanine; 4- methylphenylalanine; 4-fluorophenylalanine; 4-chlorophenylalanine; or 3-(9-anthryl)-alanine.
  • the cCPP are of Formula (I) or (A), where Ri can be the side chain of tyrosine. Ri can be the side chain of phenylalanine. Ri can be the side chain of 1 -naphthylalanine. Ri can be the side chain of 2-naphthylalanine. Ri can be the side chain of tryptophan. Ri can be the side chain of homophenylalanine. Ri can be H.
  • the cCPP are of Formula (I) or (A), where Ra can be the side chain of tyrosine.
  • Ra can be the side chain of phenylalanine.
  • Ra can be the side chain of 1 -naphthylalanine.
  • Ra can be the side chain of 2-naphthylalanine.
  • Ra can be the side chain of tryptophan.
  • Ra can be the side chain of homophenylalanine.
  • Ra can be H.
  • the cCPP are of Formula (I) or (A), where R 7 can be the side chain of tyrosine.
  • Rj can be the side chain of phenylalanine.
  • Rs can be the side chain of 1 -naphthylalanine.
  • Rs can be the side chain of 2-naphthylalanine.
  • Ra can be the side chain of tryptophan.
  • Ra can be the side chain of homophenylalanine.
  • Ra can be H.
  • the cCPP are of Formula (J) or (A), where R4 can be H, or a side chain of arginine, citrulline, serine or histidine.
  • Rt can be H.
  • R4 can be a side chain of arginine.
  • R4 can be a side chain of citrulline.
  • R4 can be a side chain of serine.
  • R4 can be a side chain of histidine.
  • the cCPP are of Formula (A), where Rs can be H, or a side chain of arginine, citrulline, serine or histidine.
  • Rs can be H.
  • Rs can be a side chain of arginine.
  • Rs can be a side chain of citrulline.
  • Rs can be a side chain of serine.
  • Rs can be a side chain of histidine.
  • the cCPP are of Formula (I) or (A), where Re can be H, or a side chain of arginine, citrulline, serine or histidine.
  • Re can be H.
  • Re can be a side chain of arginine.
  • Re can be a side chain of citrulline.
  • Re can be a side chain of serine.
  • Re can be a side chain of histidine.
  • the cCPP are of Formula (A), where R 7 can be H, or a side chain of arginine, citrulline, serine or histidine.
  • R 7 can be H.
  • R 7 can be a side chain of arginine.
  • R 7 can be a side chain of citrulline.
  • R 7 can be a side chain of serine.
  • R 7 can be a side chain of histidine.
  • the cCPP are of Formula (I) or (A), where one, two, or three of Ri, R2, R3, R4, Rs, Re, and R 7 can be H.
  • One of Ri, R2, Rs, R4, Rs, Re, and R 7 can be H.
  • Two of Ri, R2, Rs, R4, Rs, Re, and R 7 can be H.
  • Three of Ri, R2, Rs, Rs, Re, and R 7 can be H. At least one of Ri, R2, Rs, R4, Rs, Re, and R 7 can be H.
  • the cCPP are of Formula (I) or (A), where one of Ri, R2, or Rs, is H.
  • the cCPP are of Formula (I) or (A), where at least one of R», Rs, Re, and R 7 can be H or a side chain of arginine, citrulline, serine, or histidine. At least one of R4, Rs, Re, and R 7 is H. At least one of R4, Rs, Re, and R 7 can be side chain of arginine. At least one of R4, Rs, Re, and R 7 can be side chain of citrulline. At least one of R4. Rs, Re, and R 7 can be side chain of serine. At least one of R4, Rs, Re, and R 7 can be side chain of histidine.
  • R4, Rs, Re, and R 7 can be H or a side chain of arginine, citrulline, serine, or histidine.
  • One of R4, Rs, Re, and R 7 is H.
  • One of R4, Rs, Re, and R 7 can be side chain of arginine.
  • One of R4, Rs, Re, and R 7 can be side chain of citrulline.
  • One of R4, Rs, Re, and R 7 can be side chain of serine.
  • R4, Rs, Re, and R 7 can be side chain of histidine.
  • the cCPP are of Formula (I) or (A), where two of R4, Rs, Re, and R 7 can be H or a side chain of arginine, citrulline, serine, or histidine.
  • Two of Rt, Rs, Re, and R 7 can be H.
  • Two of R4, Rs, Re, and R 7 can be side chain of arginine.
  • Two of R4, Rs, Re, and R 7 can be side chain of citrulline.
  • Two of R-4, Rs, Re, and R 7 can be side chain of serine.
  • Two of R», Rs, Re, and R 7 can be side chain of histidine.
  • the cCPP are of Formula (A), where three of Rt, Rs, Re, and R 7 can be H or a side chain arginine, citrulline, serine or histidine.
  • Three of Ri, Rs, Re, and R 7 can be H.
  • Three of R4, Rs, Re, and R 7 can be side chain of arginine.
  • Three of R4, Rs, Re, and R 7 can be side chain of citrulline.
  • Three of R4, Rs, Re, and R 7 can be side chain of serine.
  • Three of R4, Rs, Re, and R 7 can be side chain of histidine.
  • the cCPP are of Formula (A), where AAsc can be a side chain of a residue of asparagine, glutamine, or homoglutamine. AAsc can be a side chain of a residue of glutamine.
  • the cCPP can further comprise a linker conjugated to the AAsc, e g., the residue of asparagine, glutamine, or homoglutamine.
  • the cCPP can further comprise a linker conjugated to the asparagine, glutamine, or homoglutamine residue.
  • the cCPP can further comprise a linker conjugated to the glutamine residue.
  • the cCPP are of Formula (A), where q can be 1, 2, or 3. q can 1 or 2. q can be 1. q can be 2. q can be 3. q can be 4.
  • the cCPP are of Formula (A), where m can be an integer from 1 to 3. m can be 1 or 2. m can be 0. m can be 1. m can be 2. m can be 3.
  • the cCPP of Formula (A) or (I) can comprise the structure of Formula (I-a) or Formula (I- b):
  • the cCPP of Formula (A) or (I) can comprise the structures of Formulae (1-1), (1-2), (1-3), (1-4), (1-5), (1-6) or (1-7): Formulae (7-1), (1-2), (1-3), (7-4), (1-5), (1-6) or (1-7) protonated form thereof, wherein AAsc and m are as defined herein relative to Formula (A) and/or Formula (F).
  • the cCPP can comprise one of the following sequences: Ff ⁇ RrRr, FGFGRGR; GfFGrGr, Ff ⁇ GRGR; FfFGRGR; Ff ⁇ GrGr; FGFGRRR; or FGFRRRR.
  • the cCPP can have one of the following sequences: Ff ⁇ DRrRrQ, FGFGRGRQ; GfFGrGrQ, Ff ⁇ GRGRQ; FfFGRGRQ; Ff ⁇ GrGrQ; FGFGRRRQ; orFGFRRRRQ.
  • the cCPP of Formula (A) or Formula (I) can have the structure of Formula (I-c):
  • the cCPP of Formula (IA) or Formula (I) can have the structure of Formula (I-d):
  • the cCPP of Formula (A) or Formula (I) can have the structure of Formula (I-e):
  • the cCPP of Formula (A) can be selected from : [0250] The cCPP of Formula (A) can be selected from:
  • the cCPP of Formula (A) can be selected from: [0252]
  • the cCPP is selected from:
  • the cCPP can be of the Formula (II)
  • R1, R2, and R3 can each independently be H or an amino acid residue having a side chain comprising an aryl or heteroaryl group; at least two of Ri, R 7 , and R3 is an aryl or heteroaryl side chain of an amino acid;
  • R4, R5, R6, R7 are independently H or an amino acid side chain; at least two of R4, Rs, Re, R7 are independently a side chain of arginine,
  • AAsc is an amino acid side chain; nx is 0 or 1; and q is 1, 2, 3 or 4.
  • the cCPP can be of the Formula (HI)
  • Ri, R2, and R3 can each independently be H or an amino acid residue having a side chain comprising an aryl or heteroaryl group; at least two of R1, R2, and R3 is an aryl or heteroaryl side chain of an amino acid;
  • R4, and R6 are independently H or an amino acid side chain
  • the cCPP is of Formula (II) or (HI) where at least one of Ri, Rz, or R3 are, independently, H.
  • the cCPP is of Formula (II) or Formula (III) where at least one of Ri, Rz, and R3 are, independently, an amino acid residue having a side chain comprising an aryl or heteroaryl group.
  • the amino acid residue having a side chain comprising an aryl or heteroaryl group is phenylalanine, beta homophenylalanine, or 3-(2-naphthyl)-alanine.
  • the cCPP is of Formula (II) where at least two of R4, Re are each independently an amino acid residue having a side chain comprising a charged group.
  • the amino acid residue having a side chain comprising a charged group is arginine.
  • the cCPP is of Formula (II) or (HI) where q is 1.
  • the cCPP is of Formula (II) where n x is 1 and where Rs and R 7 are a side chain of arginine.
  • the cCPP is of Formula (II) where nx is 1, wherein the aryl or heteroaryl group is phenylalanine, beta homophenylalanine, or 3-(2-naphthyl)-alanine, and where at least two of RA, RS, Re and R 7 are the side chain of arginine.
  • the cCPP is of Formula (II) where nx is 1 , where Ri, Rs, Re and R 7 are the side chain of arginine.
  • the cCPP is of Formula (II) where n x is 1, where Rs and R 7 are the side chain of arginine, and RA and Re are H.
  • the cCPP is of Formula (II) or (HI) where at least one of RA, RS (if present), Re, or R 7 (if present) are H or the amino acid side chain of serine or histidine.
  • the cCPP is of Formula (II) or (HI) where at least two of RA, RS (if present), Re, or R 7 (if present) are, independently, H or the amino acid side chain of serine or histidine.
  • the cCPP is of Formula (II) where at least three of RA, RS, Re, or R 7 are, independently, H or the amino acid side chain of serine or histidine. In embodiments the cCPP is of Formula (II) where at least four of RA, RS, Re, or R 7 are, independently, H or the amino acid side chain of serine or histidine.
  • At least two of Ri, R2, and Rs are independently a side chain of phenylalanine, betahomophenylalanine, or naphthylalanine; at least two of RA, Rs, Re, or R 7 are independently a side chain of arginine, at least two of RA, RS, Re, or R 7 are independently H or a side chain of arginine, serine or histidine; AAsc is an amino acid side chain; n. K is 0 or 1; and q is 1. It is understood that nx is 1 when Ri is a side chain of betahomophenylalanine.
  • At least two of Ri, R2, and Rs are independently a side chain of phenylalanine, or naphthylalanine; RA and Re are each independently H or a side chain of arginine, serine or histidine; AAsc is an amino acid side chain; nx is 0 or 1; and q is 1.
  • the cCPP is of Formula (II) or (III), where two of RA, RS (if present), Re, or R 7 (if present) are independently a side chain of serine.
  • the cCPP is of Formula (II) or (HI), where two of RA, RS (if present), Re, or R 7 (if present) are independently a side chain of histidine.
  • the cCPP is of Formula (II) or (III), where two of RA, RS (if present), Re, or R 7 (if present) are independently, H.
  • the CPP is of Formula (II) wherein: at least two of Ri, R2, and Ri are independently a side chain of phenylalanine, betahomophenylaline, or naphthylalanine; at least two of R4, Rs, Rd, or R 7 are independently a side chain of arginine; at least two of R4, Rs, Rs, or R 7 are independently H or a side chain of an uncharged non-aryl amino acid selected from histidine, threonine, serine, leucine, isoleucine, valine, neopentylglycine, alanine, homoalanine, homoserine, 3-(4-thiazolyl)-alanine, 3-(4-furanyl)-alanine, and 3-(4-thienyl)-alanine; AAsc is an amino acid side chain; n x is 0 or 1; and q is 1. It is understood that n x is 1 when Ri is
  • the CPP is of Formula (III) wherein: at least two of Ri, R2, and Ri are independently a side chain of phenylalanine, naphthylalanine, orbetahomophenylanine; R4 and R 7 are independently H or a side chain of an uncharged non-aryl amino acid selected from histidine, threonine, serine, leucine, isoleucine, valine, neopentylglycine, alanine, homoalanine, homoserine, 3-(4-thiazolyl)-alanine, 3-(4-furanyl)-alanine, and 3-(4-thienyl)-alanine; AAsc is an amino acid side chain; nx is 0 or 1; and q is 1. It is understood that n x is 1 when Ri is a side chain of betahomophenylal anine.
  • the CPP is of Formula (II) wherein: at least two of Ri, R2, and Ra are independently a side chain of phenylalanine, betahomophenylalanine, or naphthylalanine; at least two of R4, Rs, Rs, or R 7 are independently a side chain of arginine; at least two of R4, Rs, Rs, or R 7 are independently a side chain of serine or histidine; AAsc is an amino acid side chain, n x is 0 or 1; and q is 1. It is understood that n x is 1 when Ri is a side chain of betahomophenylalanine.
  • the CPP is of Formula (DI) wherein, at least two of Ri, R2, and Ra are independently a side chain of phenylalanine, betahomophenylaline, or naphthylalanine; Rt and Rs are independently a side chain of serine or histidine; AAsc is an amino acid side chain; n x is 0 or 1; and q is 1. It is understood that nx is 1 when Ri is a side chain of betahomophenylalanine.
  • Ri, R2, and Ra are independently a side chain of phenylalanine, betahomophenylaline, or naphthylalanine
  • Rt and Rs are independently a side chain of serine or histidine
  • AAsc is an amino acid side chain
  • n x is 0 or 1
  • q is 1. It is understood that nx is 1 when Ri is a side chain of betahomophenylalanine.
  • the CPP is of the general Formula (II) or (DI), wherein at least one of Ri, R2, or Ri is H.
  • the CPP is of the general Formula (II) or (III), wherein at least one of Ri, R2, or Rais a side chain of phenylalanine.
  • the CPP is of the general Formula (II) or (III), wherein at least two of Ri, R2, or Raare a side chain of phenylalanine.
  • the CPP is of the general Formula (D) or (ID), wherein at least one of Ri, R2, or Ra is a side chain of naphthylalanine.
  • the CPP is of the general Formula (II), wherein at least two of R4, Rs, Re, or R 7 are independently a side chain of serine or histidine.
  • the CPP is of the general Formula (III), wherein R4, and Re are independently H, or a side chain of serine or histidine.
  • the CPP is of the general Formula (III), wherein R4, and Re are the side chain of serine.
  • the CPP is of the general Formula (IH), wherein R4, and Re are the side chain of histidine.
  • the CPP is of the general Formula (III), wherein R4, and Re are H.
  • the CPP is of the general Formula (II) or (III), wherein at least one of R4, Rs (if present), Re, R 7 (if present) are independently an uncharged, non-aryl side chain of an amino acid.
  • the CPP is of the general Formula (II) or (III), wherein at least two of R4, Rs (if present), Re, or R 7 (if present) are independently side chains of an uncharged non-aryl amino acid (e g., histidine, threonine, serine, leucine, isoleucine, valine, neopentylglycine, alanine, homoalanine, homoserine, 3-(4-thiazolyl)-alanine, 3-(4-furanyl)-alanine, and 3-(4-thienyl)- alanine).
  • an uncharged non-aryl amino acid e g., histidine, threonine, serine, leucine, isoleucine, valine
  • the CPP is of the general Formula (II) or (III), wherein at least two of R4, Rs (if present), Rs, or R 7 (if present) are independently side chains of an uncharged non-aryl amino acid selected from histidine, threonine, serine, leucine, isoleucine, valine, neopentylglycine, alanine, homoalanine, homoserine, 3-(4-thiazolyl)-alanine, 3-(4-furanyl)-alanine, and 3-(4- thienyl)-alanine.
  • R4, Rs (if present), Rs, or R 7 (if present) are independently side chains of an uncharged non-aryl amino acid selected from histidine, threonine, serine, leucine, isoleucine, valine, neopentylglycine, alanine, homoalanine, homoserine, 3-(4-thiazolyl)-alanine, 3-(4
  • the CPP is of the general Formula (II) or (III), wherein at least two of R4, Rs (if present), Rs, or R 7 (if present) are independently side chains of an uncharged non- aryl amino acid selected from histidine, and serine.
  • the CPP is of the general Formula (II), wherein at least one of R4, Rs, Re, R 7 is, independently, H. In embodiments, the CPP is of the general Formula (II), wherein two of R4, Rs, Re, R 7 are, independently, H. In embodiments, the CPP is of the general Formula (III), wherein at least one of R4 or Re is, independently, H. In embodiments, the CPP is of the general Formula (III), wherein two of R4 and Re are H.
  • compounds that include a cyclic peptide having from 6 to 12 amino acids, wherein at least two amino acids of the cyclic peptide are charged amino acids, at least two amino acids of the cyclic peptide are aryl or heteroaryl hydrophobic amino acids and at least two amino acids of the cyclic peptide are uncharged, non-aryl amino acids.
  • at least two charged amino acids of the cyclic peptide are arginine.
  • At least two aryl or heteroaryl, hydrophobic amino acids of the cyclic peptide are phenylalanine, naphthylalanine (3-naphth-2-yl-alanine), betahomophenylalanine, or a combination thereof.
  • at least two uncharged, non-aryl amino acids of the cyclic peptide are glycine.
  • two of the uncharged amino acids are serine, histidine or a combination thereof.
  • the CPP may comprise one of the following sequences: FGFGHGH; FGFSHSH; FGFGHGHQ; or FGFSHSHQ.
  • the cCPP can comprise one of the following sequences: ⁇ hF-F ⁇ SRSR, phF-Fd>GRGR, bhF-f-d>GrGr; bhF-fd>SRSR; or FfOSrSr.
  • the cCPP can comprise one of the following sequences: ⁇ hF-Ffl>SRSRQ, ⁇ hF-F0GRGRQ, bhF- fDGrGrQ; bhF-f ⁇ SRSRQ; or FfCbSrSrQ.
  • the cCPP can comprise one of the following sequences: FfFSRSR; FGFSRSR; ⁇ hF-f-Nal-SRSR; FfFSRSRQ; FGFSRSRQ; or ⁇ hF- f-Nal-SRSRQ.
  • the cCPP of F ormula (AV) or F ormula (II) can have the structure of F ormula (II- A) or (II- B):
  • the cCPP can comprise one of the following sequences: phF-FOSRSR, phF-FCDGRGR, bhF-f- ⁇ GrGr; bhF-fd>SRSR; or FfiDSrSr.
  • the cCPP can comprise one of the following sequences: phF-F ⁇ >SRSRQ, phF-F ⁇ DGRGRQ,bhF-f ⁇ hGrGrQ; bhF-fOSRSRQ, or Ff ⁇ SrSrQ.
  • the cCPP can comprise one of the following sequences: FfFSRSR; FGFSRSR; ⁇ hF-f-Nal-SRSR; FflFSRSRQ; FGFSRSRQ; or ⁇ hF-f-Nal-SRSRQ.
  • AAsc can be wherein t can be an integer from 0 to 5. AAsc can be wherein t can be 0 or an integer from 1 to 5. t can be 1 to 5.
  • T is 2 or 3.
  • t can be 2.
  • t can be 3.
  • AAsc can be conjugated to a linker.
  • the cCPP can be selected from: Exocyclic Peptides
  • the ocular delivery construct comprises a cyclic cell penetrating peptide (cCPP) and an exocyclic peptide (EP); or an endosomal escape vehicle (EEV) that comprises a cCPP, an EP, and one or more linkers.
  • cCPP cyclic cell penetrating peptide
  • EP exocyclic peptide
  • EEV endosomal escape vehicle
  • the EP can comprise a sequence of a nuclear localization signal (NLS).
  • the exocyclic peptide (EP) can comprise from 2 to 10 amino acid residues e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues, inclusive of all ranges and values therebetween.
  • the EP can comprise from 6 to 9 amino acid residues.
  • the EP can comprise from 4 to 8 amino acid residues.
  • Each amino acid in the exocyclic peptide may be a natural or non-natural amino acid.
  • non-natural amino acid refers to an organic compound that is a congener of a natural amino acid in that it has a structure similar to a natural amino acid so that it mimics the structure and reactivity of a natural amino acid.
  • the non-natural amino acid can be a modified amino acid, and/or amino acid analog, that is not one of the 20 common naturally occurring amino acids or the rare natural amino acids selenocysteine or pyrrolysine.
  • Non-natural amino acids can also be the D- isomer of the natural amino acids.
  • amino acids examples include, but are not limited to, alanine, allosoleucine, arginine, citrulline, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, napthylalanine, phenylalanine, proline, pyroglutamic acid, serine, threonine, tryptophan, tyrosine, valine, a derivative thereof, or combinations thereof.
  • amino acids can be A, G, P, K, R, V, F, H, Nal, or citrulline.
  • the EP can comprise at least one positively charged amino acid residue, e g., at least one lysine residue and/or at least one amine acid residue comprising a side chain comprising a guanidine group, or a protonated form thereof.
  • the EP can comprise 1 or 2 amino acid residues comprising a side chain comprising a guanidine group, or a protonated form thereof.
  • the amino acid residue comprising a side chain comprising a guanidine group can be an arginine residue.
  • Protonated forms can mean salt thereof throughout the disclosure.
  • the EP can comprise at least two, at least three or at least four or more lysine residues.
  • EP can comprise 2, 3, or 4 lysine residues.
  • the amino group on the side chain of each lysine residue can be substituted with a protecting group, including, for example, trifluoroacetyl (-COCF3), allyl oxycarbonyl (Alloc), l-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde), or (4,4- dimethyl-2,6-dioxocyclohex-l-ylidene-3)-methylbutyl (ivDde) group.
  • the amino group on the side chain of each lysine residue can be substituted with a trifluoroacetyl (-COCF3) group.
  • the protecting group can be included to enable amide conjugation.
  • the protecting group can be removed after the EP is conjugated to a cCPP.
  • the EP can comprise at least 2 amino acid residues with a hydrophobic side chain.
  • the amino acid residue with a hydrophobic side chain can be selected from valine, proline, alanine, leucine, isoleucine, and methionine.
  • the amino acid residue with a hydrophobic side chain can be valine or proline.
  • the EP can comprise at least one positively charged amino acid residue, e.g., at least one lysine residue and/or at least one arginine residue.
  • the EP can comprise at least two, at least three or at least four or more lysine residues and/or arginine residues.
  • the EP can comprise KK, KR, RR, HH, UK, HR, RH, KKK, KGK, KBK, KBR, KRK, KRR, RKK, RRR KKH, KHK, HKK, HRR HRH, HHR HBH, HHH, HHHH, KHKK, KKHK, KKKH, KHKH, HKHK, KKKK, KKRK, KRKK, KRRK, RKKR RRRR, KGKK, KKGK, HBHBH, HBKBH, RRRRR KKKKK, KKKRK, RKK, KRKKK, KKRKK, KKKKR KBKBK, RKKKKG, KRKKKG, KKRKKG, KKKKRG, RKKKKB, KRKKKB, KKRKKB, KKKKRB, KKKRKV, RRRRRR, HHHH, RHRHRH, HRHRHR KRKRKR, RKRKRKRKRKR
  • the EP can comprise KK, KR RR, KKK, KGK, KBK, KBR, KRK, KRR, RKK, RRR, KKKK, KKRK, KRKK, KRRK, RKKR, RRRR KGKK, KKGK, KKKKK, KKKRK, KBKBK, KKKRKV, PKKKRKV, PGKKRKV, PKGKRKV, PKKGRKV, PKKKGKV, PKKKRGV or PKKKRKG.
  • the EP can comprise PKKKRKV, RR, RRR RHR RBR RBRBR RBHBR or HBRBH, wherein B is beta-alanine.
  • the EP can comprise PKKKRKV.
  • the amino acids in the EP can have D or L stereochemistry.
  • the EP can consist of KK, KR RR KKK, KGK, KBK, KBR KRK, KRR RKK, RRR KKKK, KKRK, KRKK, KRRK, RKKR, RRRR KGKK, KKGK, KKKKK, KKKRK, KBKBK, KKKRKV, PKKKRKV, PGKKRKV, PKGKRKV, PKKGRKV, PKKKGKV, PKKKRGV or PKKKRKG.
  • the EP can consist of PKKKRKV, RR RRR, RHR RBR RBRBR RBHBR or HBRBH, wherein B is beta-alanine.
  • the EP can consist of PKKKRKV.
  • the amino acids in the EP can have D or L stereochemistry.
  • the EP can comprise an amino acid sequence identified in the art as a nuclear localization sequence (NLS).
  • the EP can consist of an amino acid sequence identified in the art as a nuclear localization sequence (NLS).
  • the EP can comprise an NLS comprising the amino acid sequence PKKKRKV.
  • the EP can consist of an NLS comprising the amino acid sequence PKKKRKV.
  • the EP can comprise an NLS comprising an amino acid sequence selected from NLSKRPAAIKKAGQAKKKK, PAAKRVKLD, RQRRNELKRSF,
  • the EP can consist of aann NLS comprising an amino acid sequence selected from NLSKRPAAIKKAGQAKKKK, PAAKRVKLD, RQRRNELKRSF,
  • All exocyclic sequences can also contain an N-terminal acetyl group.
  • the EP can have the structure: Ac-PKKKRKV.
  • EEVs Endosomal Escape Vehicles
  • a cargo conjugate comprising a cargo and an ocular delivery construct
  • the ocular delivery construct comprises an endosomal escape vehicle (EEV).
  • the EEV can comprise at least one cell penetrating peptide (CPP), for example, a cyclic cell penetrating peptide (cCPP), which is conjugated to an exocyclic peptide (EP).
  • CCPP cell penetrating peptide
  • cCPP cyclic cell penetrating peptide
  • EP exocyclic peptide
  • the EEV comprises one cCPP.
  • the EEV comprises one or more linkers.
  • the configuration of the cargo conjugate comprising an EEV may vaiy.
  • the EP can be coupled to the cargo.
  • the EP can be coupled to the cCPP.
  • the EP can be coupled to the cargo and the cCPP.
  • the EP can be coupled to the linker.
  • the cCPP can be coupled to the cargo.
  • the cCPP can be coupled to the EP.
  • the cCPP can be coupled to the cargo and the EP.
  • the cCPP can be coupled to the linker.
  • the cargo can be coupled to the EP.
  • the cargo can be coupled to the cCPP.
  • the cargo can be coupled to the cCPP and the EP. In embodiments, the cargo can be coupled to the linker. In embodiments, the cCPP and the EP can be coupled to the linker and the cargo can be coupled to the cCPP or the EP. In embodiments, the cCPP and the cargo can be coupled to the linker and the EP can be coupled to the cCPP or the cargo. In embodiments, the EP and the cargo can be coupled to the linker and the cCPP can be coupled to the EP or the cargo. In embodiments, the cCPP, the EP, and the cargo can be coupled to the linker.
  • Coupling between the EP, cargo, cCPP, linker, or combinations thereof, may be non- covalent or covalent.
  • the EP can be attached through a peptide bond to the N- terminus of the cCPP.
  • the EP can be attached through a peptide bond to the C- terminus of the cCPP.
  • EP can be attached to the cCPP through a side chain of an amino acid in the cCPP.
  • the EP can be attached to the cCPP through a side chain of a lysine which can be conjugated to the side chain of a glutamine in the cCPP.
  • the EP when the cargo is a therapeutic oligonucleotide, the EP can be conjugated to the 5' or 3'. In embodiments, the EP can be conjugated to an amino group of the linker. In embodiments, the EP can be coupled to a linker via the C-terminus of the EP and a cCPP through a side chain on the cCPP and/or EP.
  • an EP may comprise a terminal lysine which can then be coupled to a cCPP containing a glutamine through an amide bond.
  • the side chain of the lysine can be used to conjugate the EP to the cCPP and the C- or N- terminus may be attached to the linker or the cargo.
  • One or more linkers (L) may be used to link the components of a delivery construct and/or the cargo conjugates.
  • a linker is a moiety that covalently couples two or more components of a delivery construct and/or a cargo construct; includes one or more functional group that can be used to conjugate one or more components to a linker; or both.
  • the functional group on the linker may be reacted with a functional group on a component (e.g., an EP, cCPP, or cargo) to form a reaction product and covalently couple to component to the linker and link the component to one or more other components covalently attached to the linker.
  • a component e.g., an EP, cCPP, or cargo
  • the delivery constructs and/or cargo conjugates of the disclosure can include one or more linkers.
  • the linker can link a cargo to the cCPP.
  • the linker can link an EP to the cCPP.
  • the linker can link the cCPP to the EP and the cargo.
  • the linker can be attached to the side chain of an amino acid of the cCPP, and the cargo can be attached at a suitable position on the linker.
  • the linker can be any appropriate moiety which can conjugate a cCPP to one or more additional components, e.g., an exocyclic peptide (EP) and/or a cargo.
  • EP exocyclic peptide
  • the linker Prior to conjugation (e.g., to the cCPP and/or one or more additional components), the linker has two or more functional groups, each of which is independently capable of forming a covalent bond (e.g., to the cCPP and/or one or more additional components).
  • the cargo is an oligonucleotide
  • the linker can be covalently bound to the 5' end of the cargo or the 3' end of the oligonucleotide cargo.
  • the linker can be covalently bound to the 5' end of the oligonucleotide cargo.
  • the linker can be covalently bound to the 3* end of the oligonucleotide cargo.
  • the linker can be covalently bound to the N-terminus or the C-terminus of the peptide cargo.
  • the linker can be covalently bound to the backbone (e g., somewhere in the middle and not at a terminus or termini) of the oligonucleotide or peptide cargo.
  • the linker can be any appropriate moiety that conjugates a cCPP described herein to a therapeutic moiety such as an oligonucleotide, peptide or small molecule.
  • the linkers can be chemically functionalized to influence efficacy and tolerability of the resultant construct, for example to increase efficacy.
  • efficacy is increased without decreasing tolerability.
  • the linker can comprise one or more PEG (polyethylene glycol) regions and/or the linker can vary in length.
  • the linker length (e g., between the EP and cCPP) can influence efficacy and tolerability.
  • linker length e.g., between the EP and cCPP
  • the linker length increases as linker length decreases, efficacy increases and tolerability decreases; and as linker length increases, efficacy decreases and tolerability increases.
  • the length of the linker increases (e.g., between a cargo and EEV), the tolerability increases.
  • the linker can comprise hydrocarbon linker.
  • the linker can comprise a cleavage site.
  • the cleavage site can be a disulfide, or caspasecleavage site (e.g, Val-Cit-PABC).
  • the linker can comprise: (i) one or more D or L amino acids, each of which is optionally substituted; (ii) optionally substituted alkylene; (iii) optionally substituted alkenylene; (iv) optionally substituted alkynylene; (v) optionally substituted carbocyclyl; (vi) optionally substituted heterocyclyl; (vii) one or more -(R 1 ’J-R 2 )z”- subunits, wherein each of R 1 and R 2 , at each instance, are independently selected from alkylene, alkenylene, alkynylene, carbocyclyl, and heterocyclyl, each J is independently C, NR 3 , -NR 3 C(O)-, S, and O, wherein R 3 is independently selected from H, alkyl, alkenyl, alkynyl, carbocyclyl, and heterocyclyl, each of which is optionally substituted, and z” is an integer from 1 to 50; (viii)
  • the linker can comprise one or more D or L amino acids and/or -(R ⁇ J-R ⁇ z”-, wherein each of R 1 and R 2 , at each instance, are independently alkylene, each J is independently C. NR 3 , - NR 3 C(O)-, S, and O, wherein R 4 is independently selected from H and alkyl, and z” is an integer from 1 to 50; or combinations thereof.
  • the linker can comprise a -(OCH2CH2) Z - (e g., as a spacer), wherein z' is an integer from 1 to 60, e g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 29, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60.
  • “-(OCHiCFbjz can also be referred to as polyethylene glycol (PEG).
  • the linker can comprise one or more amino acids.
  • the linker can comprise a peptide.
  • the linker can comprise a -(OCHaCHjjz-, wherein z' is an integer from 1 to 60, and a peptide .
  • the peptide can comprise from 2 to 10 amino acids.
  • the linker can further comprise a functional group (FG) capable of reacting through click chemistry. FG can be an azide or alkyne, and a triazole is formed when the ASO is conjugated to the linker.
  • the linker can further comprise a carboxylic acid functional group capable of reacting with an amine to form an amide when an ASO is conjugated to the linker.
  • the linker can comprise (i) a 0 alanine residue and lysine residue; (ii) -(J-R'jz”; or (iii) a combination thereof.
  • Each R 1 can independently be alkylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl, each J is independently C, NR 3 , -NR 3 C(O)-, S, or O, wherein R 3 is H, alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl, each of which is optionally substituted, and z” can be an integer from 1 to 50.
  • Each R 1 can be alkylene and each J can be O.
  • the linker can comprise (i) residues of 0-alanine, glycine, lysine, 4-aminobutyric acid, 5- aminopentanoic acid, 6-aminohexanoic acid or combinations thereof; and (ii) -(R ⁇ Jjz”- or -(J- R’)z”.
  • Each R 1 can independently be alkylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl, each J is independently C, NR 3 , -NR 3 C(O)-, S, or O, wherein R 3 is H, alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl, each of which is optionally substituted, and z” can be an integer from 1 to 50.
  • Each R 1 can be alkylene and each J can be O.
  • the linker can comprise glycine, beta-alanine, 4-aminobutyric acid, 5-aminopentanoic acid, 6-aminohexanoic acid, or a combination thereof.
  • the linker can be a trivalent linker.
  • the linker can have the structure: , wherein Ai, Bi, and Ci, can independently be a hydrocarbon linker (e.g., NRH-(CHz)n-COOH), a PEG linker (e.g., NRH-(CHzO)n-COOH, wherein R is H, methyl or ethyl) or one or more amino acid residue, and Z is independently a protecting group.
  • the linker can also incoiporate a cleavage site, including a disulfide [NH2- (CH2O)n-S-S-(CH2O)n-COOH], or caspase-cleavage site (Val-Cit-PABC).
  • the hydrocarbon can be a residue of glycine or beta-alanine.
  • the linker can be bivalent and link the cCPP to an AC.
  • the linker can be bivalent and link the cCPP to an exocyclic peptide (EP).
  • the linker can be trivalent and link the cCPP to an AC and to an EP.
  • the linker can be a bivalent or trivalent C1-C50 alkylene, wherein 1-25 methylene groups are optionally and independently replaced by -N(H)-, -N(CI-C4 alkyl)-, -N(cycloalkyl)-, -O-, - C(O)-, -C(O)O-, -S-, -S(O)-, -S(O)2-, -S(O)2N(CI-C4 alkyl)-, -S(O) 2 N(cycloalkyl)-, -N(H)C(O)-, -N(CI-C 4 alkyl)C(O)-, -N(cycloalkyl)C(O)-, -C(O)N(H)-, -C(O)N(Ci-C 4 alkyl), - C(O)N(cycloalkyl), aryl, heterocyclyl, heteroaiy
  • the linker can be a bivalent or trivalent C1-C50 alkylene, wherein 1-25 methylene groups are optionally and independently replaced by -N(H)-, -O-, -C(O)N(H)-, or a combination thereof.
  • the cargo can be coupled to the glutamic acid of the cyclic peptide, which converts the glutamic acid to glutamine.
  • the linker (L) can couple the cargo to the glutamine/glutamic acid of the cyclic peptide.
  • a linker (L) is covalently bound to the backbone of the cargo.
  • the linker can have the structure: , wherein: each AA is independently an amino acid residue; * is the point of attachment to the AAsc, and AAsc is side chain of an amino acid residue of the cCPP; x is an integer from 0-10; y is an integer from 1-5; and z Is an integer from 1-10.
  • x can be an integer from 0-5.
  • x can be an integer from 0-3.
  • X can be 1.
  • x can be 0.
  • y can be an integer from 2-4.
  • y can be 4.
  • z can be an integer from 1-5.
  • z can be an integer from 1-3. z can be 1.
  • Each AA can independently be selected from glycine, P-alanine, 4-aminobutyric acid, 5-aminopentanoic acid, and 6-aminohexanoic acid.
  • the cCPP can be attached to the cargo through a linker (“L”).
  • the linker can be conjugated to the cargo through a bonding group (“M”).
  • the linker can have the structure: , wherein: x is an integer from 0-10; y is an integer from 1-5; z is an integer from 1-10, each AA is independently an amino acid residue; * is the point of attachment to the AAsc, and AAsc is side chain of an amino acid residue of the cCPP; and M is a bonding group defined herein.
  • a linker has a terminal NH that is conjugated to an exocyclic peptide (EP)
  • the terminal NH of the linker may be a part of the EP.
  • the terminal NH of the linker may be a part of an amide bond formed between the reaction of the carboxylic acid of the C-terminal amino acid of the EP with an amine of the linker.
  • the terminal NH of the linker may be covalently bonded to the carbonyl of the C-terminal amino acid of the EP.
  • the stated side chain includes a covalent bond between an atom of the side chain and the linker.
  • the linker is covalently attached to the side chain of glutamine
  • the glutamine side chain includes a covalent bond between the nitrogen of the side chain of glutamine and the linker.
  • the linker can have the structure: wherein: y is an integer from 1-5; z' is an integer from 1-60; A is the point of attachment to the EP; * is the point of attachment to the AAsc, and AAsc is a side chain of an amino acid residue of the cCPP; and M is a bonding group defined herein.
  • the linker can have the structure:
  • the terminal NH of the linker may be conjugated to an EP via the terminal C of the EP.
  • the linker can have the structure: wherein: x' is an integer from 1-60; y is an integer from 1 -5; and z' is an integer from 1-60; * is the point of attachment to the AAsc, and AAsc is a side chain of an amino acid residue of the cCPP.
  • the terminal NH of the linker may be conjugated to an EP.
  • x' can be an integer from 0-60, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 29, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60, inclusive of all ranges and subranges therebetween, x' can Be an integer from 5-15. x' can Be an integer from 9-13. x' can be an integer from 1-5. x' can be 1.
  • y can be an integer from 1-5, e.g., 1, 2, 3, 4, or 5, inclusive of all ranges and subranges therebetween, y can be an integer from 2-5. y can be an integer from 3-5. y can be 3 or 4. y can be 4 or 5. y can be 3. y can be 4. y can be 5.
  • z can be an integer from 1-10, e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, inclusive of all ranges and subranges therebetween.
  • z' can be an integer from 1-60, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 29, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60, inclusive of all ranges and subranges therebetween, z' can Be an integer from 5-15. z' can Be an integer from 9-13. z' can be 11.
  • the linker or M (wherein M is part of the linker) can be covalently bound to cargo at any suitable location on the cargo.
  • the linker or M (wherein M is part of the linker) can be covalently bound to the 3' end of oligonucleotide cargo or the 5' end of an oligonucleotide cargo.
  • the linker or M (wherein M is part of the linker) can be covalently bound to the N-terminus or the C-terminus of a peptide cargo.
  • the linker or M (wherein M is part of the linker) can be covalently bound to the backbone of an oligonucleotide or a peptide cargo.
  • the linker can be bound to the side chain of aspartic acid, glutamic acid, glutamine, asparagine, or lysine, or a modified side chain of glutamine or asparagine (e.g., a reduced side chain having an amino group), on the cCPP.
  • the linker can be bound to the side chain of lysine on the cCPP.
  • the linker can have a structure: wherein:
  • M is a group that conjugates L to an AC
  • AAs is a side chain or terminus of an amino acid on the cCPP; each AAx is independently an amino acid residue; o is an integer from 0 to 10; and p is an integer from 0 to 5.
  • the linker can have a structure: wherein:
  • M is a group that conjugates L to a cargo (e.g., an ASO);
  • AAs is a side chain or terminus of an amino acid on the cCPP; each AAx is independently an amino acid residue; o is an integer from 0 to 10; and p is an integer from 0 to 5.
  • the linker can have a structure: wherein:
  • M is a group that conjugates L to a cargo (e.g., an ASO);
  • M can comprise an alkylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl, each of which is optionally substituted.
  • M can comprise or be selected from: , wherein
  • R is alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl.
  • M can comprise or be selected from: wherein: B is a nucleobase of an oligonucleotide cargo (e g., the 3' base of an oligonucleotide cargo); and R 10 is alkylene, cycloalkyl, or wherein a is 0 to 10.
  • o is an integer from 0 to 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10); s' is an integer from 0 to 10 (e g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10); c' is an integer from 0 to 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10); and B is a nucleobase of an oligonucleotide cargo (e.g., the 3' base of an oligonucleotide cargo).
  • M can be and a is O to 10.
  • M can be
  • M ean be a heterobifunctional crosslinker, e g., which is disclosed in Williams et al. Curr. Protoc Nucleic Acid Chem. 2010, 42, 4.41.1-4.41.20, incorporated herein by reference its entirety.
  • M ean be -C(O)-.
  • M can be or comprise can be or comprise wherein f is 0 to 10.
  • M can be .
  • M can be or comprise wherein B is a nucleobase of an oligonucleotide cargo (e.g., the 3' base of an oligonucleotide cargo such as a PMO).
  • AAs can be a side chain or terminus of an amino acid on the cCPP.
  • Non-limiting examples of AA S include aspartic acid, glutamic acid, glutamine, asparagine, or lysine, or a modified side chain of glutamine or asparagine (e.g., a reduced side chain having an amino group).
  • AAs can be an AAsc as defined herein.
  • Each AAx is independently a natural or non-natural amino acid.
  • One or more AAx can be a natural amino acid.
  • One or more AAx can be a non-natural amino acid.
  • One or more AAx can be a p-amino acid.
  • the p-amino acid can be p-alanine.
  • o can be an integer from 0 to 10, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. o can be 0, 1, 2, or
  • o can be 0. o can be 1. o can be 2. o can be 3.
  • p can be 0 to 5, e.g., 0, 1, 2, 3, 4, or 5.
  • p can be 0.
  • p can be 1.
  • p can be 2.
  • p can be 3.
  • p can be 4.
  • p can be 5.
  • the linker can have the structure: wherein M, AAs. each -(R 1- J-R 2 )z”-, o and z” are defined herein; r can be 0 or 1.
  • r is 0. In embodiments, r is 1 [0340]
  • the linker can have the structure: wherein each of M, AA$, o, p, q, r and z” can be as defined herein.
  • z" can be an integer from 1 to 50, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50, inclusive of all ranges And values therebetween, z " can Be an integer from 5-20. z " can be an integer from 10-15.
  • the linker can have the structure: wherein:
  • M, AAs and o are as defined herein.
  • a compound comprising a cCPP and an AC that is complementary to a target in a pre-mRNA sequence further comprising L, wherein the linker is conjugated to the AC through a bonding group (M), wherein
  • a compound comprising a cCPP and an antisense compound (AC), for example, an antisense oligonucleotide, that is complementary to a target in a pre-mRNA sequence, wherein the compound further comprises L, wherein the linker is conjugated to the AC through a bonding group (M), wherein M comprises or is selected from: wherein t' is 0 to 10 wherein each R is independently an alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl, wherein R 1 is , and t' is 2.
  • AC antisense compound
  • M bonding group
  • M can , wherein t' is O to 10.
  • M ean be
  • the linker can have the structure: wherein AAs is as defined herein, and m' is 0-10. [0348] The linker can be of the formula. wherein z' is defined herein. [0349] The linker can be of the formula: , wherein
  • base is a nucleobase of an oligonucleotide cargo (e g., the 3' base of an oligonucleotide cargo such as PMO); and z' is defined herein.
  • the linker can be of the formula:
  • base is a nucleobase of an oligonucleotide cargo (e.g., the 3' base of an oligonucleotide cargo such as PMO); and z' is defined herein.
  • the linker can be of the formula: , wherein “base” is a nucleobase of an oligonucleotide cargo (e.g., the 3' base of an oligonucleotide cargo such as PMO).
  • the linker can be of the formula:
  • base is a nucleobase of an oligonucleotide cargo (e g., the 3' base of an oligonucleotide cargo such as PMO).
  • the linker can be of the formula:
  • base is a nucleobase of an oligonucleotide cargo (e.g., the 3' base of an oligonucleotide cargo such as a PMO), and z' is defined herein.
  • the linker can comprise: wherein:
  • base a nucleobase of an oligonucleotide cargo (e g., the 3' base of an oligonucleotide cargo) base is a nucleobase of an oligonucleotide cargo.
  • the linker can comprise: wherein: “base” is a nucleobase of an oligonucleotide cargo (e g., the 3' base of an oligonucleotide cargo); and z' is defined herein.
  • the linker can be covalently bound to a cargo at any suitable location on the oligonucleotide cargo.
  • the linker is covalently bound to the 3' end of cargo or the 5' end of an oligonucleotide cargo.
  • the linker can be covalently bound to the backbone of an oligonucleotide cargo.
  • the linker can be bound to the side chain of aspartic acid, glutamic acid, glutamine, asparagine, or lysine, or a modified side chain of glutamine or asparagine (e.g., a reduced side chain having an amino group), on the cCPP.
  • the linker can be bound to the side chain of lysine on the cCPP.
  • the components of an ocular cargo conjugate can be conjugated to a linker defined herein.
  • An ocular delivery construct can be linked to a cargo to from a cargo conjugate.
  • the cargo can be linked to the ocular delivery construct through a linker.
  • the cargo can be conjugated to the linker using any conjugation reaction disclosed herein to form a bonding group (M).
  • a cargo is directly conjugated to the cCPP of an ocular delivery' construct to form a cargo conjugate.
  • at least one atom of the cCPP can be replaced by a cargo or at least one lone pair can form a bond to a cargo.
  • at least one atom of an amino acid side chain of the cCPP is replaced by a cargo or at least one lone pair of the atom forms a bond to a cargo.
  • a hydrogen atom on the NHj group of the carboxamide side chain of a cCPP can be replaced by a bond to the cargo.
  • a hydrogen atom on the NH? group of the carboxamide of a glutamine side chain of the cCPP can be replaced by a bond to the cargo.
  • a cargo is linked to an ocular delivery construct through a linker to form a cargo conjugate.
  • the ocular delivery construct comprises a cCPP and a linker.
  • the AAscof a cCPP is conjugated to a linker and the cargo is conjugated to the linker thereby forming a cargo conjugate.
  • the cargo can be any therapeutic moiety such as a peptide, oligonucleotide, or small molecule.
  • the cargo can be a peptide sequence or a non-peptidyl therapeutic agent.
  • the cargo can be a therapeutic moiety that is an antibody or an antigen binding fragment thereof, including, but not limited to an scFv or nanobody.
  • the cCPP can be conjugated to a linker defined herein.
  • the linker can be conjugated to an AAsc of the cCPP as defined herein.
  • the linker can comprise a -(OCIfcCEbjz- subunit (e.g., as a spacer), wherein z' is an integer from 1 to 60, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 29, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60.
  • “-(OCHzCHa),' is also referred to as PEG.
  • z' is 11.
  • z' is 1.
  • the linker can comprise a -(OCEbCHk)/- subunit (e.g., as a spacer), wherein z' is an integer from 1 to 23. e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23. “-(OCH2CH2)Z' is also referred to as PEG. In embodiments, z' is 11. In embodiments, z" is 1.
  • An endosomal escape vehicle can comprise a cyclic cell penetrating peptide (cCPP), an exocyclic peptide (EP) and linker, and can be conjugated to a cargo to form an EEV-conjugate (cargo conjugate) comprising the structure of Formula (Z):
  • EEVs comprising a cyclic cell penetrating peptide (cCPP), linker and exocyclic peptide (EP) are provided.
  • a cargo construct comprising an EEV can comprise the structure of Formula (X):
  • cCPP is a cCPP of Formula (I).
  • cCPP is a cCPP of Formula (II).
  • a cargo construct comprising an EEV can comprise the structure of Formula (B ):
  • Ri, Rz, and R3 are each independently H or an aryl or heteroaryl side chain of an amino acid
  • Rt and R6 are independently H or an amino acid side chain; peptide is an exocyclic peptide as defined herein;
  • M is a bonding group as defined herein; each m is independently an integer from 0-3; cargo is a therapeutic moiety as defined herein; n is an integer from 0-2; x' is an integer from 1 -20; y is an integer from 1-5; q is 1-4; and z' is an integer from 1-20.
  • the cargo conjugate comprises the structure of Formula (B) or a protonated form thereof, wherein: Ri, R2, and Rs are each independently H or an aryl or heteroaryl side chain of an amino acid; at least two of Ri, R2, and Rs are an aryl or heteroaryl side chain of an amino acid;
  • R4 and R6 are independently H or an amino acid side chain; peptide is an exocydic peptide (EP); each m is independently an integer from 0-3; cargo is a therapeutic moiety as defined herein; n is an integer from 0-2; x' is an integer from 0-20; y is an integer from 1-5; q is 1-4; and z' is an integer from 0-20.
  • the cargo conjugate comprises the structure of Formula (B) or a protonated form thereof wherein:
  • Ri, R2, and Rs can each independently be H or an amino acid residue having a side chain comprising an aryl group
  • R4 and Rs are independently H or an amino acid side chain; peptide is an exocydic peptide as defined herein;
  • M is a bonding group as defined herein; cargo is a therapeutic moiety as defined herein; each m is independently an integer from 0-3; n is an integer from 0-2; x' is an integer from 1 -20; y is an integer from 1-5; q is an integer from 1-4; and z' is an integer from 1-20.
  • a cargo conjugate comprising an EEV can comprise the structure of Formula (B-l) or
  • Ri, R2, and Rs are each independently H or an aryl or heteroaryl side chain of an amino acid
  • R4 is H or an amino acid side chain
  • EP is an exocyclic peptide as defined herein; each m is independently an integer from 0-3; cargo is a therapeutic moiety as defined herein; x' is an integer from 1-20; y is an integer from 1-5, and z' is an integer from 1-20.
  • a cargo conjugate comprising an EEV can comprise the structure of Formula (C): Formula (C): or a protonated form thereof, wherein EP, R 1 , R 2 , R 3 , R 4 , and m are as defined above in Formula (B); cargo is a therapeutic moiety as defined herein; AA can be an amino acid as defined herein; n can be an integer from 0-2, x can be an integer from 0-10; y can be an integer from 1-5; and z can be an integer from 1-10.
  • a cargo construct comprising an EEV can comprise the structure of Formula the structure of Formula (D):
  • RI, Rz, and R3 can each independently be H or an amino acid residue having a side chain comprising an aryl or heteroaryl group; at least two of Ri, Ra, and Ra is an aryl or heteroaryl side chain of an amino acid;
  • R4, and Rs are independently H or an amino acid side chain
  • M is a bonding group as defined herein.
  • q is 1, 2, 3 or 4; each m is independently an integer from 0-3; n is an integer from 0-2; x' is an integer from 1-20; y is an integer from 1-5; q is an integer from 1-4; and z' is an integer from 1-20.
  • cCPP is a cCPP of Formula (X), (B), (B-l), (B-2), (C), or (D) wherein x' is 1.
  • cCPP is a cCPP of Formula (X), (B), (B-l), (B-2), (C), or (D) wherein y is 1.
  • cCPP is a cCPP of Formula (X), (B), (B-l ), (B-2), (C), or (D) wherein z' is 2.
  • cCPP is a cCPP of Formula (X), (B), (B-l), (B-2), (C), or (D) wherein z' is 11.
  • cCPP is a cCPP of Formula (X), (B), (B-l), (B-2), (C), or (D) wherein M comprises embodiments, cCPP is a cCPP of Formula (B), (C), or (D) wherein M is or comprises , wherein t' is 0 to 10.
  • cCPP is a cCPP of
  • Delivery constructs having a structure prior to conjugation to a cargo are provided.
  • the delivery constructs can include a cCPP, a cCPP and a linker, or an EEV (cCPP, EP, and linker).
  • the linker Prior to conjugation to the cargo, the linker can include a reactive group.
  • the reactive group can react with a reactive group on a cargo to form at least a portion of a bonding group M as described herein. Examples of reactive groups include an azide (Ns) and a carboxylic acid.
  • click chemistry may be used to conjugate the delivery construct to a cargo.
  • an azide reactive group may be incorporated in a delivery construct as an amino acid derivative, for example, azidolysine (K(N3)).
  • an amino acid derivative for example, azidolysine (K(N3)).
  • amid bond forming chemistry may be used to conjugate the delivery construct to a cargo.
  • a delivery construct prior to conjugation to a cargo can be selected from cyclo[Ff-Nal-RrRrQ]-PEGz-OH; and cyclo[Ff-Nal-RrRrQ]-PEG z -K(N3)-NH2, wherein x' and z' are, independently, an integer from 0 to 12.
  • a delivery construct prior to conjugation to a cargo can be selected from Ac-PKKKRKV-PEGx-K(cyclo[GfFGrGrQ])-PEGz-OH; and Ac-PKKKRKV-PEGx-K(cyclo[GfFGrGrQ])-PEGz-K(N3)-NH2 wherein x' and z' are, independently, an integer from 0 to 12.
  • a delivery construct prior to conjugation to a cargo can be selected from Ac-PKKKRKV-PEGx-K(cyclo[FfFGRGRQ])-PEGz'-OH; and Ac-PKKKRKV-PEGx'-K(cyclo[FfFGRGRQ])-PEGz’-K(N3)-NH2 wherein x' and z' are, independently, an integer from 0 to 12.
  • a delivery construct prior to conjugation to a cargo can be selected from Ac-PKKKRKV-PEGx , -K(cyclo[Ff-Nal-GrGrQ])-PEGz-OH; and Ac-PKKKRKV- PEGx-K(cyclo[Ff-Nal-GrGrQ])-PEGz-K(N3)-NH2 wherein x' and z' are, independently, an integer from 0 to 12.
  • a delivery construct prior to conjugation to a cargo can be selected from Ac-PKKKRKV-PEGx -K(cyclo[FGFGRGRQ])-PEGz-OH; and Ac-PKKKRKV-PEGx'-K(cyclo[FGFGRGRQ])-PEGz-K(N3)-NH2 wherein x' and z' are, independently, an integer from 0 to 12.
  • a delivery construct prior to conjugation to a cargo can be selected from Ac-KKKRK-PEGx-K(cyclo(FGFGRGRQ))-PEGz'-OH ; and Ac-KKKRK-PEGx-K(cyclo(FGFGRGRQ))-PEGz’-K(N3)-NH2 wherein x' and z' are, independently, an integer from 0 to 12.
  • a delivery construct prior to conjugation to a cargo can be selected from Ac-PKKKRKV-PEGx-K(cyclo[FGFRRRRQ])-PEGz-OH; and Ac-PKKKRKV-PEGx--K(cyclo[FGFRRRRQ])-PEGz’- K(N3)-NH 2 wherein x' and z' are, independently, an integer from 0 to 12.
  • a delivery construct prior to conjugation to a cargo can be selected from Ac-PKKKRKV-PEGx-K(cyclo[FF ⁇ PGRGRQ])-PEGz-OH; and Ac-PKKKRKV-PEGx-K(cyclo[FF ⁇ PGRGRQ])-PEG/-K(N3)-NH 2 wherein x' and z' are, independently, an integer from 0 to 12.
  • a delivery construct prior to conjugation to a cargo can be selected from Ac-PKKKRKV-PEGx-K(cyclo[ ⁇ hF-F0SRSRQ])-PEGz -OH; and Ac-PKKKRKV-PEGx-K(cyclo[ ⁇ hF-FOSRSRQ])-PEGz- K(N3)-NH2 wherein x' and z' are, independently, an integer from 0 to 12.
  • a delivery construct prior to conjugation to a cargo can be selected from Ac-PKKKRKV-PEGx-K(cyclo[ ⁇ hF-- ⁇ GRGRQ])-PEGz-OH; and Ac-PKKKRKV-PEGx’-K(cyclo[ ⁇ hF-F0GRGRQ])-PEG 2 - K(N3)-NH2, wherein x' and z' are, independently, an integer from 0 to 12.
  • a delivery construct prior to conjugation to a cargo can be selected from
  • a delivery construct prior to conjugation to a cargo can be selected from Ac-PKKKRKV-PEGx-K(cyclo[bhF-f- ⁇ DGrGrQ])-PEGz-OH, and Ac-PKKKRKV-PEGx-K(cyclo[bhF-f- ⁇ SRSRQ])-PEGz'- K(N3)-NH2, wherein x' and z' are, independently, an integer from 0 to 12.
  • K(N 3 ) is azidolysine; and a terminal OH indicates a terminal carboxylic acid.
  • PEG2 is miniPEGi or miniPEG.
  • x' is 0. In embodiments, x' is 2. In embodiments, x' is 4. In embodiments, x' is 8. In embodiments, x' is 12. In embodiments, z' is 0. In embodiments, z' is 2. In embodiments, z' is 4. In embodiments, z' is 8. In embodiments, z' is 12.
  • a delivery construct prior to conjugation to a cargo can be selected from cyclo[Ff-Nal-RrRrQ]-PEGi2-OH; cyclo[Ff-Nal-RrRrQ]-PEGi2-K(N3)-NH2;
  • a delivery construct prior to conjugation to a cargo can be selected from
  • a delivery construct prior to conjugation to a cargo can be: Ac-KKKRK-miniPEG2-K(cyclo(FGFGRGRQ))-miniPEG 2 -K(N3)-NH 2 .
  • a delivery construct prior to conjugation to a cargo can be selected from Ac-PKKKRKV-miniPEG 2 -K(cyclo[ ⁇ hF-f- ⁇ I SRSRQ])-PEGi 2 -OH; and Ac-PKKKRKV-miniPEG2-K(cyclo[ ⁇ hF-F ⁇ I>GRGRQ])-PEGi 2 -OH.
  • PEG 2 is miniPEGz or miniPEG.
  • a delivery construct having an azide group that can be used in a click chemistry reaction to conjugate a cargo to the delivery construct can be selected from cyclo[Ff-Nal-RrRrQ]-PEGz’-K(N3)-NH2 Ac-PKKKRKV-PEG x'-K(cy cl o[GfFGrGrQ])-PEGz'-K(N3)-NH 2 ; Ac-PKKKRKV-PEG x-'K(cyclo[FfFGRGRQ])-PEG z-K(N3)-NH2; Ac-PKKKRK V-PEG x-K(cyclo[Ff-Nal-GrGrQ])-PEG z’-K(N3)-NH 2 ; Ac-PKKKRKV-PEG x-K(cyclo[FGFGRGRQ])-PEGz-K(N3)-NH2; Ac-PKKKRKV-PEG x-K(cyclo
  • x' is 0. In embodiments, x' is 2. In embodiments, x' is 4. In embodiments, x' is 8. In embodiments, x' is 12. In embodiments, z' is 0. In embodiments, z' is 2. In embodiments, z' is 4. In embodiments, z' is 8. In embodiments, z' is 12. In embodiments, PEGt* is miniPEG. In embodiments, PEG?/ is miniPEG or miniPEG.
  • a delivery construct having an azide group that can be used in a click chemistry reaction to conjugate a cargo to the delivery construct can be cyclo[Ff-Nal-RrRrQ]- PEG2-K(N3)-NHz.
  • the delivery construct can be cyclo[Ff-Nal-RrRrQ]-PEGi?- K(N3)-NH2.
  • a delivery construct having an azide group that can be used in a click chemistry reaction to conjugate a cargo to the delivery construct can be Ac-PKKKRKV-PEG- K(cyclo[GfFGrGrQ])-PEG2-K(N3)-NH? where each PEG2 may, independently, be miniPEG2.
  • the delivery construct can be Ac-PKKKRKV-PEG-K(cyclo[GfFGGrQ])-PEGi?- K(N3)-NH2 where PEG2 may be miniPEG2.
  • a delivery construct having an azide group that can be used in a clickchemistry reaction to conjugate a cargo to the delivery construct can be Ac-PKKKRKV-PEG- K(cyclo[FfFGRGRQ])-PEG-K(N3)-NH? where each PEG2 may, independently, be miniPEG2.
  • the delivery construct can be Ac-PKKKRKV-PEG-K(cyclo[FfFGRGRQ])-PEGi?- K(N 3 )-NH2 where PEG2 may be miniPEG2.
  • the delivery construct can be Ac-PKKKRKV-PEG-K(cyclo[Ff-Nal- GrGrQ])-PEG- K(N3)-NH? where each PEG? may, independently, be miniPEG?.
  • the delivery construct can be Ac-PKKKRKV-PEG-K(cyclo[Ff-Nal-GrGQ])-PEGi?-K(N3)-NH? where PEG? may be miniPEG2.
  • the delivery ccoonnssttrruucctt ccaann bbee Ac-PKKKRKV-PEG2- K(cyclo[FGFGRGRQ])-PEG2 K(N3)-NH2 where each PEG? may, independently, be miniPEG2.
  • the delivery construct can be Ac-PKKKRKV-PEG2-K(cyclo[FGFGRGRQ])-PEGi2 K(N3)-NH? where PEG2 may be miniPEG2.
  • the delivery construct can be Ac-PKKKRKV-PEG- K(cyclo[FGFRRRRQ])-PEG K(N3)-NH? where each PEG2 may, independently, be miniPEG2.
  • the delivery construct can be Ac-PKKKRKV-PEG-K(cyclo[FGFRRRRQ])-PEGi? K(N3>NH2 where PEG may be miniPEG.
  • the delivery construct can be Ac-PKKKRKV-K(cyclo[Ff-Nal-G-r-G- rQ])-PEG2 K(N3)-NH? where each PEG may, independently, be miniPEG2.
  • the delivery construct can be Ac-PKKKRKV-K(cyclo[Ff-Nal-G-r-G-rQ])-PEGi2 K(Ns)-NH2 where PEG may be miniPEG.
  • the delivery construct can be Ac-PKKKRKV-K(cyclo[Ff-Nal-G-r-G- rQ])-PEG K(N3)-NH2 where each PEG? may, independently, be miniPEG.
  • the delivery construct can be Ac-PKKKRKV-K(cyclo[f-Nal-G-r-G-rQ])-PEGi2 K(N3)-NH? where PEG may be miniPEG.
  • tthhee delivery construct can be Ac-PKKKRKV-PEG-
  • the delivery construct can be Ac-PKKKRKV-PEG-K(cyclo[FGFGRGRQ])- PEGi2-K(N3)-NH2 where PEG2 may be miniPEG2.
  • the delivery construct ccaann bbee Ac-PKKKRKV-PEGz- K(cyclo[FfFGRGRQ])- PEG-K(N3)-NH2 where each PEG2 may, independently, be miniPEG.
  • the delivery construct can be Ac-PKKKRKV-PEG-K(cyclo[FfFGRGRQ])- PEGi2-K(N3)-NH2 where PEG2 may be miniPEGz.
  • the delivery construct can be Ac-KKKRK-PEG-K(cyclo(F GF GRGRQ)- PEG-K(N3)-NH2 where each PEG2 may, independently, be miniPEG2.
  • the delivery construct can be Ac-KKKRK-PEG-K(cyclo(FGFGRGRQ))-PEGi2-K(N3)-NH2 where PEG2 may be miniPEG?.
  • the delivery construct can be Ac-PKKKRKV-PEG- K(cyclo[FF ⁇ GRGRQ])- PEG-K(N3)-NH? where each PEG? may, independently, be miniPEG.
  • the delivery' construct can be Ac-PKKKRKV-PEG2-K(cy'clo[FFC>GRGRQ])- PEGi?-K(N3)-NH2 where PEG may be miniPEG.
  • the delivery construct can be Ac-PKKKRKV-PEG-K(cyclo[phF- F0SRSRQ])-PEG-K(N3)-NH2 where each PEG may, independently, be miniPEG.
  • the delivery construct can be Ac-PKKKRKV-PEG2-K(cyclo[piiF-F ⁇ I’SRSRQ])- PEG12-K(N 3 )-NH2 where PEG may be miniPEG.
  • the delivery construct can be Ac-PKKKRKV-PEG?-K(cyclo[ ⁇ hF- F ⁇ GRGRQ])-PEG-K(N3)-NH2 where each PEG may, independently, be miniPEG.
  • the delivery construct can be Ac-PKKKRKV-PEG2-K(cyclo[phF-F ⁇ GRGRQ])- PEGi2-K(N 3 )-NH 2 where PEG2 may be miniPEG.
  • the delivery construct ccaann bbee Ac-PKKKRKV-PEG2- K(cyclo[FGFGRGRQ])- PEG2-K(N3)-NH2 where each PEG2 may, independently, be miniPEGz.
  • the delivery construct can be Ac-PKKKRKV-PEGz-K(cyclo[FGFGRGRQ])- PEG12-K(N3)-NH2 where PEGz may be mini PEG.
  • a delivery construct having an azide group that can be used in a click chemistry reaction to conjugate a cargo to the delivery construct can be selected from wherein OH indicates a terminal carboxylic acid; and x' and z' are, independently, an integer from 0 to 12. In embodiments, x' is 0. In embodiments, x' is 2. In embodiments, x' is 4. In embodiments, x' is 8. In embodiments, x' is 12. In embodiments, z' is 0. In embodiments, z' is 2. In embodiments, z' is 4. In embodiments, z' is 8. In embodiments, z' is 12. In embodiments, PEG/ is miniPEGz. In embodiments, PEG/ is miniPEG.
  • a delivery construct having an azide group that can be used in a click chemistry reaction to conjugate a cargo to the delivery construct can be cyclo[Ff-Nal-RrRrQ]- PEG2-OH.
  • the delivery construct can be cyclo[Ff-Nal-RrRrQ]-PEGi2-OH.
  • a delivery construct having an azide group that can be used in a click chemistry reaction to conjugate a cargo to the delivery construct can be Ac-PKKKRKV-PEG- K(cyclo[GfFGGQ])-PEG-OH where each PEG may, independently, be miniPEG.
  • the delivery construct can be Ac-PKKKRKV-PEG-K(cyclo[GfFGGrQ])-PEGi2- OH where PEG may be miniPEGz.
  • a delivery construct having an azide group that can be used in a click chemistry reaction to conjugate a cargo to the delivery construct can be Ac-PKKKRKV-PEG- K(cyclo[FfFGRGRQ])-PEG2-OH where each PEGz may, independently, be miniPEGz.
  • the delivery construct can be Ac-PKKKRKV-PEG-K(cyclo[FfFGRGRQ])-PEGi2- OH where PEGz may be miniPEGz.
  • the delivery construct can be Ac-PKKKRKV-PEG-K(cyclo[Ff-Nal- GrGrQ])-PEG-OH where each PEG may. independently, be miniPEGz.
  • the delivery construct can be Ac-PKKKRKV-PEG-K(cyclo[Ff-Nal-GrGrQ])-PEGi2-OH where PEGz may be miniPEG.
  • tthhee delivery ccoonnssttrruucctt ccaann bbee Ac-PKKKRKV-PEG- K(cyclo[FGFGRGRQ])-PEGz-OH where each PEGz may, independently, be miniPEG.
  • the delivery construct can be Ac-PKKKRKV-PEGz-K(cyclo[FGFGRGRQ])- PEG12-OH where PEG may be mini PEG?.
  • the delivery ccoonnssttrruucctt ccaann bbee Ac-PKKKRKV-PEGz- K(cyclo[FGFRRRRQ])-PEG-OH where each PEG may, independently, be miniPEG.
  • the delivery construct can be Ac-PKKKRKV-PEG-K(cyclo[FGFRRRRQ])- PEGiz-OH where PEG may be miniPEG.
  • the delivery construct can be Ac-PKKKRKV-K(cyclo[Ff-Nal-G-r-G- rQ])-PEGz-OH where each PEG may, independently, be miniPEGz.
  • the delivery construct can be Ac-PKKKRKV-K(cyclo[Ff-Nal-G-r-G-rQ])-PEGi2-OH where PEG may be miniPEGz.
  • the delivery construct can be Ac-PKKKRKV-K(cyclo[Ff-Nal-G-r-G- rQ])-PEG K(N3)-NHZ where each PEG may, independently, be miniPEGz.
  • the delivery construct can be Ac-PKKKRKV-K(cyclo[Ff-Nal-G-r-G-rQ])-PEGiz-OH where PEG may be miniPEGz.
  • the delivery construct can be Ac-PKKKRKV-PEGz-K(cyclo[FGFGRGRQ])- PEG12-OH where PEG may be miniPEG.
  • tthhee delivery construct can be Ac-PKKKRKV-PEG2-
  • the delivery construct can be Ac-PKKKRKV-PEG2-K(cyclo[FfFGRGRQ])- PEG12-OH where PEG2 may be miniPEG2.
  • the delivery construct can be Ac-KKKRK-PEG2-K(cyclo(FGFGRGRQ)- PEG2-OH where each PEG2 may, independently, be miniPEG2.
  • the delivery construct can be Ac-KKKRK-PEG2-K(cyclo[FGFGRGRQ])-PEGi?-OH where PEG2 may be miniPEG2.
  • the delivery construct can be Ac-PKKKRKV-PEG2-
  • the delivery construct can be Ac-PKKKRKV-PEG2.-K(cyclo[FFOGRGRQ])- PEG12-OH where PEG2 may be miniPEG2.
  • the delivery construct can be Ac-PKKKRKV-PEG2-K(cyclo[ ⁇ hF- FOSRSRQD-PEG2-OH where eachPEG2 may, independently, be miniPEG2.
  • the delivery construct can be Ac-PKKKRKV-PEG2-K(cyclo[ ⁇ hF-F ⁇ DSRSRQ])-PEGi2-OH where PEG2 may be miniPEG2.
  • the delivery construct can be Ac-PKKKRKV-PEG2-K(cyclo[phF- F ⁇ GRGRQ])-PEG2-OH where each PEG2 may, independently, be miniPEG2.
  • the delivery construct can be Ac-PKKKRKV-PEG2-K(cyclo[phF-F ⁇ DGRGRQ])-PEGi?-OH where PEG2 may be miniPEG2.
  • tthhee delivery construct can be Ac-PKKKRKV-PEG2-
  • the deliveiy construct can be Ac-PKKKRKV-PEG2-K(cyclo[FGFGRGRQ])- PEG12-OH where PEG2 may be miniPEG2.
  • the cargo conjugates and the components of the cargo conjugates can be prepared in a variety of ways known to one skilled in the art of organic synthesis or variations thereon as appreciated by those skilled in the art.
  • the compounds can be prepared from readily available starting materials. Reaction conditions can vary with the particular reactants or solvents used, but such conditions can be determined by one skilled in the art.
  • Variations on the compounds described herein include the addition, subtraction, or movement of the various constituents as described for each compound. Similarly, when one or more chiral centers are present in a molecule, the chirality of the molecule can be changed. Additionally, compound synthesis can involve the protection and deprotection of various chemical groups.
  • protecting groups can be determined by one skilled in the art.
  • the chemistry of protecting groups can be found, for example, in Wuts and Greene, Protective Groups in Organic Synthesis, 4 th Ed., Wiley & Sons, 2006, which is incorporated herein by reference in its entirety.
  • the starting materials and reagents used in preparing the disclosed compounds and compositions are either available from commercial suppliers such as Aldrich Chemical Co., (Milwaukee, WI), Acros Organics (Morris Plains, NJ), Fisher Scientific (Pittsburgh, PA), Sigma (St.
  • Reactions to produce the compounds described herein can be carried out in solvents, which can be selected by one of skill in the art of organic synthesis. Solvents can be substantially nonreactive with the starting materials (reactants), the intermediates, or products under the conditions at which the reactions are carried out, e.g., temperature and pressure. Reactions can be carried out in one solvent or a mixture of more than one solvent. Product or intermediate formation can be monitored according to any suitable method known in the art.
  • product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e g., ‘H or 13 C) infrared spectroscopy, spectrophotometry (e g., UV-visible), or mass spectrometry, or by chromatography such as high-performance liquid chromatography (HPLC) or thin layer chromatography.
  • spectroscopic means such as nuclear magnetic resonance spectroscopy (e g., ‘H or 13 C) infrared spectroscopy, spectrophotometry (e g., UV-visible), or mass spectrometry
  • chromatography such as high-performance liquid chromatography (HPLC) or thin layer chromatography.
  • portions of the cargo constructs can be prepared by solid phase peptide synthesis wherein the amino acid a-N-terminus is protected by an acid or base protecting group.
  • Such protecting groups should have the properties of being stable to the conditions of peptide linkage formation while being readily removable without destruction of the growing peptide chain or racemization of any of the chiral centers contained therein.
  • Suitable protecting groups are 9- fluorenylmethyloxycarbonyl (Fmoc), t-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz), biphenylisopropyloxycarbonyl, t-amyloxycarbonyl, isobomyloxycarbonyl, a,a-dimethyl-3,5- dimethoxybenzyloxycarbonyl, o-nitrophenylsulfenyl, 2-cyano-t-butyloxycarbonyl, and the like.
  • the 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group can be used for the synthesis of the disclosed compounds.
  • side chain protecting groups are, for side chain amino groups like lysine and arginine, 2,2,5,7,8-pentamethylchroman-6-sulfonyl (pmc), nitro, p-toluenesulfonyl, 4- methoxybenzene- sulfonyl, Cbz, Boc, and adamantyloxycarbonyl, for tyrosine, benzyl, o- bromobenzyloxy-carbonyl, 2,6-dichlorobenzyl, isopropyl, t-butyl (t-Bu), cyclohexyl, cyclopenyl and acetyl (Ac); for serine, t-butyl, benzyl and tetrahydropyranyl; for histidine, trityl, benzyl, Cbz, p-toluenesulfonyl and 2,4-dinitrophenyl; for tryptophan,
  • the a-C-terminal amino acid is attached to a suitable solid support or resin.
  • suitable solid supports useful for the above synthesis are those materials which are inert to the reagents and reaction conditions of the stepwise condensationdeprotection reactions, as well as being insoluble in the media used.
  • Solid supports for synthesis of a-C-terminal carboxy peptides is 4-hydroxymethylphenoxymethyl-copoly(styrene-l% divinylbenzene) or 4-(2',4*-dimethoxyphenyl-Fmoc-aminomethyl)phenoxyacetamidoethyl resin available from Applied Biosystems (Foster City. Calif.).
  • the a-C-terminal amino acid is coupled to the resin by means of N,N'-dicyclohexylcarbodiimide (DCC), N.N 1 -diisopropylcarbodiimide (DIC) or O-benzotriazol-l-yl-N,N,N',N'-tetramethyluroniumhexafluorophosphate (HBTU), with or without 4-dimethylaminopyridine (DMAP), 1 -hydroxybenzotriazole (HOBT), benzotriazol- 1- yloxy-tris(dimethylamino)phosphoniumhexafluorophosphate (BOP) oorr bis(2-oxo-3- oxazolidinyl)phosphine chloride (BOPCI), mediated coupling for from about 1 to about 24 hours at a temperature of between 10°C and 50°C in a solvent such as dichloromethane or DMF.
  • DCC N,N'-dicyclohe
  • the Fmoc group is cleaved with a secondary amine, for example, piperidine, prior to coupling with the a-C-terminal amino acid as described above.
  • One method for coupling to the deprotected 4 (2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy-acetamidoethyl resin is O-benzotriazol-1- yl-N,N,N',N'-tetramethyluroniumhexafluorophosphate (HBTU, 1 equiv.) and 1- hydroxybenzotriazole (HOBT, 1 equiv.) in DMF.
  • the coupling of successive protected amino acids can be carried out in an automatic polypeptide synthesizer.
  • the a-N-terminus in the amino acids of the growing peptide chain are protected with Fmoc.
  • the removal of the Fmoc protecting group from the a-N-terminal side of the growing peptide is accomplished by treatment with a secondary amine, for example, piperidine.
  • a secondary amine for example, piperidine.
  • Each protected amino acid is then introduced in about 3 -fold molar excess, and the coupling can be carried out in DMF.
  • the coupling agent can be O-benzotriazol-l-yl-N,N,N',N , -tetramethyluroniumhexafluorophosphate (HBTU, 1 equiv.) and 1- hydroxybenzotriazole (HOBT, 1 equiv.).
  • HBTU O-benzotriazol-l-yl-N,N,N',N , -tetramethyluroniumhexafluorophosphate
  • HOBT 1- hydroxybenzotriazole
  • Removal of the polypeptide and deprotection can be accomplished in a single operation by treating the resinbound polypeptide with a cleavage reagent comprising thianisole, water, ethanedithiol and trifluoroacetic acid.
  • a cleavage reagent comprising thianisole, water, ethanedithiol and trifluoroacetic acid.
  • the resin is cleaved by aminolysis with an alkylamine.
  • the peptide can be removed by transesterification, e.g. with methanol, followed by aminolysis or by direct transamidation.
  • the protected peptide can be purified at this point or taken to the next step directly.
  • the removal of the side chain protecting groups can be accomplished using the cleavage cocktail described above.
  • the fully deprotected peptide can be purified by a sequence of chromatographic steps employing any or all of the following types: ion exchange on a weakly basic resin (acetate form); hydrophobic adsorption chromatography on underivitized polystyrene-divinylbenzene (for example, Amberlite XAD); silica gel adsorption chromatography; ion exchange chromatography on carboxymethylcellulose; partition chromatography, e.g. on Sephadex G-25, LH-20 or countercurrent distribution; high performance liquid chromatography (HPLC), especially reversephase HPLC on octyl- or octadecylsilyl-silica bonded phase column packing.
  • HPLC high performance liquid chromatography
  • Polymers such as PEG groups
  • an oligonucleotide such as an ASO, a cCCP, EP, or ocular delivery construct under any suitable conditions. Any means known in the art can be used, including via acylation, reductive alkylation, Michael addition, thiol alkylation or other chemoselective conjugation/ligation methods through a reactive group on the PEG moiety (e g., an aldehyde, amino, ester, thiol, a-haloacetyl, maleimido or hydrazino group) to a reactive group on the ASO a cCCP, an EEV, or a compound comprising an EEV (e.g., an aldehyde, amino, ester, thiol, a-haloacetyl, maleimido or hydrazino group).
  • an EEV e.g., an aldehyde, amino, ester, thiol, a-halo
  • Activating groups which can be used to link the water soluble polymer to one or more proteins include without limitation sulfone, maleimide, sulfhydryl, thiol, triflate, tresylate, azidirine, oxirane, 5-pyridyl, and alpha-halogenated acyl group (e.g., a-iodo acetic acid, a-bromoacetic acid, a-chloroacetic acid).
  • the polymer selected should have a single reactive aldehyde so that the degree of polymerization is controlled.
  • amino acid residues of the CPP may be reacted with an organic derivatizing agent that is capable of reacting with a selected side chain or theN- or C-termini of an amino acids.
  • Reactive groups on the peptide or conjugate moiety include, e.g., an aldehyde, amino, ester, thiol, a-haloacetyl, maleimido or hydrazino group.
  • Derivatizing agents include, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N -hydroxy succinimide (through lysine residues), glutaraldehyde, succinic anhydride or other agents known in the art.
  • Non-limiting examples of compounds that include a CPPs and a reactive group useful for conjugation to an therapeutic moiety are shown in Table 3.
  • Example linker groups are also shown.
  • Example reactive groups include tetrafluorophenyl ester (TFP), free carboxylic acid (COOH), an azide (N3) and an alkyne (e.g., a cyclooctyne).
  • n is an integer from 0 to 20;
  • Pipa6 is AcRXRRBRRXRYQFLIRXRBRXRB wherein B is P-Alanine and X is aminohexanoic acid; Dap is 2,3-diaminopropionic acid; NLS is a nuclear localization sequence;
  • the cCPPs have free carboxylic acid groups that may be utilized for conjugation to a therapeutic moiety, such as an oligonucleotide, a polypeptide, or a small molecule.
  • the EEVs have free carboxylic acid groups that may be utilized for conjugation to the therapeutic moiety.
  • FIGS. 3A-3D and FIG. 4 show example conjugation chemistry schemes.
  • the CPPs of the examples may be modified with linker that have reactive groups allowing conjugation.
  • the linkers in each example scheme may not be fully shown and/or are non-limiting.
  • Two example schemes showing conjugation of a CPP or a modified CPP to the modified 5' end of an oligonucleotide via an amide bond is shown in FIG. 3A.
  • FIG. 3B An example scheme showing the conjugation of a CPP or modified CPP to the modified 3' end of an oligonucleotide via an amide bond.
  • FIG. 3C An example scheme showing the conjugation of an azide modified CPP to a 5 '-cyclooctyne modified PMO via strain-promoted azide-alkyne cycloaddition is shown in FIG. 3C.
  • FIG. 3D Several azide-alkyne conjugation reactions for conjugating a modified 3' of an oligonucleotide to a modified CPP are shown in FIG. 3D.
  • FIG. 4 Various examples showing conjugation chemistries using a bifunctional compound that includes a PEG moiety and two reactive handles (e g., activated tetrafluorophenyl ester, alkyne, and N -hydroxysuccinimide ester) are shown in FIG. 4.
  • One of the reactive handles is used to conjugate the AC to the bifunctional molecule and the second reactive handle is used to conjugate the CPP to the bifunctional molecule thereby conjugating the oligonucleotide to the CPP.
  • compositions are provided that include one or more cargo conjugates.
  • the composition is a pharmaceutical composition; that is, a composition designed for administration to a subject.
  • pharmaceutically acceptable salts and/or prodrugs of the disclosed cargo conjugates are disclosed.
  • Pharmaceutically acceptable salts include salts of the disclosed cargo conjugates that are prepared with acids or bases, depending on the particular substituents found on the cargo conjugates. Under conditions where the cargo conjugates are sufficiently basic or acidic to form stable nontoxic acid or base salts, administration of the cargo conjugates as salts can be appropriate.
  • pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, or magnesium salt.
  • physiologically acceptable acid addition salts include hydrochloric, hydrobromic, nitric, phosphoric, carbonic, sulfuric, and organic acids like acetic, propionic, benzoic, succinic, fumaric, mandelic, oxalic, citric, tartaric, malonic, ascorbic, alpha-ketoglutaric, alpha-glycophosphoric, maleic, tosyl acid, methanesulfonic, and the like.
  • Pharmaceutically acceptable salts of a cargo conjugate can be obtained using standard procedures well known in the art, for example, by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion.
  • Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example calcium) salts of carboxylic acids can also be made.
  • the disclosed cargo conjugates can be formulated in a physiologically- or pharmaceutically-acceptable form and administered by any suitable route known in the art including, for example, compositions for dropping on the eye or for injecting into the eye (intraocular administration).
  • Administration of the disclosed cargo conjugates or compositions can be a single administration, or at continuous or distinct intervals as can be readily determined by a person skilled in the art.
  • the cargo conjugates, and compositions comprising them can also be administered utilizing liposome technology. These delivery methods can, advantageously, provide a uniform dosage over an extended period of time.
  • the cargo conjugates can also be administered in their salt derivative forms or crystalline forms.
  • the cargo conjugates can be formulated according to known methods for preparing pharmaceutically acceptable compositions (pharmaceutical compositions). Formulations are described in detail in a number of sources which are well known and readily available to those skilled in the art. For example, Remington's Pharmaceutical Science by E.W. Martin (1995) describes formulations that can be used in connection with the disclosed methods. In general, the cargo conjugates can be formulated such that an effective amount of the cargo conjugates is combined with a suitable carrier in order to facilitate effective administration of the cargo conjugates.
  • the compositions used can also be in a variety of forms. These include, for example, liquid dosage forms, such as liquid solutions or suspension, and injectable and infusible solutions. The form depends on the intended mode of administration and therapeutic application.
  • compositions can also include conventional pharmaceutically-acceptable carriers and diluents which are known to those skilled in the art.
  • carriers or diluents for use with the compounds include water, ethanol, dimethyl sulfoxide, glycerol, alumina, starch, saline, and equivalent carriers and diluents.
  • compositions can advantageously comprise between about 0.1% and 100% by weight of the total of one or more of the subject cargo conjugates based on the weight of the total composition including carrier or diluent.
  • Formulations suitable for administration include, for example, aqueous sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the interstitial fluid of the eye of the intended recipient; and aqueous and nonaqueous sterile suspensions, which can include suspending agents and thickening agents.
  • the formulations can be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and can be stored in a freeze dried (lyophilized) condition requiring only the condition of the sterile liquid carrier, for example, water for injections, prior to use.
  • Extemporaneous injection solutions and suspensions can be prepared from sterile powder, granules, tablets, etc. It should be understood that in addition to the ingredients mentioned above, the compositions disclosed herein can include other agents conventional in the art having regard to the type of formulation in question.
  • Cargo conjugates, and compositions comprising them can be delivered to a cell either through direct contact with the cell or via a carrier means.
  • Carrier means for delivering compounds and compositions to cells are known in the art and include, for example, encapsulating the composition in a liposome moiety.
  • Another means for delivery of compounds and compositions to a cell can comprise attaching the cargo conjugates to a protein or nucleic acid that is targeted for delivery to the target cell.
  • U.S. Patent No. 6,960,648 and U.S. Application Publication Nos. 20030032594 and 20020120100 disclose amino acid sequences that can be coupled to another composition and that allows the composition to be translocated across biological membranes.
  • compositions for transporting biological moieties across cell membranes for intracellular delivery can also be incorporated into polymers, examples of which include poly (D-L lactide-co-glycolide) polymer for intracranial tumors; poly[bis(p-carboxyphenoxy) propane: sebacic acid] in a 20:80 molar ratio (as used in GLIADEL); chondroitin; chitin; and chitosan.
  • the cargo conjugate can be administered to a patient in need of treatment in combination with other active agents designed to treat the disease. These other substances or treatments can be given at the same as or at different times from the cargo conjugates.
  • a pharmaceutical composition comprising a cargo conjugate can further include a second active agent designed to treat the disease.
  • Cargo conjugates and compositions containing the same, including pharmaceutically acceptable salts or prodrugs thereof, can be administered by drop, infusion, or injection.
  • the compounds and compositions can be administered parenterally.
  • the compounds and compositions can be administered intravenously.
  • the compounds and compositions can be administered subcutaneously.
  • the compounds and compositions disclosed herein can be administered intraocularly.
  • the compounds and compositions can be administered intravitreally.
  • Solutions of the active agent or its salts can be prepared in water, optionally mixed with a nontoxic surfactant Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations can contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient, which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes.
  • the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage.
  • the liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various other antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • Isotonic agents can be included, for example, sugars, buffers or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the inclusion of agents that delay absorption, for example, aluminum monostearate and gelatin
  • Sterile injectable solutions are prepared by incorporating a cargo conjugate and/or other agent in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filter sterilization.
  • the methods of preparation include vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.
  • cargo conjugates and compositions containing the same can be applied in as a liquid or solid.
  • a ophthalmologically acceptable carrier which can be a solid or a liquid.
  • compounds are formulated into a solution or suspension for topical application to a surface of the eye, such as to a surface of the cornea.
  • Useful liquid carriers include w z ater, alcohols or glycols or water-alcohol/glycol blends, in which the compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants.
  • Adjuvants such as fragrances and additional antimicrobial agents can be added to modify the properties for a given use.
  • the resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers, for example.
  • Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the surface of the eye.
  • compositions that comprise a compound disclosed herein in combination with a pharmaceutically acceptable carrier, including, but not limited to, pharmaceutical compositions adapted for oral, topical, parenteral or intraocular administration, comprising an amount of a compound
  • a pharmaceutically acceptable carrier including, but not limited to, pharmaceutical compositions adapted for oral, topical, parenteral or intraocular administration, comprising an amount of a compound
  • the dose administered to a patient, particularly a human should be sufficient to achieve a therapeutic response in the patient over a reasonable time frame, without lethal toxicity, and causing no more than an acceptable level of side effects or morbidity.
  • dosage will depend upon a variety of factors including the condition (health) of the subject, the body weight of the subject, kind of concurrent treatment, if any, frequency of treatment, therapeutic ratio, as well as the severity and stage of the pathological condition.
  • kits that comprise a cargo conjugate or composition containing the same in one or more containers.
  • the disclosed kits can optionally include pharmaceutically acceptable carriers and/or diluents.
  • a kit can include one or more other components, adjuncts, or adjuvants.
  • a kit includes one or more additional active agents for treating an eye disease.
  • a kit can include instructions or packaging materials that describe how to administer a cargo conjugate or composition of the kit.
  • Containers of the kit can be of any suitable material, e.g., glass, plastic, metal, etc., and of any suitable size, shape, or configuration.
  • a cargo conjugate can be provided in the kit as a solid, such as a tablet, pill, or powder form.
  • a cargo conjugate can be provided in the kit as a liquid or solution.
  • a kit can comprise an ampoule or syringe containing a cargo conjugate or composition containing the same in liquid or solution form.
  • the method comprises identifying a subject having a disease or disorder of the eye and administering a cargo conjugate or a composition comprising one or more cargo conjugates to the subject.
  • the cargo conjugate includes a therapeutic moiety designed to treat an ocular disease.
  • the method may be a method of treatment of an ocular disease.
  • Identifying a subject includes identifying a subject that has an ocular disease.
  • the subject may be a mammal.
  • the subject may be human.
  • a subject that has an ocular disease may display one or more symptoms or clinical signs associated with an ocular disease; may have a genetic signature associated with an ocular disease, for example, may have a mutation in a gene associated with an ocular disease; or both.
  • Identification of a subject having an ocular disease may include diagnosing the subject with an ocular disease. Diagnosis of an ocular disease may be by way of a physician or other health care provider conducting tests and exams to identify a cause of symptoms displayed by a subject.
  • Genetic testing may be used to diagnose a subject with an ocular disease. Genetic testing may include sequencing a portion of the subjects genome to identify genetic anomalies in one or more genes associated with an ocular disease. To identify genetic anomalies, the sequenced portion of a subject’s genome can be compared to a standard genome that does not contain genetic anomalies in the genes associated with the eye. Sequencing technologies and analysis methods are well known.
  • genetic testing following diagnosis of an ocular disease may be used to identify a particular genetic anomaly causing the disease. Identification of the particular genetic anomaly, such as a mutation that results in the displayed phenotype, may allow one to choose a particular therapeutic moiety designed to have a biological effect given the genetic anomaly. For example, some therapeutic moieties are designed to be effective when a subject displays a particular genetic anomaly of a disease and may not be effective or as effective if the subject displays a different anomaly of the same disease. For example, a subject diagnosed with an ocular disease characterized by one or both of two independent mutations may undergo genetic testing to determine if they have the first mutation, the second mutation, or both.
  • a first cargo conjugate having a first therapeutic moiety designed to be effective in a disease state of the first mutation may be administered to the subject.
  • a second cargo conjugate having a second therapeutic moiety designed to be effective in a disease state of the second mutation may be administered to the subject.
  • the first cargo conjugate may not be effective or as effective if the subject displays the second mutation and the second cargo conjugate may not be effective or as effective if the subject displays the first mutation.
  • the method of administration further includes administering a cargo conjugate or a composition comprising one or more cargo conjugates to the subject.
  • the cargo conjugate or a pharmaceutical composition directly to the eye of the subject; that is, the cargo conjugate or pharmaceutical composition is ocularly administered to the subject.
  • the composition is administered directly to a tissue, such as, for example, to the cornea, sclera, lens, iris, ciliary body, optic nerve, choroid, or retina.
  • the cargo conjugate or pharmaceutical composition is topically administered.
  • the cargo conjugate or pharmaceutical composition is delivered to the cornea.
  • the pharmaceutical composition may be an eye drop formulation that is contacted with the cornea of the subject’s eye.
  • Other routes of topical administration include drug delivery contacts soaked or coated with a cargo conjugate or pharmaceutical composition. The contact may be designed for and allow a delayed and/or sustained release of the cargo conjugate.
  • the cargo conjugate or pharmaceutical composition is periocularly administered.
  • Periocular administration includes drug deposition on the scleral external surface. The drug can diffuse through the scelara.
  • Examples of periocular administration include, but are not limited to, subconjunctival administration, subTenon injection, retrobulbar administration, peribulbar juxtascleral administration, posterior juxtascleral administration, and iontophoresis techniques such as the EYEGATE U system from Kiora Pharmaceuticals, Inc. (Encinitas, CA).
  • the cargo conjugate or pharmaceutical composition is intravitreally delivered.
  • Intravitreal injection delivers the injected species into the vitreous humor.
  • Techniques that may be used for intravitreal administration include intravitreal injection (also called intraocular injection) and intravitreal infusion.
  • the cargo conjugate or pharmaceutical composition is subretinally administered.
  • Subretinal administration includes delivery of drug or composition to the retina. Examples of subretinal administration include retinal injection (subretinal injection) and implantation of biodegradable drug delivery devices or non-biodegradable medical devices.
  • Subretinal administration may involve subjecting the subject to a pars plana vitrectomy and a retinotomy. Subretinal administration may deliver the drug or composition to the space between or including the retinal pigment epithelium layer and the photoreceptor layer.
  • the cargo conjugate or pharmaceutical composition is suprachoroidally administered.
  • Suprachoroidal administration involves depositing a drug or composition in the suprachoroidal space between the scelara and the choroid.
  • Techniques that may be used for suprachoroidal administration include injection and infusion using, for example, a micro needle, a cannulas, or a catheter.
  • the cargo conjugate or pharmaceutical composition is intracamerally administered.
  • Intracameral administration involves depositing a drug or composition into the anterior chamber of the eye. Intracameral administration may be accomplished by intracameral injection.
  • the cargo conjugate or pharmaceutical composition is delivered to the eye through the use of a medical device.
  • the medical device may be biodegradable or non- biodegradable.
  • the medical device may be implanted at any location within the eye.
  • a cargo conjugate or pharmaceutical composition may be a part of an intravitreal implant, a Cul- de-sac implant, or an episcleral implant.
  • Therapeutically effective dosages of the cargo conjugates and pharmaceutical compositions can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art.
  • Therapeutically effective dosage ranges for the administration of the cargo conjugates and compositions containing the same are those large enough to produce the desired effect in which the symptoms or disorder are affected.
  • the dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like.
  • the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art.
  • the dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days.
  • the type and origin of the molecule are not limited by the case and/or typeface (e.g., bold, italics, etc.).
  • a specific name of gene, transcript, or protein may be denoted as unitalicized and upper case, italicized and upper case, unitalicized and upper case, or unitalicized and lower case.
  • the specific type and origin of the molecule are to be understood in the context of the use of the stated molecule.
  • cyclic cell penetrating peptide refers to a peptide that facilitates the delivery of a cargo, e.g., a therapeutic moiety, into a cell.
  • EEV endosomal escape vehicle
  • EEV-conjugate refers to an endosomal escape vehicle defined herein conjugated by a chemical linkage (i.e., a covalent bond or non-covalent interaction) to a cargo.
  • the cargo can be a therapeutic moiety (e.g., an oligonucleotide, peptide or small molecule) that can be delivered into a cell by the EEV.
  • EP exocyclic peptide
  • cCPP cyclic cell penetrating peptide
  • the EP when a part of an ocular delivery construct, may alter the tissue distribution and/or retention of the compound.
  • the EP comprises at least one positively charged amino acid residue, e.g , at least one lysine residue and/or at least one arginine residue.
  • NLS nuclear localization sequence
  • linker refers to a moiety that covalently couples two or more components of a delivery construct and/or a cargo construct; includes one or more functional groups that can be used to conjugate one or more components to the linker; or both.
  • a linker can covalently couple one or more moieties (e.g., an exocyclic peptide (EP) and a cargo; a cCPP and an EP; a cCPP and a cargo; a cCPP, an EP, and a cargo) to the cyclic cell penetrating peptide (cCPP).
  • a linker can covalently link a cargo and a delivery construct and/or two or more components of a delivery construct.
  • the linker can comprise a natural or non-natural amino acid or polypeptide.
  • the linker can be a synthetic compound containing two or more appropriate functional groups suitable to conjugate one or more components of a delivery construct and/or a delivery construct to a cargo.
  • the linker can comprise a polyethylene glycol (PEG) moiety.
  • the linker can comprise one or more amino acids.
  • the cCPP may be covalently bound to a cargo via a linker.
  • oligonucleotide refers to an oligomeric compound comprising a plurality of linked nucleotides or nucleosides.
  • One or more nucleotides of an oligonucleotide can be modified.
  • An oligonucleotide can comprise ribonucleic acid (RNA) or deoxyribonucleic acid (DNA).
  • Oligonucleotides can be composed of natural and/or modified nucleobases, sugars and covalent intemucleoside linkages, and can further include non- nucleic acid conjugates.
  • peptide “protein,” and “polypeptide” are used interchangeably to refer to a natural or synthetic molecule comprising two or more amino acids linked by the carboxyl group of one amino acid to the alpha amino group of another. Two or more amino acid residues can be linked by the carboxyl group of one amino acid to the alpha amino group. Two or more amino acids of the polypeptide can be joined by a peptide bond.
  • the polypeptide can include a peptide backbone modification in which two or more amino acids are covalently attached by a bond other than a peptide bond.
  • the polypeptide can include one or more non-natural amino acids, amino acid analogs, or other synthetic molecules that are capable of integrating into a polypeptide.
  • polypeptide includes naturally occurring and artificially occurring amino acids.
  • polypeptide includes peptides, for example, that include from about 2 to about 100 amino acid residues as well as proteins, that include more than about 100 amino acid residues, or more than about 1000 amino acid residues, including, but not limited to therapeutic proteins such as antibodies, enzymes, receptors, soluble proteins and the like.
  • the term “therapeutic agent” or “therapeutic moiety” can be used to refer to a cargo that has therapeutic, prophylactic or other biological activity.
  • the therapeutic agent can be a peptide, oligonucleotide or a small molecule.
  • the therapeutic agent can be an enzyme.
  • the therapeutic agent can be an antibody or antigen binding fragment.
  • the therapeutic agent can be an oligonucleotide.
  • the therapeutic agent can be an antisense oligonucleotide.
  • the therapeutic agent can be one or more components of a gene editing machinery (GEM).
  • the therapeutic agent can be an oligonucleotide encoding one or more components of a gene editing machinery (GEM).
  • the therapeutic agent can include gRNA.
  • the therapeutic agent can include a nuclease or an oligonucleotide encoding a nuclease.
  • the nuclease is a Cas nuclease.
  • the nuclease is a Cas9 nuclease.
  • the therapeutic agent can include a ribonucleoprotein (RNP) or aann oligonucleotide encoding a RNP.
  • RNP ribonucleoprotein
  • small molecule refers to an organic compound with pharmacological activity and a molecular weight of less than about 2000 Daltons, or less than about 1000 Daltons, or less than about 500 Daltons. Small molecule therapeutics are typically manufactured by chemical synthesis.
  • the term “contiguous” refers to two amino acids, which are connected by a covalent bond.
  • the term “contiguous” refers to two amino acids, which are connected by a covalent bond.
  • cCPP such as , AA1/AA2, AA2/AA3.
  • AA3/AA4,and AA5/AA1 exemplify pairs of contiguous amino acids.
  • a residue of a chemical species refers to a derivative of the chemical species that is present in a particular product.
  • the product at least one atom of the species is replaced by a bond to another moiety, such that the product contains a derivative, or residue, of the chemical species.
  • the cyclic cell penetrating peptides can have amino acids (e g., arginine) incorporated therein through formation of one or more peptide bonds.
  • the amino acids incorporated into the cCPP may be referred to residues, or simply as an amino acid.
  • arginine or an arginine residue refers to [0484]
  • the term “protonated form thereof' refers to a protonated form of an amino acid.
  • the guanidine group on the side chain of arginine may be protonated to form a guanidinium group.
  • the structure of a protonated form of arginine is
  • the term “chirality” refers to a molecule that has more than one stereoisomer that differs in the three-dimensional spatial arrangement of atoms, in which one stereoisomer is a non-superimposable mirror image of the other. Amino acids, except for glycine, have a chiral carbon atom adjacent to the carboxyl group.
  • the term “enantiomer” refers to stereoisomers that are chiral.
  • the chiral molecule can be an amino acid residue having a “D” and “L” enantiomer.
  • Hydrophobic refers to a moiety that is not soluble in water or has limited solubility in water. Generally, neutral moieties and/or non-polar moieties, or moieties that are predominately neutral and/or non-polar are hydrophobic. Hydrophobicity can be measured by one of the methods disclosed herein.
  • aromatic refers to an unsaturated cyclic molecule having 4n + 2 n electrons, wherein n is any integer.
  • non-aromatic refers to any unsaturated cyclic molecule which does not fall within the definition of aromatic.
  • non-aromatic amino acids include, but are not limited to, glycine and citrulline.
  • Alkyl refers to a fully saturated, straight or branched hydrocarbon chain radical having from one to forty carbon atoms, and which is attached to the rest of the molecule by a single bond. Alkyls comprising any number of carbon atoms from 1 to 40 are included. An alkyl comprising up to 40 carbon atoms is a C1-C40 alkyl, an alkyl comprising up to 10 carbon atoms is a C1-C10 alkyl, an alkyl comprising up to 6 carbon atoms is a C1-C6 alkyl and an alkyl comprising up to 5 carbon atoms is a C1-C5 alkyl.
  • a C1-C5 alkyl includes Cs alkyls, C* alkyls, C3 alkyls, C2 alkyls and Ci alkyl (z.e., methyl).
  • a Ci-Ce alkyl includes all moieties described above for C1-C5 alkyls but also includes Ce alkyls.
  • a C1-C10 alkyl includes all moieties described above for C1-C5 alkyls and Ci-Ce alkyls, but also includes Ci, Cs, C9 and C10 alkyls.
  • a Ci-Cu alkyl includes all the foregoing moieties, but also includes Cn and C12 alkyls.
  • Non-limiting examples of C1-C12 alkyl include methyl, ethyl, zz-propyl, z-propyl, sec-propyl, zz-butyl, z-butyl, sec-butyl, Z-butyl, zz-pentyl, /-amyl, w-hexyl, zz-heptyl, w-octyl, rz-nonyl, zz-decyl, zz-undecyl, andzz- dodecyl.
  • an alkyl group can be optionally substituted.
  • Alkylene refers to a fully saturated, straight or branched divalent hydrocarbon chain radical, having from one to forty carbon atoms.
  • C2-C40 alkylene include ethylene, propylene, zz-butylene, ethenylene, propenylene, zz-butenylene, propynylene, zz-butynylene, and the like. Unless stated otherwise specifically in the specification, an alkylene chain can be optionally substituted.
  • alkenyl refers to a straight or branched hydrocarbon chain radical having from two to forty carbon atoms and having one or more carbon-carbon double bonds. Each alkenyl group is attached to the rest of the molecule by a single bond. Alkenyl groups comprising any number of carbon atoms from 2 to 40 are included.
  • An alkenyl group comprising up to 40 carbon atoms is a C2-C40 alkenyl
  • an alkenyl comprising up to 10 carbon atoms is a C2- Cio alkenyl
  • an alkenyl group comprising up to 6 carbon atoms is a C2-C6 alkenyl
  • an alkenyl comprising up to 5 carbon atoms is a C2-C5 alkenyl.
  • a C2-C5 alkenyl includes Cs alkenyls, C4 alkenyls, C3 alkenyls, and C2 alkenyls.
  • a C2-C6 alkenyl includes all moieties described above for C2-C5 alkenyls but also includes C6 alkenyls.
  • a C2-C10 alkenyl includes all moieties described above for C2-C5 alkenyls and C2-C6 alkenyls, but also includes C?, Cs, C9 and C10 alkenyls.
  • a C2-C12 alkenyl includes all the foregoing moieties, but also includes Cu and C12 alkenyls.
  • Non-limiting examples of C2-C12 alkenyl include ethenyl (vinyl), 1 -propenyl, 2-propenyl (allyl), iso-propenyl, 2-methyl-l -propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1 -pentenyl, 2- pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1- heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-heptenyl.
  • 6-heptenyl 1-octenyl, 2-octenyl, 3- octenyl, 4-octenyl, 5-octenyl, 6-octenyl, 7-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 4-nonenyl, 5-nonenyl, 6-nonenyl, 7-nonenyl, 8-nonenyl, 1-decenyl, 2-decenyl, 3-decenyl, 4-decenyl, 5- decenyl, 6-decenyl, 7-decenyl, 8-decenyl, 9-decenyl, 1 -undecenyl, 2-undecenyl, 3-undecenyl, 4- undecenyl, 5-undecenyl, 6-undecenyl, 7-undecenyl, 8-undecenyl, 9-undecen
  • alkenylene refers to a straight or branched divalent hydrocarbon chain radical, having from two to forty carbon atoms, and having one or more carbon-carbon double bonds.
  • C2-C40 alkenylene include ethene, propene, butene, and the like. Unless stated otherwise specifically in the specification, an alkenylene chain can be optionally.
  • Alkoxy or “alkoxy group” refers to the group -OR, where R is alkyl, alkenyl, alkynyl, cycloalkyl, or heterocyclyl as defined herein. Unless stated otherwise specifically in the specification, an alkoxy group can be optionally substituted.
  • acyl or “acyl group” refers to groups -C(O)R, where R is hydrogen, alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl, as defined herein Unless stated otherwise specifically in the specification, acyl can be optionally substituted.
  • Alkylcarbamoyl or “alkylcarbamoyl group” refers to the group -O-C(O)-NRaRb, where Ra and Rb are the same or different and are independently an alkyl, alkenyl, alkynyl, aryl, heteroaryl, as defined herein, or RaRb can be taken together to form a cycloalkyl group or heterocyclyl group, as defined herein. Unless stated otherwise specifically in the specification, an alkylcarbamoyl group can be optionally substituted.
  • Alkylcarboxamidyl or “alkylcarboxamidyl group” refers to the group -C(O)-NRaRb, where Ra and Rb are the same or different and are independently an alkyl, alkenyl, alkynyl, aryl, heteroaiyl, cycloalkyl, cycloalkenyl, cycloalkynyl, or heterocyclyl group, as defined herein, or RaRb can be taken together to form a cycloalkyl group, as defined herein. Unless stated otherwise specifically in the specification, an alkylcarboxamidyl group can be optionally substituted.
  • Aryl refers to a hydrocarbon ring system radical comprising hydrogen, 6 to 18 carbon atoms and at least one aromatic ring.
  • the aryl radical can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which can include fused or bridged ring systems.
  • Aryl radicals include, but are not limited to, aryl radicals derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene, as-indacene, s-indacene, indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene, and triphenylene.
  • aryl is meant to include aryl radicals that are optionally substituted.
  • Heteroaryl refers to a 5- to 20-membered ring system radical comprising hydrogen atoms, one to thirteen carbon atoms, one to six heteroatoms selected from nitrogen, oxygen and sulfur, and at least one aromatic ring
  • the heteroaryl radical can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which can include fused or bridged ring systems; and the nitrogen, carbon or sulfur atoms in the heteroaryl radical can be optionally oxidized; the nitrogen atom can be optionally quatemized.
  • Examples include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, benzooxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[6][l,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl (benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[l,2-a]pyridinyl, carbazolyl, cirmolinyl, dibenzofuranyl, dibenzothiophenyl, fur
  • substituted means any of the above groups (i.e., alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, acyl, alkylcarbamoyl, alkylcarboxamidyl, alkoxycarbonyl, alkylthio, or arylthio) wherein at least one atom is replaced by a non-hydrogen atoms such as, but not limited to: a halogen atom such as F, Cl, Br, and I; an oxygen atom in groups such as hydroxyl groups, alkoxy groups, and ester groups; a sulfur atom in groups such as thiol groups, thioalkyl groups, sulfone groups, sulfonyl groups, and sulfoxide groups; a nitrogen atom in groups
  • “Substituted” also means any of the above groups in which one or more atoms are replaced by a higher-order bond (e g., a double- or triple-bond) to a heteroatom such as oxygen in oxo, carbonyl, carboxyl, and ester groups; and nitrogen in groups such as imines, oximes, hydrazones, and nitriles.
  • a higher-order bond e g., a double- or triple-bond
  • nitrogen in groups such as imines, oximes, hydrazones, and nitriles.
  • Rg and Rh are the same or different and independently hydrogen, alkyd, alkenyl, alkynyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, haloalkyl, haloalkenyl, haloalkynyl, heterocyclyl, A-heterocyclyl, heterocyclylalkyl, heteroaryl, yV-heteroaryl and/or heteroarylalkyl.
  • “Substituted” further means any of the above groups in which one or more atoms are replaced by an amino, cyano, hydroxyl, imino, nitro, oxo, thioxo, halo, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, haloalkyl, haloalkenyl, haloalkynyl, heterocyclyl, JV-heterocyclyl, heterocyclylalkyl, heteroaryl, A-heteroaryl and/or heteroarylalkyl group.
  • “Substituted” can also mean an amino acid in which one or more atoms on the side chain are replaced by alkyl, alkenyl, alkynyl, acyl, alkylcarboxamidyl, alkoxy carbonyl, carbocyclyl, heterocyclyl, aryl, or heteroaiyl.
  • each of the foregoing substituents can also be optionally substituted with one or more of the above substituents.
  • a “subject” is meant an individual.
  • the subject can be a mammal.
  • the “subject” can include domesticated animals (e.g., cats, dogs, etc ), livestock (e.g. , cattle, horses, pigs, sheep, goats, etc ), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc ), and birds.
  • “Subject” can also include a mammal, such as a primate or a human.
  • the subject can be a human or veterinary' patient.
  • patient refers to a subject under the treatment of a clinician, e.g., physician.
  • the term “inhibit” refers to a decrease in an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This can also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels. [0501] By “reduce” or other forms of the word, such as “reducing” or “reduction,” is meant lowering of an event or characteristic (e.g., tumor growth).
  • “reduces tumor growth” means reducing the rate of growth of a tumor relative to a standard or a control (e.g., an untreated tumor).
  • treatment refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder.
  • This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder.
  • this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to reducing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
  • terapéuticaally effective refers to the amount of the compound or composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.
  • pharmaceutically acceptable refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio.
  • carrier means a compound, composition, substance, or structure that, when in combination with a compound or composition, aids or facilitates preparation, storage, administration, delivery, effectiveness, selectivity, or any other feature of the compound or composition for its intended use or purpose.
  • a carrier can be selected to reduce any degradation of the active ingredient and any adverse side effects in the subject.
  • pharmaceutically acceptable carrier refers to sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use.
  • aqueous and nonaqueous carriers, diluents, solvents or vehicles examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol and the like
  • carboxymethylcellulose and suitable mixtures thereof such as vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.
  • These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.
  • the injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable media just prior to use.
  • Suitable inert carriers can include sugars such as lactose.
  • amino acid refers to an organic compound that includes an amino group and a carboxylic acid group and has the general formula where R can be any organic group.
  • An amino acid may be a naturally occurring amino acid or non-naturally occurring amino acid.
  • An amino acid may be a proteogenic amino acid or a non-proteogenic amino acid.
  • An amino acid can be an L-amino acid or a D- amino acid.
  • amino acid side chain or “side chain” refers to the characterizing substituent (“R”) bound to the a-carbon of a natural or non-natural a-amino acid.
  • An amino acid may be incorporated into a polypeptide via a peptide bond.
  • an “uncharged” amino acid is an amino acid having a side chain that has a net neutral charge at pH 7.35 to 7.45.
  • Examples of uncharged amino acids include, but are not limited to, glycine and citrulline.
  • a “charged” amino acid is an amino acid having a side chain having a net charge at a pH of 7.35 to 7.45.
  • An example of a charged amino acid is arginine.
  • “polyethylene glycol” and “PEG” are used interchangeably.
  • PEGm,” and “PEGm,” are, or are derived from, a molecule of the formula HO(CO)-(CIh)n-(OCH2CH2)m- NH2 where n is any integer from 1 to 5 and m is any integer from 1 to 23.
  • n is 1 or 2.
  • n is 1.
  • n is 2.
  • n is 1 and m is 2.
  • n is 2 and m is 2 In embodiments, n is 1 and m is 4. In embodiments, n is 2 and m is 4. In embodiments, n is 1 and m is 12. In embodiments, n is 2 and m is 12.
  • miniPEGm or “miniPEGm” are, or are derived from, a molecule of the formula HO(CO)-(CH2)n-(OCH2CH2)m-NH2 where n is i and m is any integer from 1 to 23.
  • miniPEG2 or “miniPEG?” is, or is derived from, (2-[2-[2-aminoethoxy]ethoxy]acetic acid)
  • miniPEG4 or “miniPEGi” is, or is derived from, HO(CO)-(CH2)n-(OCH2CH2)m- NH2 where n is 1 and m is 4.
  • “2-[2-[2-aminoethoxy]ethoxy]acetic acid” is also referred to as AEEA, miniPEG or PEG2.
  • the term “target” refers to a macromolecule associated with a disease.
  • the target is a macromolecule implicated in a disease or pathology of the eye.
  • the target is a polypeptide or protein.
  • the target is an oligonucleotide.
  • the oligonucleotide target comprises DNA.
  • the oligonucleotide target comprises RNA.
  • the oligonucleotide target comprises mRNA.
  • the target is associated with an ocular disease.
  • the terms “targeting” or “targeted to” refer to selective association of a therapeutic moiety with a target molecule.
  • an antisense oligonucleotide may selectively bind with a target nucleic acid molecule or a region of a target nucleic acid molecule.
  • a peptide may selectively bind with a target protein or region of a target protein.
  • selective binding of a therapeutic moiety with a target molecule is useful for the treatment of a disease, pathologies or other abnormal states or conditions of the eye.
  • the therapeutic moiety includes an antisense oligonucleotide that is capable of hybridizing to a target nucleic acid under physiological conditions.
  • the antisense compound targets a specific portion or site within the target nucleic acid, for example, a portion of the target nucleic acid having at least one identifiable structure, function, or characteristic such as a particular exon or intron, or selected nucleobases or motifs within an exon or intron.
  • a therapeutic oligonucleotide is “specific” for a target oligonucleotide if the therapeutic oligonucleotide has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with a portion of the nucleic acid sequence of the target oligonucleotide when the nucleic acid sequences of the therapeutic oligonucleotide and the target oligonucleotide are aligned.
  • a therapeutic oligonucleotide that is “selectively binds” or “specifically binds” to a target oligonucleotide is one that is capable of hybridizing to the target oligonucleotide of interest and not substantially hybridizing to other oligonucleotide sequences which are not of interest under stringent hybridization/washing conditions.
  • An oligonucleotide which is “gene-specific” is specific for a target oligonucleotide sequence in a gene of interest and does not substantially hybridize to other genes.
  • hybridize or “specifically hybridize” refers to a process where two complementary nucleic acid strands anneal to each in accordance with Watson-Crick base pairing rules. Hybridizations are typically and preferably conducted with probe-length nucleic acid molecules, preferably 20-100 nucleotides in length. Nucleic acid hybridization techniques are well known in the art. See, e.g., Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview, N.Y.
  • hybridization/washing conditions such that sequences having at least a desired level of complementarity will stably hybridize, while those having lower complementarity will not.
  • hybridization conditions and parameters see, e.g., Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview, N. Y.; Ausubel, F. M. et al. 1994, Current Protocols in Molecular Biology. John Wiley & Sons, Secaucus, N.J.
  • a binding protein or antibody or antigen-binding fragment thereof specifically binds to a target with a KD greater than lO ⁇ M, for example, with a KD between 10"*M and 10" 12 M.
  • target nucleic acid sequence refers to the nucleic acid sequence or the nucleotide sequence to which a therapeutic moiety, such as an antisense oligonucleotide, binds or hybridizes.
  • Target nucleic acids include, but are not limited, to a portion of a target transcript, target RNA (including, but not limited to pre-mRNA and mRNA or portions thereof), as well as a portion of target nontranslated RNA, such as miRNA.
  • a target nucleic acid can be a portion of a target cellular gene (or mRNA transcribed from such gene) whose expression is associated with a particular disorder or disease state.
  • portion refers to a defined number of contiguous (i.e., linked) nucleotides of a nucleic acid.
  • RNA transcript refers an RNA molecule transcribed from DNA and includes, but is not limited to mRNA, pre -mRNA, and partially processed RNA.
  • target transcript and “target RNA” refer to the pre-mRNA or mRNA transcript that is bound by the therapeutic moiety.
  • the target transcript may include a target nucleotide sequence.
  • the target transcript includes a splice site.
  • the target RNA includes a polyadenylation site or a portion thereof.
  • target gene and “gene of interest 5 ’ refer to the gene of which modulation of the expression and/or activity is desired or intended.
  • the target gene may be transcribed into a target transcript that includes a target nucleotide sequence.
  • the target transcript may be translated into a protein of interest.
  • target protein refers to the polypeptide or protein encoded by the target transcript (e.g., target mRNA).
  • the term “expression,” “gene expression,” “expression of a gene,” or the like refers to all the functions and steps by which information encoded in a gene is converted into a functional gene product, such as a polypeptide or a non-coding RNA, in a cell.
  • noncoding RNA include transfer RNA (tRNA) and ribosomal RNA.
  • Gene expression of a polypeptide includes transcription of the gene to form a pre-mRNA, processing of the pre-mRNA to form a mature mRNA, translocating the mature mRNA from the nucleus to the cytoplasm, translation of the mature mRNA into the polypeptide, and assembly of the encoded polypeptide.
  • Expression includes partial expression
  • expression of a gene may be referred to herein as generation of a gene transcript.
  • Translation of a mature mRNA may be referred to herein as expression of the mature mRNA.
  • modulate or “modulation” refers to an increase (e g., upregulation) or decrease (e.g., downregulation) of an activity of interest.
  • modulation of gene expression refers to modulation of one or more of the processes associated with gene expression.
  • modification of gene expression may include modification of one or more of gene transcription, RNA processing, RNA translocation from the nucleus to the cytoplasm, and translation of mRNA into a protein.
  • modulation of protein activity refers to modulation of protein activity for example, by increasing or decreasing protein expression, increasing or decreasing expression of a fragment of a protein having a biological activity, or by blocking one or more protein-protein or protein-substrate interactions.
  • EEVs described herein may effectively deliver cargo (e.g., a therapeutic moiety) intracellularly when delivered directly to the eye.
  • cargo e.g., a therapeutic moiety
  • an EEV-PMO conjugate may achieve broad biodistribution throughout retina after intravitreal injection, including delivery to photoreceptors.
  • reagents used in the Examples are provided as follows HATU (1- [Bis(dimethylamino)methylene]-lH-l,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate); DMSO (dimethylsulfoxide); DIPEA (N,N-Diisopropylethylamine); and DAPI (4',6-diamidino-2-phenylindole).
  • compositions comprising vehicle, Cy5, or EEVI -Cy5 were injected into the retina of the left and/or right eyes of six C57BL/6 male mice. After 24 hours, the eyes were harvested, embedded, sectioned, and imaged for Cy5 (red) fluorescence and counterstained with DAPI (blue). No detectable signal above background was detected in untreated, vehicle, and Cy5 groups (data not shown). Distribution throughout the inner and outer nuclear layer, and in one animal retinal pigment epithelium (RPE) was observed for the EEV1-Cy5 group, demonstrating that the EEV effectively intracellularly delivered the Cy5.
  • RPE retinal pigment epithelium
  • EEVI The structure of the EEVI was Ac-PKKKRKV-K(cyclo[Ffd>GrGrQ])-PEGi 2-K(N3)-NH 2 ).
  • Cy5-DBCO Cy5-azadibenzocyclooctyne was conjugated to EEVI using strain-promoted azidealkyne cycloaddition, purified via reverse-phase HPLC and lyophilized.
  • EEV2 EEV2 conjugated to an oligonucleotide (PM01) and to Cy5 and (ii) the PMO conjugated to Cy5.
  • the structure of EEV2 was as follows: Ac-PKKKRKV-miniPEG2-K(cyclo[FGFGRGRQ]-PEGi2-OH.
  • PM01 had a nucleotide sequence of 5'-ATATTGCTATTACCTTAACCCAGAA-3'. The 3' end of the PM01 was attached to the linker (PEG12-OH) of EEV2 via amide conjugation.
  • EEV2 and PM01 were dissolved in DMSO, then predissolved HATU and DIPEA were added, and the solution was allowed to react at room temperature (RT) until complete After the reaction was complete, the reaction was quenched with water/acetonitrile before purification and subsequent lyophilization.
  • RT room temperature
  • the Cy5-DBCO was conjugated to the 5' end of the PMO using strain-promoted alkyne-azide cycloaddition using standard protocols, followed by purification and lyophilization.
  • Cy5-DBCO was attached to the 5' end of the PMO using strain-promoted alkyne-azide cycloaddition using standard protocols, followed by purification and lyophilization.
  • EEV2-PMO-Cy5 and PMO-Cy5 formulation was injected into the retinas of the mice. No detectable signal above background was detectable in vehicle and Cy5 groups (data not shown). Weak distribution was observed into ganglion cell layer (GCL)/inner nuclear layer (INL) for PMO- Cy5. Broad distribution was observed throughout the retina for EEV2-PMO-Cy5, demonstrating that EEV2 effectively intracellularly delivered the PMO when injected into the eye.
  • GCL ganglion cell layer
  • INL inner nuclear layer
  • mice were injected via bilateral intravitreally injection with vehicle, or 25 ⁇ g an EEV conjugated to a PMO designed to induce exon skipping in mRNA produced in retinal cells.
  • the EEV used was EEV2 (Ac-PKKKRKV-miniPEGi- K(cyclo[FGFGRGRQ]-PEGi2-OH).
  • the 3' end of the PMO was attached to the linker (PEG12-OH) of EEV2 via amide conjugation. Seven days after injection, the mouse eyes were for histology analyzation.
  • the PMO-EEV2 conjugate was distributed in the outer nuclear lever of the retina, the inner nuclear layer of the retina, and the ganglion cell layer of the retina as well as the layers between those explicitly labeled (not all retinal layers could be discreetly determined with the staining method used; FIG. 6). Since EEV2 was able to deliver a PMO to the retina, EEV2 may be useful for delivering PMOs for treating various ocular diseases associated with the retina.
  • mice were injected via intravitreally injection with vehicle, or an EEV conjugated to a PMO designed to induce exon skipping in mRNA produced in retinal cells
  • the EEV used was EEV5 (Ac-PKKKRKV-miniPEG2-K(cyclo[FfFGRGRQ])-miniPEG2-K(N3)- NHz).
  • the 3' end of the PMO was attached to the linker (K(N3)) of EEV5 via click chemistry.
  • the mouse eyes were harvested and sectioned for histology analyzation.
  • FIG. 7 shows the results. Eye tissue treated with DAPI and a GFAP dye.
  • DAPI stains nuclei.
  • the GFAP dye stains glial fibrillary acidic protein, a protein in astrocytes. Intravitreal dosing of the PMO-EEV5 conjugate was well tolerated in mice with no evidence in changes in retinal structure. Additionally, lack of astrocyte infiltration into the retina suggests no inflammation after treatment.
  • Example 5 Evaluation of various delivery constructs conjugated to a PMO
  • C57BL/6 mice were injected via bilateral intravitreally injection with vehicle, 25 pg of a control, PMO, or PMO conjugated with different EEVs (EEV2-8 listed in Table 5). Each treatment group included 4 mice (8 eyes).
  • the control was an ASO (not a PMO) known to induce exon skipping the gene targeted by the PMO. Seven days post injection, mouse eyes were harvested, exon skipping in six eyes was evaluated by RT-PCR.
  • FIG. 8 shows the results. Conjugates having the delivery constructs EEV5, EEV6, and EEV7 induced the greatest amount of exon skipping.
  • Example 6 Evaluation of various delivery constructs conjugated to a PMO
  • Delivery constructs can be conjugated to cargo that includes therapeutic moiety such as a PMO designed to modulate expression of a gene associated with an ocular disease.
  • the cargo can be conjugated to various delivery' constructs using amide chemistry when the delivery construct includes a terminal carboxylic acid group (-PEGx-OH) or click chemistry when the delivery construct includes a terminal azide (Ns).
  • amide chemistry when the delivery construct includes a terminal carboxylic acid group (-PEGx-OH) or click chemistry when the delivery construct includes a terminal azide (Ns).
  • the structure of the delivery constructs (EEVs) that may be tested are provided in Table 6 below.
  • C57BL/6 mice may be injected via bilateral intravitreally injection with vehicle, 25 pg of a control, PMO, or PMO conjugated with different EEVs Table 6).
  • a control may be included, for example, an ASO (not a PMO) known to induce exon skipping the gene targeted by the therapeutic moiety.
  • mouse eyes may be harvested and the impact of the therapeutic moiety evaluated.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Epidemiology (AREA)
  • Molecular Biology (AREA)
  • Ophthalmology & Optometry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

The present disclosure is directed to compounds and compositions that contain cell penetrating peptides and therapeutic moieties for treating a disease of the eye. The compounds and compositions may be delivered to the eye for treating a disease of the eye. Method of administering the compounds and compositions to the eye for treating a disease of the eye are also provided.

Description

OCULAR DELIVERY OF THERAPEUTIC AGENTS
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63/411,840, filed September 30, 2022, and U.S. Provisional Patent Application No. 63/425,807, filed November 16, 2022, each of which is incorporated herein by reference in its entirety.
INTRODUCTION
[0002] A number of therapeutic agents for treating ocular diseases exist or have been proposed. Some ocular therapeutic agents, such as oligonucleotides, polypeptides, or intracellularly acting small molecule therapeutics, may have limited effectiveness unless they are delivered to the correct eye location, cell-type, and/or to an intracellular compartment where their effects may be realized.
[0003] The anatomy and physiology of the eye presents challenges to delivering therapeutic agents to a desired eye location and/or specific cells of the eye. The eye includes static and dynamic barriers that tend to exclude entry of xenobiotics and discourage active absorption of therapeutic agents (see, for example, Gote, et al., J Pharm Exp Ther, September 2019, 370(3): 602-624). Static barriers include tissue that provides a passive physical barrier to therapeutic agent penetration (see Rowe-Rendleman, et al., Invest Opthamol Vis Sci, 2014 Apr, 55(4):2714-2730). Dynamic barriers include physiological clearance mechanisms such as aqueous turnover, vitreous flow, ocular drug metabolism, and eye metabolizing enzymes (see, id).
[0004] For different routes of administration, the eye presents distinct barriers for achieving effective levels of therapeutic agents. For example, barriers for intraocular administration, such as intravitreal injection, includes static barriers such as the vitreous humor and dynamic barriers such as anterior aqueous humor flow, posterior trans-retinal flow, and efflux transporters (see, id). Barriers of suprachoroidal administration, such as via suprachoroidal injection, includes static barriers, such as the choriod, Bruch’s membrane, PRE tight junctions, and the retina, as well as dynamic barriers such as choroidal circulation and retinal circulation (see, id). Barriers for periocular administration, such as sub-tenon’s injection, includes static barriers, such as the sclera, the choriod, Bruch’s membrane, RPE tight junctions, the retina, as well as dynamic barriers such as subconjunctival-episcleral lymph and blood vessels, choriodal circulation, and retinal circulation (see, id). Barriers for topical administration, such as eye drops, includes static barriers, such as the cornea, the conjunctiva, the sclera, the choriod, Bruch’s membrane, RPE tight junctions, and the retina, as well as dynamic barriers, such as tears and lacrimal drainage, subconjunctival-espisleral lymph and blood vessels, choroidal circulation, and retinal circulation (see, id).
[0005] To achieve a therapeutic effect, a therapeutic agent not only needs to be delivered to an appropriate location of the eye, but also needs to be delivered to the appropriate cells and cellular compartments of the eye. Carrier systems, such as polymers, cationic liposomes, or chemical modifications, for example, by the covalent attachment of cholesterol molecules, have been used to facilitate intracellular delivery. Still, intracellular delivery efficiency by these approaches is often low and improved delivery systems to increase efficacy of intracellular delivery have remained elusive.
[0006] In the late 1980s, it was discovered that the highly positively charged HIV Tat peptide could translocate across the mammalian cell membrane. Subsequently, other “cell penetrating peptides” (CPPs) have been discovered that are capable of penetrating the cell membrane at low micromolar concentrations without causing significant membrane damage. However, effective cytosolic delivery by many of these CPPs is limited by poor endosomal escape efficiency, and some may have undesirably high toxicity levels.
[0007] Accordingly, new cell penetrating peptides having endosomal escape properties and compositions comprising such cell penetrating peptides and therapeutic agents suitable for treating ocular diseases are needed.
SUMMARY
[0008] The present disclosure relates to, among other things, cargo conjugates and compositions containing the same, for delivering a cargo to the eye. The cargo conjugate may include a cyclic cell penetrating peptide and a therapeutic agent. The therapeutic agent may be designed to have a biological effect, for example, to treat a disease of the eye. As such, the cargo conjugates and compositions containing the same, may be used to treat ocular diseases.
[0009] The present di sclosure is related to, among other things, methods of admi ni storing the cargo conjugates and compositions containing the same to a subject in need thereof. The method may include identifying a subject that may benefit from the delivery of a therapeutic agent to the eye. For example, the subject may have, or be at risk of having, an ocular disease. The method may further include delivering the cargo conjugate or composition containing the same directly to the eye. In embodiments, cargo conjugate and/or compositions containing the same are delivered to the surface of the eye. In embodiments, cargo conjugate and/or compositions containing the same are intraocularly injected. In embodiments, cargo conjugate and/or compositions containing the same are injected into the retina. In embodiments, cargo conjugate and/or compositions containing the same are delivered intravitreally.
[0010] In embodiments, a method of delivering a therapeutic agent to the eye of a subject comprises administering a therapeutically effective amount of a cargo conjugate to the eye of the subject. The cargo conjugate comprises (i) a cargo comprising the therapeutic agent that selectively binds to a target molecule associated with a disease of the eye; and (ii) an ocular delivery' construct comprising an exocyclic peptide (EP), a first cyclic cell penetrating peptide (cCPP) and one or more linkers. The ocular delivery construct has the structure:
Figure imgf000005_0001
, or a protonated form thereof, wherein:
R1, R2, and R3 are each independently H or an aryl or heteroaryl side chain of an amino acid; at least two of R1, R2, and R3 are an aryl or heteroaryl side chain of an amino acid; R4 and Re are independently H or an amino acid side chain; peptide is an exocyclic peptide (EP) comprising from 2 to 10 amino acids ;
M is a bonding group; each m is independently an integer from 0-3; n is an integer from 0-2; x' is an integer from 0-20; y is an integer from 1-5; q is 1-4; z' is an integer from 1-23; and
Cargo is a therapeutic moiety.
[0011] In embodiments, the amino acid residue comprising an aryl or heteroaryl group is phenylalanine or 3-(2-naphthyl)-alanine. In embodiments, Ri and Re are, independently, H or a side chain of an amino acid selected from arginine, citrulline, serine or histidine. In embodiments, at least one of R4 and Re are H. In embodiments, at least one of R» and Re are an amino acid side chain of arginine. In embodiments, at least one of R4 and Re are an amino acid side chain of serine. In embodiments, at least one of Rt and Re are an amino acid side chain of histidine.
[0012] In embodiments, the cCPP of the ocular delivery construct has a sequence selected from: FfORrRrQ, FGFGRGRQ; GfFGrGrQ, FfOGRGRQ; FfFGRGRQ; FfrDGrGrQ; FGFGRRRQ; and FGFRRRRQ.
[0013] In embodiments, a method of delivering a therapeutic agent to an eye of a subject is provided, the method comprising administering a therapeutically effective amount of a cargo conjugate to the eye of the subject. The cargo conjugate comprises (i) a cargo comprising the therapeutic agent that selectively binds to a target molecule associated with a disease of the eye; and (ii) an ocular delivery construct comprising an exocyclic peptide (EP), a first cyclic cell penetrating peptide (cCPP) and one or more linkers, wherein the ocular delivery construct has the structure:
Figure imgf000007_0001
, or a protonated form thereof, wherein:
Ri, Rz, and R3 can each independently be H or an amino acid residue having a side chain comprising an aryl or heteroaryl group; at least two of Ri, Rz, and R? is an aryl or heteroaryl side chain of an amino acid;
R4, and R6 are independently H or an amino acid side chain;
AAscis an amino acid side chain; nx is 1; q is 1, 2, 3 or 4; peptide is an exocyclic peptide (EP) comprising from 2 to 10 amino acids;
M is a bonding group; each m is independently an integer from 0-3; n is an integer from 0-2; x' is an integer from 0-20; y is an integer from 1-5; z' is an integer from 1-23; and
Cargo is a therapeutic moiety.
[0014] In embodiments, the amino acid residue comprising an aryl or heteroaryl group is phenylalanine, beta homophenylalanine, or 3-(2-naphthyl)-alanine. In embodiments, R.4 and Rs are, independently, H or a side chain of an amino acid selected from arginine, citrulline, serine or histidine. In embodiments, at least one of R4 and Rr> are H. In embodiments, at least one of R4 and Re are an amino acid side chain of arginine. In embodiments, at least one of R.4 and Rs are an amino acid side chain of serine. In embodiments, at least one of R» and Rs are an amino acid side chain of histidine.
[0015] In embodiments, the cCPP of the ocular delivery construct has a sequence selected from: βhF-FOSRSRQ and βhF-FΦDGRGRQ.
[0016] In embodiments, M comprises
Figure imgf000008_0005
,or
Figure imgf000008_0001
, wherein t' is 0 to 10.
In embodiments, M comprises . In embodiments, M comprises
Figure imgf000008_0002
Figure imgf000008_0004
wherein t' is 0 to 10. In embodiments, M comprises w
Figure imgf000008_0003
[0017] In embodiments, the EP comprises 1 or 2 amino acids comprising a side chain comprising a guanidine group, or a protonated form thereof. In embodiments, the EP comprises 1, 2, 3, or 4 lysine residues. In embodiments, the EP comprises one of the following sequences: PKKKRKV; KR, RR, KKK; KGK; KBK; KBR; KKK; KRR; RKK; RRR; KKKK; KKRK; KRKK; KRRK; RKKR; RRRR; KGKK; KKGK; KKKKK; KKKRK; KBKBK; KKKRKV; PGKKRKV; PKGKRKV; PKKGRKV; PKKKGKV; PKKKRGV; or PKKKRKG. In embodiments, EP comprises PKGKRKV.
[0018] In embodiments, the cargo comprises a therapeutic oligonucleotide. In embodiments, the therapeutic oligonucleotide is an antisense oligonucleotide. In embodiments, the antisense oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO). In embodiments, the cargo comprises a therapeutic peptide.
[0019] In embodiments, the disease of the eye is selected from one or more of diabetic retinopathy; glaucoma; retinitis pigmentosa; Usher syndrome; retinal tears or holes; retinal detachment; retinal ischemia; damage associated with laser therapy including photodynamic therapy; surgical light induced iatrogenic retinopathy; drug-induced retinopathies; autosomal dominant optic atrophy; toxic and/or nutritional amblyopias; Leber's hereditary optic neuropathy; atypical retinitis pigmentosa; Bardet-Biedl syndrome; blue-cone monochromacy; cataracts; central areolar choroidal dystrophy; choroideremia; cone dystrophy; rod dystrophy; rod-cone dystrophy; congenital stationary nightblindness; cytomegalovirus retinitis, diabetic macular edema; dominant drusen; giant cell arteritis; Goldmann Favre dystrophy; graves' ophthalmopathy; gyrate atrophy; iritis; juvenile retinoschisis; Kearns-Sayre syndrome; Lawrence-Moon syndrome; Leber Congenital Amaurosa, wet macular degeneration; dry macular degeneration; macular dystrophy; ocularhistoplasmosis syndrome; Oguchi disease; oxidative damage; proliferative vitreoretinopathy; refsum disease; retinitis punctata albescens; retinopathy of prematurity; rod monochromatism; Usher syndrome such as Usher Syndrome type 2A; scleritis; Sjogren-Larsson syndrome; Sorsby fundus dystrophy; Stargardt disease; choroideremia; optic neuropathy; Bietti crystalline dystrophy; Alport syndrome; X-linked retinoschisis; Macula dystrophy; Achromatopsia; congenital stationary night blindness; Best disease; Pattern dystrophy; and Doyne's honeycomb dystrophy.
[0020] In embodiments, administering a cargo conjugate to directly to an eye of the subject comprises local administration. In embodiments, local administration is selected from topical administration, intravitreal administration, periocular administration, subretinal administration, suprachoroidal administration, and intracameral administration.
[0021] In embodiments, the method further comprises identifying a subject having an ocular disease. In embodiments, the subject is suffering from an ocular disease or disorder, or is at risk of developing, an ocular disease or disorder. In embodiments, the subject has a genetic disease or disorder of the eye. Tn embodiments, the subject is a mammal In embodiments, the mammal is a human.
[0022] In embodiments, administration of the compound modulates expression or activity of a target molecule. In embodiments, administration of the compound downregulates expression or activity of the target molecule. In embodiments, administration of the compound upregulates expression or activity of the target molecule. In embodiments, the target molecule is a protein. In embodiments, the target molecule is an oligonucleotide.
[0023] In embodiments, the cargo conjugate is administered in a pharmaceutical composition.
BRIEF DESCRIPTION OF THE DRAWINGS [0024] FIG. 1A is a schematic drawing of the eye with a section of the retina shown as schematically enlarged. Adapted from Simple Anatomy of the Retina by Helga Kolb, Webvision, the Organization of the Retina and Visual System (webvision.med.utah.edu/book/part-i- foundations/simple-anatomy-of-the-retina/).
[0025] FIG. IB is a schematic drawing of layers and cell components of the retina. Adapted from Koeppen & Stanton: Berne and Levy Physiology, 6th Edition, 2008 by Mosby, an imprint of Elsevier, Inc
[0026] FIG. 2 shows examples of modified nucleotides used in antisense oligonucleotides described herein. Structures 1-3 (1 = Phosphorothioate; 2 = (SC5-Rp)-a,0-CAN; 3 = PMO) are phosphate backbone modifications, 4 (2-thio-dT) is a base modification; 5-8 (5 = 2'-0Me-RNA, 6 = 2'0-M0E-RNA; 7 = 2T-RNA; 8 = 2T-ANA) are 2' sugar modifications; 9-11 are constrained nucleotides; 12-14 (9 = LNA; 10 = (S)-cET; 11 = tcDNA; 12 = FHNA; 13 = (S)5'-C-methyl; 14 = UNA) are additional sugar modification; and 15-18 (15= E-VP; 16 = Methyl phosphonate; 17 = 5' phosphorothioate; 18 = (S)-5'-C-methyl with phosphate) are 5' phosphate stabilization modifications; 19= a morpholino sugar. 20 is a representation of a phosphorodiamidate morpholino oligomer backbone. Reformatted from Khvorova, A., et al., Nat. Biotechnol. 2017 Mar; 35(3): 238-248.
[0027] FIGS. 3A-3D illustrate examples of conjugation chemistries for connecting an antisense compound (AC) , such as an antisense oligonucleotide (ASO), to a cyclic cell penetrating peptide. FIG. 3A shows amide bond formation between peptides with a carboxylic acid group or with TFP activated ester and primary amine residues at the 5' end of an AC. FIG. 3B shows conjugation of secondary amine or primary amine modified AC at 3' and peptide-TFP ester through amide bond formation. FIG. 3C shows conjugation of a peptide-azide to the 5' cyclooctyne modified AC via copper-free azide-alkyne cycloaddition. FIG. 3D demonstrates another example of conjugation between a 3' modified cyclooctyne ACs or 3' modified azide ACs and CPP containing linker-azide or linker-alkyne/cyclooctyne moiety, via a copper-free azide-alkyne cycloaddition or cupper catalyzed azide-alkyne cycloaddition, respectively (click reaction).
[0028] FIG. 4 shows an example of conjugation chemistry for connecting an ASO and cCPP with an additional linker modality containing a polyethylene glycol (PEG) moiety. [0029] FIG. 5 shows images of sectioned mouse eye tissue treated with a vehicle or a PMO-EEV conjugate. Tissue was stained for nuclear material (DAPI, blue) and for PMOs (anti-PMO antibody, green).
[0030] FIG. 6 shows images of sectioned mouse retinal tissue treated with a vehicle or a PMO- EEV conjugate. The tissue was stained for nuclear material (DAPI, blue) and for PMOs (anti-PMO antibody, green). The top images show both the DAPI and anti-PMO stains and the bottom images show only the anti-PMO stains. RPE = retinal pigment epithelium; nuclear layer; INL = inner nuclear layer; GCL = ganglion cell layer
[0031] FIG. 7 shows images of sectioned mouse retinal tissue treated with a vehicle or a PMO- EEV conjugate. The tissue was stained for nuclear material (DAPI, blue) and for glial fibrillary acidic protein (GFAP; pink).
[0032] FIG. 8 is a plot showing the percent exon skipping after mice were treated with various cargo conjugates.
DETAILED DESCRIPTION
[0033] Methods and compositions are provided herein that can be used to transport a therapeutic moiety or therapeutic agent to the cells of the eye of a subject. Methods and compositions are provided herein may be used to treat ocular diseases.
[0034] Disclosed herein are cargo conjugates comprising an ocular delivery construct conjugated to a cargo. The cargo may be a therapeutic moiety for which delivery to a cell of the eye is desired. The therapeutic moiety may be a molecule useful for treating a disease of the eye. In embodiments, the therapeutic moiety selectively binds to a target molecule associated with an ocular disease. In embodiments, the target molecule is a macromolecule implicated in a disease or pathology of the eye. In embodiments, the target molecule is a polypeptide or protein. In embodiments, the target molecule is an oligonucleotide. In embodiments, the oligonucleotide target comprises DNA. In embodiments, the oligonucleotide target comprises genomic DNA. In embodiments, the oligonucleotide target comprises RNA. In embodiments, the oligonucleotide target comprises mRNA. In embodiments, the target is associated with an ocular disease. In embodiments, selective binding of the therapeutic moiety with the target molecule is useful for the treatment of a disease, pathology or other abnormal state or condition of the eye. In embodiments, selective binding of the therapeutic moiety to the target molecule upregulates expression or activity of the target molecule. In embodiments, selective binding of the therapeutic moiety to the target molecule downregulates expression or activity of the target molecule.
[0035] As used herein, the term “ocular delivery construct” refers to a compound that, when conjugated to cargo and ophthalmically administered, enhances uptake of the cargo in a cell of the eye relative to uptake of the cargo alone (when not conjugated to the ocular delivery construct). The ocular delivery construct may preferentially targets a tissue of the eye. In embodiments, the ocular delivery construct targets one or more structures, tissues, layers, and/or cells of eye. In embodiments, the ocular delivery construct comprises a cyclic cell penetrating peptide (cCPP); a compound comprising a cCPP and a linker; a compound comprising a cCPP and an exocyclic peptide; or a compound comprising an endosomal escape vehicle (EEV) which comprises a cCPP, an exocyclic peptide, and a linker.
[0036] In embodiments, an ocular delivery construct provided herein can be used to transport a cargo, such as a therapeutic agent, to a cell of the eye of subject. In embodiments, the ocular delivery construct transports a therapeutic moiety to an intracellular compartment of a cell. In embodiments, an ocular delivery construct transports a therapeutic moiety to the retina of a subject. In embodiments, the ocular delivery construct transports a therapeutic moiety intracellularly to cells of one or more cell types within the retina. In embodiments, the ocular delivery construct preferentially transports a therapeutic agent intracellularly to cells of one or more cell type within the retina.
[0037] In embodiments, a cargo conjugate or a composition comprising a cargo conjugate may be ophthalmically administered. As used herein, the term “ophthalmically administered,” “ophthalmic administration,” “ocularly administered,” and “ocular administration” refer to local delivery of a compound or composition directly to one or more components of the eye (described in detail later herein). Such terms exclude systematic administration of the cargo conjugate.
[0038] For ocular diseases, ocular administration of a therapeutic may allow for a more effective treatment than systematic administration. Systemic administration, in some cases, may not achieve effective concentrations of therapeutics in the eye due in part to systemic clearance mechanisms and blood-ocular barriers. Ocular administration may avoid, mitigate, or circumvent one or more systemic clearance mechanisms, such as first-pass metabolism, and/or blood-ocular barriers, such as the blood-aqueous barrier and the blood-retinal barrier to achieve therapeutically effective concentrations in cells of the eye. The blood-ocular barriers may hinder a therapeutic from penetrating the eye and/or reaching portions of the eye at a therapeutically relevant concentration when systemically administered. Additionally, ocular administration may result in fewer side effects than systemic administration of a therapeutic because ocular administration provides local delivery of the therapeutic to the tissue to be treated.
[0039] In embodiments, a cargo conjugate may be targeted to and/or localize to one or more structures, layers, and/or cell types of the eye. In embodiments, an ocularly administered cargo conjugate may be targeted to and/or localize to one or more structures, layers, and/or cell types of the eye. As used herein in the context of biodistribution of a cargo conjugate or delivery construct, the term “targeted to x” (where x is a system, such as the neuromuscular system, an organ, a structure, a layer, a tissue type, or a cell type) refers to the cargo conjugate or delivery construct preferentially interacting with x. It is understood that a cargo conjugate or delivery construct target to a particular system, organ, structure, layer, tissue type, or cell type may interact with other one or more additional systems, on or more additional organs, one or more additional tissue types, one or more additional cell types, or any combination thereof. Reference to a cargo conjugate or delivery construct targeted to x includes all the components comprising x. For example, a cargo conjugate targeted to the eye includes the cargo conjugate being targeted to one or more structures, tissues, layers, and/or cells that make up the eye.
[0040] As used herein in the context of biodistribution of a delivery construct or a cargo conjugate, the terms “localize” and “localized” refer to the physical position of the cargo conjugate within a body, an organ, a tissue, or a cell. Reference to a cargo conjugate or delivery construct being localized within a particular locale includes all the sublocales comprising the locale. For example, a cargo conjugate or delivery construct localized to the eye includes the cargo conjugate or delivery construct being localized to one or more structures, tissues, layers, and/or cells that make up the eye and can be referred to herein as an “ocular delivery construct.” An ocular delivery construct localized to the retina may be further localized to one or more of the layers and/or cells that comprise the retina.
Anatomy of the Eye
[0041] The anatomy, physiology, and chemical composition of the eye provide barriers to delivery of therapeutic agents. Many therapeutics struggle to penetrate target tissues in the eye, such as the cornea or retina, which can result in poor bioavailability. The eye has static and dynamic barriers that inhibit therapeutic agents from reaching target tissues at therapeutically effective concentrations. Static barriers of the eye include tissues that provide a passive physical barrier to therapeutic agent penetration. Dynamic barriers include physiological clearance mechanisms and barriers that include aqueous turnover, vitreous flow, ocular drug metabolism, and eye metabolizing enzymes.
[0042] As shown in FIG. 1A, main structures in a mammalian eye include, the cornea, iris, pupil, aqueous humor, lens, vitreous humor, retina, and optic nerve. The retina is a layered structure containing distinct layers of neurons interconnected by synapses, some of which are shown in FIG. IB. Retina layers from the anterior to posterior surface include: inner limiting membrane, nerve fiber layer (NFL), ganglion cell layer (GCL), inner plexiform layer, inner nuclear layer (INL), middle limiting membrane, outer plexiform layer, outer nuclear layer (ONL), outer (external) limiting membrane, photoreceptor layer, and the retinal pigment epithelium (RPE). The cells in the retina can be subdivided into several cell types shown in FIG. IB: ganglion cells, Muller cells (Muller glia), bipolar cells, amacrine cells, horizontal cells, photoreceptor cells (rods and cones), and pigment cells. The majority of cell types in the retina span one, two, or three retinal layers (see FIG. IB). Some cell types are elongated such that a first part of the cells of a cell type is in a first retinal layer, a second part of the cells of the cell type is in a second retinal layer, and a third part of the cells in the cell type is in a third retinal layer. For example, the nuclei of the photoreceptor cells are generally in the outer nuclear layer, the rods/cones of the photoreceptor cells are generally in the photoreceptor layer, and the synapses of the photoreceptor cells are generally in the outer plexiform layer.
[0043] In embodiments, an ocular delivery construct can be used to transport a therapeutic moiety (as a conjugate construct) to a structure of the eye. In embodiments, an ocular delivery construct may be used to transport a therapeutic moiety to the retina. In embodiments, the ocular delivery construct may be used to transport a therapeutic moiety to one or more retinal layer. In embodiments, the ocular delivery construct may be used to transport a therapeutic moiety to one or more of the inner limiting membrane, nerve fiber layer (NFL), ganglion cell layer (GCL), inner plexiform layer, inner nuclear layer (INL), middle limiting membrane, outer plexiform layer, outer nuclear layer (ONL), outer (external) limiting membrane, photoreceptor layer, and the retinal pigment epithelium (RPE). In embodiments, the ocular delivery construct may be used to transport a therapeutic moiety to and within one or more of a ganglion cell, a Muller cell (Muller glia), a bipolar cell, an amacrine cell, a horizontal cell, a photoreceptor cell (rods and cones), and a pigment cell.
[0044] In embodiments, when ophthalmically administered, a cargo conjugate may be targeted to and/or localize in the retina. In embodiments, the cargo conjugate may be targeted to and/or localize in one or more of the layers of the retina such as the inner limiting membrane, nerve fiber layer (NFL), ganglion cell layer, inner plexiform layer, inner nuclear layer, middle limiting membrane, outer plexiform layer, outer nuclear layer, outer (external) limiting membrane, photoreceptor layer, and retinal pigment epithelium, and/or one or more of the cell types of the retina such as a ganglion cell, a Muller cell, a bipolar cell, an amacrine cell, a horizontal cell, a photoreceptor cell (rods and cones), and a pigment cell.
Ocular Diseases and Mechanisms of Therapeutic Moieties
[0045] The ocular delivery constructs provided herein may be conjugated to a therapeutic moiety for which delivery to the eye is desired. For example, the cargo conjugate may include a therapeutic moiety designed to provide a therapeutic effect to a subject inflicted with an ocular disease. In embodiments, the cargo conjugate includes a therapeutic moiety useful for treating a disease of the eye (i.e., an ocular disease). In embodiments, the therapeutic moiety targets a protein or gene associated with a disease of the eye. As such, the cargo conjugates and compositions may be used to treat an ocular disease. In embodiments, the therapeutic moiety modulates expression of a protein or gene associated with a disease of the eye. In embodiments, the therapeutic moiety upregulates expression of a protein or gene associated with a disease of the eye. In embodiments, the therapeutic moiety downregulates expression of a protein or gene associated with a disease of the eye. In embodiments, the therapeutic moiety modulates the composition of mRNA. In embodiments, the therapeutic moiety modulates processing of the mRNA. In embodiments, the therapeutic moiety modulates splicing of a pre-mRNA target transcript. In embodiments, the therapeutic moiety modulates alternative splicing of a pre-mRNA target transcript. In embodiments, modulation of splicing or alternative splicing induces exon skipping and/or exon inclusion.
[0046] Non-limiting examples of ocular diseases that a cargo conjugate may be used to treat include, diabetic retinopathy; glaucoma; retinitis pigmentosa (RP), such as autosomal dominant RP, autosomal recessive RP, sector RP, and RP associated with Usher syndrome; retinal tears or holes; retinal detachment; retinal ischemia; damage associated with laser therapy including photodynamic therapy; surgical light induced iatrogenic retinopathy; drug-induced retinopathies; autosomal dominant optic atrophy; toxic and/or nutritional amblyopias; Leber’s hereditary optic neuropathy; atypical retinitis pigmentosa; Bardet-Biedl syndrome; blue-cone monochromacy; cataracts; central areolar choroidal dystrophy; choroideremia; cone dystrophy; rod dystrophy; rodcone dystrophy; congenital stationary’ night blindness; cytomegalovirus retinitis; diabetic macular edema; dominant drusen; giant cell arteritis; Goldmann Favre dystrophy; graves’ ophthalmopathy; gyrate atrophy; iritis; juvenile retinoschisis; Kearns-Sayre syndrome; Lawrence-Moon syndrome; Leber Congenital Amaurosa; wet macular degeneration; dry macular degeneration; macular dystrophy; ocularhistoplasmosis syndrome; Oguchi disease; oxidative damage; proliferative vitreoretinopathy; refsum disease; retinitis punctata albescens; retinopathy of prematurity; rod monochromatism; Usher syndrome such as Usher Syndrome type 2A; scleritis; Sjogren-Larsson syndrome; Sorsby fundus dystrophy; Stargardt disease; choroideremia; optic neuropathy; Bietti crystalline dystrophy; Alport syndrome; X-linked retinoschisis; Macula dystrophy; Achromatopsia; congenital stationary night blindness; Best disease; Pattern dystrophy; and Doyne’s honeycomb dystrophy.
[0047] In embodiments, a cargo conjugate having a therapeutic moiety designed to treat an ocular disease is provided. In embodiments, a composition comprising such a cargo conjugate is ocularly delivered to a subject in need thereof. In embodiments, the composition is administered directly the retina. In embodiments, the composition is administered intravitreally.
Mechanisms of Action of Therapeutic Moieties
[0048] The cargo conjugate may include a therapeutic moiety (TM) for which delivery to the eye is desired, for example, to treat an ocular disease TMs can include antisense oligonucleotides (ASO), polypeptides, and small molecules.
[0049] In embodiments, a TM is an effector that modulates target gene expression and/or target protein activity. A target gene is the gene of which modulation of gene expression and/or protein activity is desired. A target transcript is the pre-mRNA or mRNA transcript that is transcribed from the target gene. A target protein is the polypeptide or protein encoded by the target transcript. The target gene, target transcript, or target protein may be associated with a disease state. The target gene, target transcript, or target protein may be associated with an ocular disease. For example, a target gene may be a mutated gene associated with the disease state; a target transcript may have a coding sequence associated with the disease state, may be aberrantly spliced in the disease state, may be present in reduced or increased levels in the disease state, or the like; a target protein may be present in reduced or increased levels in the disease state, may have aberrant function, reduced function, or no function in the disease state, or the like; or any combination thereof. As used herein, an “ocular target” or “target molecule” can be a target gene, a target transcript, or a target protein associated with an ocular disease. In embodiments, the target gene, target transcript, and target protein are a gene, transcript, and protein associated with a disease of the retina.
[0050] A TM may exert a therapeutic effect through any suitable mechanism. In embodiments, a cargo conjugate includes a TM that modulates one or more aspects of target gene expression, such as transcription and translation, and/or target protein activity. For example, a TM may function, relative to the disease phenotype, to downregulate target gene expression; upregulate target gene expression; inhibit target protein function; increase target protein function; or any combination thereof.
[0051] In embodiments, the cargo conjugate includes a TM that modulates one or more aspects of pre-mRNA processing of a target transcript. Pre-mRNA molecules are made in the nucleus and are processed before or during transport to the cytoplasm for translation. Processing of the pre- mRNAs includes addition of a 5' methylated cap and an approximately 200-250 base poly(A) tail to the 3' end of the transcript. mRNA processing also includes splicing of the pre-mRNA, which occurs in the maturation of 90-95% of mammalian mRNAs. Introns (or intervening sequences) are regions of a primary transcript (or the DNA encoding it) that are not included in the coding sequence of the mature mRNA. Exons are regions of a primary transcript that remain in the mature mRNA. The exons are spliced together to form the mature mRNA sequence. Splice junctions are also referred to as splice sites with the 5' side of the junction often called the “5* splice site,” or “splice donor site” and the 3' side called the “3' splice site” or “splice acceptor site.” In splicing, the 3 ' end of an upstream exon is j oined to the 5 ' end of the downstream exon. Thus, the unspliced RNA (or pre-mRNA) has an exon/intron junction at the 5' end of an intron and an intron/exon junction at the 3' end of an intron. After the intron is removed, the exons are contiguous at what is sometimes referred to as the exon/exon junction or boundary in the mature mRNA. Cryptic splice sites are those which are less often used but may be used when the usual splice site is blocked or unavailable. Cryptic splice sites may result from one or more mutations in an intron. Such mutations may result in a high occurrence of splicing at the cryptic splice site. Many eye diseases are characterized by intronic mutations that result in a high occurrence of cryptic splice site use Alternative splicing, defined as the splicing together of different combinations of exons, often results in multiple mRNA transcripts from a single gene. A TM may modulate alternative splicing of a pre-mRNA transcript by, for example, inducing exon skipping and/or inducing exon inclusion. Induction of exon skipping and/or exon inclusion may result in a mRNA that has a different combination of exons than is observed in a mRNA transcript of a diseased state.
[0052] Some genetic mutations alter splicing which can result in the inclusion of exons not normally present in the processed mRNA and/or exclusion of exons normally present in the processed mRNA. Such inclusions and exclusions may result in a truncated protein, a protein with decreased function, a protein with no function, an unstable protein, or any combination thereof. Similarly, mutations in exons that do not necessarily affect splicing, may result in a truncated protein, a protein with decreased function, a protein with no function, an unstable protein, or any combination thereof.
[0053] In embodiments, the TM is an antisense oligonucleotide (ASO). An ASO is an oligonucleotide that binds (e.g., hybridizes) to a portion of an RNA transcript. The ASO may comprise natural or modified nucleotides. The ASO may comprise one or more modified nucleosides, one or more modified intemucleoside linkages, one or more conjugate groups, or combinations thereof. In embodiments, the ASO may be used to modulate pre-mRNA processing by modulating splicing of the pre-mRNA target transcript. As used herein, “modulation of splicing,” “splicing modulation,” “modulating splicing,” and “modulate splicing” refer to altering the processing of a pre-mRNA target transcript such that the spliced mRNA transcript contains either a different combination of exons as a result of exon skipping or exon inclusion, a deletion in one or more exons, or the deletion or addition of a sequence not normally found in the spliced mRNA of the disease state (e.g., an intron sequence). Modulation of splicing may result in alternative splicing. As such modulation of splicing may result in modulation of alternative splicing. In embodiments, ASO hybridization to a target sequence in a pre-mRNA target transcript modifies splicing to produce a mature mRNA encoding a fully functional protein, a partially functional protein, or a non-functional protein. In embodiments, ASO hybridization to a target sequence in a pre-mRNA target transcript modifies splicing of a pre-mRNA in a diseased cell to produce a mature mRNA encoding a protein that is present in a non-diseased cell (e g., a wild-type protein). In embodiments, ASO hybridization results in alternative splicing of the target pre- mRNA.
[0054] In embodiments, ASO hybridization results in skipping of one or more exons. The term “exon skipping” refers to modulation of splicing in a cell to produce a mature mRNA lacking one or more exons relative to a mature mRNA in the cell for which exon skipping has not occurred. In embodiments, an ASO induces exon skipping to produce a mature mRNA that lacks one or more exons associated with a disease state. In embodiments, an ASO induces exon skipping to produce a mature mRNA that lacks one or more pseudo-exons associated with a disease state. In embodiments, an ASO induces exon skipping to produce a mature mRNA that lacks one or more exons associated with the disease state but, as a result of the exon skipping includes one or more exons that is not present in a mature mRNA of the disease state. The one or more exons that are included may be exons present in wild-type mature mRNA that are missing in a mature mRNA in the disease state. In embodiments, the skipped exon comprises a frameshift mutation, a nonsense mutation, or a missense mutation. In embodiments, the skipped exon sequence comprises a nucleic acid deletion, substitution, or insertion. In embodiments, the skipped exon itself does not comprise a sequence mutation, but a neighboring intron or exon comprises a mutation.
[0055] In embodiments, ASO hybridization to a target sequence within a target pre-mRNA prevents inclusion of an exon sequence in the mature mRNA molecule. In embodiments, antisense oligonucleotides hybridization to a target sequence within a target pre-mRNA results in preferential expression of a wild type target protein isomer. In embodiments, antisense oligonucleotides hybridization to a target sequence within a target pre-mRNA results in expression of a re-spliced target protein comprising an active fragment of a wild type target protein.
[0056] Depending on the disease and the desired phenotype, ASOs can be designed to promote exon skipping in a target transcript to result in, for example, a protein of the healthy phenotype (full length and fully functional), a full-length protein having at least some function, a nonfunctional full-length protein, a fully functional truncated protein, a truncated protein having at least some function, or a non-functional truncated protein. Exon skipping may also be used to introduce a premature stop codon in a target transcript. mRNA comprising a premature stop codon may undergo nonsense mediated decay. Inducing nonsense mediate decay of a target transcript may be beneficial to treat diseases characterized by expression of a deleterious target protein or high concentrations of a target protein and/or target transcript. [0057] An ASO can induce exon skipping by binding to and sterically inhibiting splicing of a target transcript (see, e.g., International Patent Application No. International Patent Application No. PCT/US22/28357, filed on 9 May 2022, and entitled COMPOSITIONS AND METHODS FOR MODULATING mRNA SPLICING, which application is hereby incorporated herein by reference in its entirety).
[0058] In embodiments, ASO hybridization results in exon inclusion. The term “exon inclusion” refers to modulation of splicing in a cell to produce a mature mRNA that includes one or more exons relative to a mature mRNA in the cell for which exon inclusion has not occurred. In embodiments, an ASO induces exon inclusion to produce a mature mRNA that includes one or more exons that are deleted in a disease state. In embodiments, an ASO induces exon inclusion to produce a mature mRNA that includes one or more exons whose deletion is associated with the disease state, but as a result of the exon inclusion, the mature mRNA includes the exons not present in the mature mRNA of the disease state. The one or more exons that are included may be exons present in wild-type mature mRNA that are missing in a mature mRNA in the disease state.
[0059] In embodiments, ASO hybridization to a target sequence within a pre-mRNA target transcript induces inclusion of one or more exon sequences in the mature mRNA molecule. In embodiments, ASO hybridization to a target sequence within a pre-mRNA target transcript results in preferential expression of a wild type target protein isomer. In embodiments, antisense oligonucleotides hybridization to a target sequence within a target pre-mRNA results in correction of alternative splicing observed in a disease state.
[0060] Depending on the disease and the desired phenotype, ASOs can be designed to promote intron inclusion in a target transcript to result in, for example, a protein of the healthy phenotype (full length and fully functional), a full-length protein having at least some function, a nonfunctional full-length protein, a fully functional truncated protein, a truncated protein having at least some function, or a non-functional truncated protein.
[0061] In embodiments, the TM may be an ASO that modulates pre-mRNA processing by inhibiting the poly adenylation of a target transcript. Inhibition of addition of a poly(A) tail to target transcript may decrease the levels of the target transcript and/or target protein levels. ASOs conjugated to cyclic peptides for modulating polyadenylation of mRNA is disclosed in International Patent Application No. PCT/US22/28354, filed on 9 May 2022, and entitled COMPOSITIONS AND METHODS FOR MODULATING GENE EXPRESSION, which application is hereby incorporated herein by reference in its entirety.
[0062] In embodiments, the TM may modulate one or more aspects of mRNA translation. In embodiments, the TM may be an ASO that prevents degradation of a mRNA target transcript. Since a single mRNA transcript is used as a translation template to produce multiple protein copies, stabilizing a target mRNA transcript may increase the number of times the transcript is used as a template, thereby increasing the levels of the protein encoded by the target transcript. Stabilization of a target mRNA transcript may increase the resistance of the target transcript to degradation (e.g., 5 "-3' and/or 3 '-5' exonuclease degradation), thereby increasing the half-life of the target transcript. Therapeutic moieties may stabilize an mRNA target transcript by binding to, for example, the 3' untranslated region, the 5' untranslated region, or both. Increasing the stability of an mRNA target transcript may be beneficial to treat diseases characterized by low concentrations of a protein
[0063] In embodiments, the TM may modulate target gene expression by decreasing the stability of a mRNA target transcript and/or preventing translation of the mRNA target transcript. For example, a TM may be an ASO that sterically prevents translation of an mRNA target transcript and/or increases the susceptibility of the mRNA target transcript to decay. In embodiments, the TM may induce nonsense mediated decay of the target transcript. In embodiments the ASO comprises DNA, and binding of the ASO to the target transcript creates a DNA/RNA hybrid that can be degraded by the enzyme RNaseH. Decreasing the stability and/or preventing translation of an mRNA target transcript may be beneficial to treat diseases where reducing the amount of a target protein is desired.
[0064] In embodiments, the ASO modulates one or more aspects of target gene transcription or translation through steric blocking. The following review article describes the mechanisms of steric blocking and applications thereof and is incorporated by reference herein in its entirety: Roberts et al. Nature Reviews Drug Discoveiy (2020) 19: 673-694. The ASO may modulate one or more aspects of gene expression and/or protein function through various mechanisms some of which are disclosed herein.
[0065] In embodiments, the TM may modulate the activity of a target protein. In embodiments, the TM may be a polypeptide or a small molecule that functions to inhibit or increase the function of a target protein. Usher Syndrome
[0066] In embodiments, a cargo conjugate having a TM designed to treat Usher syndrome such as Usher syndrome type II or Usher syndrome type Ila is provided. In embodiments, a composition comprising such a cargo conjugate is ocularly delivered to a subject in need thereof, for example to treat Usher syndrome. In embodiments, the composition is administered directly the retina. In embodiments, the composition is administered intravitreally.
[0067] Usher syndrome type HA (OMIM 276901) is the most common type of Usher syndrome and is characterized by progressive vision loss and congenital moderate hearing loss. Usherin (USH2A; NCBI Gene ID:7399), a protein implicated in Usher syndrome type ILA, regulates the long-term maintenance of retinal photoreceptors. Pathogenic mutations in the gene that encodes usherin that disrupt usherin production, for example, by introducing a premature stop codon that results in a truncated protein and/or non-functional protein, can lead to degeneration of photoreceptors. There are currently no approved disease-modifying therapies for Usher syndrome type HA.
[0068] Mutations in exon 13 of the USH2A gene are the most recurring mutations in Usher syndrome type IIA and include, for example, c.2299delG and C.2276G > T (See Dulla, et al., Molecular Therapy, 4 August 2021, 29(8):2441-2455). Skipping of exon 13 in USH2A pre-mRNA represents a potential therapeutic strategy for treating Usher syndrome type IIA. Dulla, et al. illustrate that a functional truncated usherin results from exon skipping in the USH2A transcripts of zebrafish using antisense oligonucleotides. Furthermore, expression of human usherin (in which exon 13 of USH2A transcripts was deleted) in mouse cells in which USH2A was knocked out, resulted in a truncated version of usherin that partially restored function (see, Pendse, et al., “Exon 13-skipped USH2A protein retains functional integrity in mice, suggesting an exo-skipping therapeutic approach to treat USH2A-associated disease,” bioRxiv, 4 February 2020, doi: doi.org/10.1101/2020.02.04.934240).
[0069] The following proteins and genes, which are associated with Usher syndrome, may be targeted to treat Usher syndrome: myosin VILA (MY07A), harmonin (USH1C), cadherin 23 (CDH23), protocadherin 15 (PDCH15), USH1G, SANS, usherin (USH2A), VLGR1, GRP98, whirlin (DBFB3), and clarin-1 (USH3A).
[0070] In embodiments, a cargo conjugate includes a TM that modulates target gene expression and/or target protein activity related to one or more target genes and/or target proteins associated with Usher syndrome type I I A. In embodiments, the target gene is USH2A and the target protein is usherin. In embodiments, the TM is an ASO that modulates one or more aspects of transcription and/or translation of one or more target genes associated with Usher syndrome type IIA. In embodiments, the ASO is directed to a target nucleotide sequence within USH2A pre-mRNA and modulates one or more aspects of USH2A pre-mRNA splicing. In embodiments, ASO induces exon skipping of one or more exons of a USH2A target transcript. In embodiments, the cargo conjugate comprising the ASO induces exon skipping of exon 13 of a human USH2A target transcript. In embodiments, skipping of an exon in the USH2A pre-mRNA results in an in-frame deletion to produce a truncated usherin protein that has at least partial activity. In embodiments, skipping of exon 13 of the USH2A target transcript results in an in-frame deletion to produce a truncated usherin protein that has at least partial activity. One of skill in the art may design suitable ASO targeting a human USH2A transcript based on the sequence of the gene, which is available at ncbi.nlm.nih.gov/gene/7399, “USH2A usherin [Homo sapiens (human)],” Gene ID: 7399. In embodiments, the ASO is a phosphorodiamidate morpholino oligomer (PMO). In embodiments, the cargo conjugate comprising the ocular delivery construct and the ASO that results in skipping of one or more exons of a human USH2A target transcript is ocularly delivered to a subject in need thereof. In embodiments, the composition is administered directly the retina. In embodiments, the composition is administered intravitreally.
Diabetic retinopathy
[0071] In embodiments, a cargo conjugate has a TM designed to treat diabetic retinopathy (DR). In embodiments, a composition comprising such a cargo conjugate is ocularly delivered to a subject in need thereof, for example, to treat diabetic retinopathy In embodiments, the composition is administered directly the retina. In embodiments, the composition is administered intravitreally.
[0072] Diabetic retinopathy (DR) is the leading cause of blindness in Americans between 20 and 74 years of age. Clinical and epidemiological studies have identified a genetic component to DR, but data from studies aimed at identifying genes or genome regions associated with DR have been inconsistent (see, e.g., Cho and Sobrin, Curr Diab Rep, 2014 Aug, 14(8):515). Some data suggests that erythropoietin (EPO), an angiogenic factor observed in the eye, may be linked with DR (see, ibid). Other studies suggest that transcription factor 7-like 2 (TCF7L2) may be linked with DR (see, ibid). [0073] In embodiments, a cargo conjugate includes a TM that modulates target gene expression and/or target protein activity related to one or more target genes and/or target proteins associated with DR. In embodiments, a cargo conjugate may include a TM that modulates one or more aspects of gene expression and/or target protein activity of one or both of EPO and TCF7L2. In embodiments, the TM is an ASO.
Glaucoma
[0074] In embodiments, a cargo conjugate has a TM designed to treat glaucoma. In embodiments, a composition comprising such a cargo conjugate is ocularly delivered to a subject in need thereof, for example, to treat glaucoma. In embodiments, the composition is administered directly the retina. In embodiments, the composition is administered intravitreally.
[0075] Glaucoma is the leading cause of blindness in the United States. Glaucoma is generally caused by damage to the optic nerve by increases in eye pressure. A number of proteins and genes have been associated with glaucoma including myocilin (MYOC), optineurin (OPTN); tankbinding kinase 1 (TBK1); ATP-binding cassette transporter Al (ABCA1); actin filament associated protein 1 (AFAP1), GDP-Mannose 4,6-Dehydratase (GMDS); phosphomannomutase 2 (PMM2); transforming growth factor beta receptor 3 (TGFBR3); fibronectin type III domain containing 3B (FNDC3B); rho guanine nucleotide exchange factor 12 (ARHGEF 12); growth arrest specific 7 (GAS7); forkhead Box Cl (FOXCI); ataxin 2 (ATXN2); thioredoxin reductase 2 (TXNRD2); ependymin related 1 (EPDRl); choline O-acetyltransferase (CHAT); GUIS family zinc finger 3 (GLIS3); FERM domain containing kindlin 2 (FERMT2); DPM2-FAM102, and P/Q- type or CaV2.1 voltage-gated calcium channel (CACNA1A) (see, e.g., Wiggs and Pasquale, Human Mol Genet, 2017 Aug 1, 26(R1):R21-R27) In embodiments, a cargo conjugate includes a TM that modulates target gene expression and/or target protein activity related to one or more target genes and/or target proteins associated with glaucoma.
Retinitis piemenlosa
[0076] In embodiments, a cargo conjugate has a TM designed to treat retinitis pigmentosa (RP). In embodiments, a composition comprising such a cargo conjugate is ocularly delivered to a subject in need thereof, for example to treat RP. In embodiments, the composition is administered directly to the retina. In embodiments, the composition is administered intravitreally. [0077] RP is characterized by the loss of photoreceptor cells over time, leading to progressive vision loss. A number of genes have been associated with RP, with the majority of them expressed in either the photoreceptors or the retinal pigment epithelium of the eye (See, e.g., Ferari et al., Curr Genomics, 2011 Jun, 12(4):238-249). For example, the following proteins and genes are associated with autosomal dominant RP. bestrophin-1 (BEST1); carbonic anhydrase IV (CA4); cone-rod homeobox protein (CRX); fascin homolog 2; actin-bundling protein (FSCN2); guanylate cyclase activator IB (GUCA1B); inosine 5 '-monophosphate dehydrogenase 1 (IMPDH1); kelch- like 7 (KLHL7); nuclear receptor subfamily 2, group E, member 3 (NR2E3); neural retina leucine zipper protein (NRL); PRP3 pre-mRNA processing factor 3 homolog (PRPF3); PRP8 pre-mRNA processing factor 8 homolog (PRPF8); PRP31 pre-mRNA processing factor 31 homolog (PRPF31); peripherin 2 (PRPH2. RDS); retinol dehydrogenase 12 (RDH12); rhodopsin (RHO), retinal outer segment membrane protein 1 (R0M1); retinitis pigmentosa 1 (RPq); RP-9 (RP9), sema domain, immunoglobulin domain (Ig) transmembrane domain I and short cytoplasmic domain of semiphorin 4A (SEMA4A); small nuclear ribonucleoprotein 200kDa (SNRNP200); and topoisomerase I binding, arginine/serine-rich (TOPORS). In embodiments, a cargo conjugate includes a TM that modulates target gene expression and/or target protein activity related to one or more target genes and/or target proteins associated with RP.
[0078] Retinitis pigmentosa may be autosomal recessive. The following proteins and genes are associated with autosomal recessive RP: RP22; RP29; RP32, ATP-binding cassette; subfamily A (ABC1), member 4 (ABCA4); bestrophin-1 (BEST1); chromosome 2 open reading frame 71 (C2ORF71); ceramide kinase-like protein (CERKL); cyclic nucleotide gated channel alphal (CNGA1); cyclic nucleotide gated channel betal (CNGB1); crumbs homolog 1 (CRB1); eyes shut homolog (EYS, RP25); family with sequence similarity 161 member A (FAM161A); NAD(+)- specific isocitrate dehydrogenase 3 beta (IDH3B); interphotoreceptor matrix proteoglycan 2 (IMPG2); lecithin retinol acyltransferase (LRAT); C-mer proto-oncogene tyrosine kinase (MERTK); nuclear receptor subfamily 2; group E, member 3 (NR2E3); neural retina leucine zipper protein (NRL); phosphodiesterase 6A, cGMP-specific, rod alpha (PDE6A); phosphodiesterase 6B, cGMP-specific, rod beta (PDE6B); phosphodiesterase 6G, cGMP-specific, rod gamma (PDE6G); progressive rod-cone degeneration (PRCD); prominin 1 (PROMI); retinol binding protein 3 (RBP3), retinal G protein-coupled receptor (RGR); rhodopsin (RHO); retinaldehyde-binding protein 1 (RLBP1); RP-1 protein (RP1); retinal pigment epithelium-specific 65kDa protein (RPE65); S-antigen; retina and pineal gland (arrestin) (SAG); spermatogenesis associated protein 7 (SPATA7); tetratricopeptide repeat domain 8 (TTC8); tubby-like protein 1 (TULP1); usher syndrome 2a (USH2A); and zinc finger protein 513 (ZNF513). In embodiments, a cargo conjugate includes a TM that modulates target gene expression and/or target protein activity related to one or more target genes and/or target proteins associated with autosomal recessive RP.
[0079] Retinitis pigmentosa may be X-linked. The following proteins and genes are associated with X-linked RP: retinitis pigmentosa 2 protein (RP2); RP6; RP23; RP24; RP34; and retinitis pigmentosa GTPase regulator (RPGR). In embodiments, a cargo conjugate includes a TM that modulates target gene expression and/or target protein activity related to one or more target genes and/or target proteins associated with X-linked RP.
[0080] Retinitis pigmentosa may be autosomal dominant Autosomal dominate RP (adRP) is caused by heterozygous mutations in the PRFPF31 gene. The PRPF31 gene encodes human pre- mRNA processing factor 3 (PRPF31). PRPF31 is a ubiquitous pre-mRNA splicing factor that is a part of the small nuclear ribonucleoprotein complex of the spliceosome. More than 130 genetic PRPF31 mutations are known, some of which are loss of function variants. Penetrance of PRPF31 - assoacited adRP is incomplete; that is, carriers may be asymptomatic but still pass the disease to offspring. The major determinant of PRPF31 mutation penetrance is thought to be the expression level of the nonmutant PRPF31 allele (See Ali-Nasser Et. al., Mol Vis, 2022; 28, pg359-368, PM1D: 36338669, PMCID: PMC9603903). Carriers that have a high expression level of the nonmutant PRPF31 protein may not be symptomatic. Wild-type PRPF31 gene expression is highly variable and impacted by several factors such as CCR4-NOT.
[0081] CCR4-NOT (transcription complex subunit 3; also called CNOT3) is a multi-protein structure that is a negative regulator of PRPF31 transcription (see, PLoS Genet 8(11): el003040. doi:10.1371/journal.pgen.l003040). CCR4-NOT binds to the promoter of PRPF31 inhibiting transcription of PRPF31. CCR4-NOT is encoded by the CNOT3 gene. Modulating the activity and/or level of the CCR4-NOT t protein and/or the level of CNOT3 transcripts may be used to modulate PRPF31 gene expression.
[0082] In embodiments, a cargo conjugate is provided that includes a TM that modulates target gene expression and/or target protein activity related to one or more target genes and/or target proteins associated with adRP. In embodiments, the target gene or target protein may be PRPF31 (PRPF31), CNOT3 (CCR4-NOT), or both. In embodiments, the TM increases PRPF31 target transcript and/or protein levels. In embodiments, the TM decreases CCR4-NOT target protein levels and/or CNOT3 target transcript levels which may result in the increase of PRPF31 transcript levels and/or PRPF31 protein levels. In embodiments, the TM induces nonsense mediate decay of CNOT3 target transcripts thereby reducing the level of CNOT3 target transcripts and/or CCR4- NOT target protein levels. In embodiments, the TM is an ASO that hybridizes to at least a portion of a CNOT3 target transcript to induce nonsense mediated decay of the CNOT3 target transcript.
Bardet-Biedl syndrome
[0083] In embodiments, a cargo conjugate has a TM designed to treat Bardet-Biedl syndrome. In embodiments, a composition comprising such a cargo conjugate is ocularly delivered to a subject in need thereof, for example to treat Bardet-Biedl syndrome. In embodiments, the composition is administered directly the retina. In embodiments, the composition is administered intravitreally
[0084] Bardet-Biedl syndrome is characterized rode and/or cone dystrophy. A number of proteins and genes have been associated with Bardet-Biedl syndrome including BBS1, BBS2, ARL6 (BBS3), BBS4, BBSS, MKKS (BBS7), TTCB (BBSS), PTHB1 (BBS9), BBS10, TRIM32 (BBS11), BBS12, MKS1 (BBS13), CEP290/NPNP6/LCA10 (BBS14), WDPCP/FRITZ (BBS15), SDCCAG8 (BBS16), LZTFL1 (BBS17), BBIP1/10 (BBS18), IFT27 (BBS19), and AZI1/CEP131 (BBS20) (See, Priya, et al., Indian J Opthamol, 2016 Sept, 64(9):620-627). In embodiments, a cargo conjugate includes a TM that modulates target gene expression and/or target protein activity related to one or more target genes and/or target proteins associated with Bardet-Biedl syndrome.
Autosomal dominant optic atrophy
[0085] In embodiments, a cargo conjugate has a TM designed to treat autosomal dominant optic atrophy (ADOA). In embodiments, a composition comprising such a cargo conjugate is ocularly delivered to a subject in need thereof, for example, to treat ADOA. In embodiments, the composition is administered directly the retina. In embodiments, the composition is administered intravitreally.
[0086] Autosomal dominant optic atrophy (ADOA) is the most common hereditary optic neuropathy (heritable in an autosomal dominant manner). Symptoms of ADOA commonly present in early childhood, and vision loss progresses throughout the lifetime of the patient. In addition to progressive visual failure, ADOA has also been associated with sensorineural deafness, ataxia, myopathy, and external ophthalmoplegia. ADOA often impacts retinal ganglion cells and the axon forming the optic nerve.
[0087] ADOA is associated with mutations in the OPA1 gene. The OPA1 (3q38-q29) gene encodes the OPA1 protein, a ubiquitously expressed mitochondrial GTPase that is involved in regulating mitochondrial function such as, for example, oxidative phosphorylation, mitochondrial DNA maintenance, and apoptosis. The OP Al gene includes 30 exons which encode OP Al precursor proteins. The OPA1 pre-cursor proteins are targeted to the mitochondria and cleaved into OPA1 long-form proteins which become anchored to the inner mitochondrial membrane, or OPA1 short-form proteins which are soluble within the cell. In humans, there are at least eight OP Al protein variants due to differential splicing of exon 4, 4b, and 5b.
[0088] Over 400 OP Al genetic mutations have been reported, some of which result in the expression of a less-functional or non-functional OPA1 protein. The truncated proteins often lack a complete GTPase domain. Some OPA1 genetic mutations are thought to result in OPA1 haploinsufficiency (see Kushnareva, et al., Cell Death Dis. 2016 Jul; 7(7): e2309, dor 10.1038/cddis.2016.160). Increasing the global expression of OPA1 (from both alleles) may allow for an increase in the levels of full-length functional OPAl.
[0089] In embodiments, a cargo conjugate includes a TM that modulates target gene expression and/or target protein activity related to one or more target genes and/or target proteins associated with ADOA. In embodiments, the target gene is OPAl. In embodiments, the I'M acts to increase OPAl target transcript levels which may increase in functional OPAl protein levels. In embodiments, the TM is an ASO. In embodiments, the ASO is a PMO. In embodiments, the TM may be used to target the 5' UTR and/or the 3' UTR of a OPAl target transcript to stabilize the transcript. One of skill in the art may design suitable antisense oligonucleotides targeting the gene and/or the transcript of human OPAl based on the sequence of the gene, which is available at ncbi.nlm.nih.gov/gene/4976, “OPAl mitochondrial dynamin like GTPase [ Homo sapiens (human) ],” Gene ID: 4976.
Leber congenital amaurosis
[0090] In embodiments, a cargo conjugate has a TM designed to treat Leber congenital amaurosis (LCA). In embodiments, a composition comprising such a cargo conjugate is ocularly delivered to a subject in need thereof, for example to treat LCA. In embodiments, the composition is administered directly the retina. In embodiments, the composition is administered intravitreally. [0091] Leber congenital amaurosis (LCA) is a form of inherited retinal degradation causing blindness or visual impairment, often before the age of one. LCA is a heterogenous disease in which mutants of at least 15 genes are associated with LCA. CEP290 (15%), GUCY2D (12%) and CRB1 (10%) are the most frequently mutated LCA genes (See Prog Retin Eye Res. 2008 Jul;27(4):391-419. Doi. 10.1016/j.preteyeres.2008.05.003). At least 35 different mutations have been identified in The CEP290 gene that are associated with LCA. The gene product of CEP290 is the centrosomal protein 290 kDA protein (CEP290; also known as nephrovystin-6 (NPHP6), MKS3, and BBS14). CEP290 plays a role in the centrosome. CEP290 also plays a role in cilia development, such as the primary cilia of the photoreceptor cells of the retina. In embodiments, a cargo conjugate includes a TM that modulates target gene expression and/or target protein activity related to one or more target genes and/or target proteins associated with LCA. In embodiments, the target gene is CEP290.
[0092] A common hypomorphic (partial loss of gene function through reduced transcription and/or translation) is located within intron 26 of a CEP290 transcript (c.2991+1655A>G) (see Collin, et al., Molecular Therapy-Nucleic Acids (2012) 1, el4; doi:10.1038/mtna.2012.3). This mutation creates a cryptic slice donor site within intron 26. The cryptic splice site results in the inclusion of an aberrant 128 base pair exon into the transcript and introduces a pre-mature stop codon (p.C998X) into the transcript Ultimately, patients with this mutation have low levels for none) of the full-length functional CEP290 protein.
[0093] In embodiments, the TM acts to increase full-length CEP290 transcript levels. The full- length CEP290 transcripts lack the aberrant 128 base pair exon addition and the pre-mature stop codon. In embodiments, the TM induces exon skipping of the aberrant 128 base pair exon in a CEP290 target transcript to restore normal splicing and result in an increase of full-length CEP290 transcript levels and/or protein levels. In embodiments, the TM is an ASO that induces exon skipping by targeting the cryptic splice donor site to sterically block splicing factors from binding to the CEP290 target transcript to complete the cryptic splicing. In embodiments, the ASO is a PMO. One of skill in the art may design suitable antisense oligonucleotides targeting the transcript or gene of human CEP290 based on the sequence of the gene, which is available at ncbi.nlm.nih.gov/gene/80184, “entrosomal protein 290 [ Homo sapiens (human) ],” Gene ID: 80184. Macular dystrovhy/Stargardt disease
[0094] In embodiments, a cargo conjugate has a TM designed to treat macular dystrophy. In embodiments, a composition comprising such a cargo conjugate is ocularly delivered to a subject in need thereof, for example to treat glaucoma. In embodiments, the composition is administered directly the retina. In embodiments, the composition is administered intravitreally.
[0095] Macular dystrophy is a group of diseases that cause deterioration of the retina. Examples of macular dystrophy diseases include Stargardt disease, Vitelliform macular dystrophy, and North Carolina macular dystrophy. Stargardt disease (STGD) is the most common form of macular dystrophy. STGD is characterized by the buildup of lipofuscin (lipid containing residues of lysosomal digestion) on the macula. The accumulation of lipofuscin damages the cells responsible for sharp central vision.
[0096] There are three known subtypes of STGD; STGD 1 , STGD2, and STDG3. STDG1 is caused by mutations in the ABCA4 gene. The ABCA4 gene encodes an ATP-binding cassette transporter subfamily A member 4 protein (ABCA4; See ABCA4 ATP binding cassette subfamily A member 4 [ Homo sapiens (human)], Gene ID NO; 24, ncbi.nlm.nih.gov/gene/24). ABCA4 is involved in the visual cycle. More specifically, ABC4A is involved in the clearance of trans-retinal and excess 11 -ci s retinal/11-cis-retinal Schiff-base conjugates from photoreceptor cells to prevent the buildup of toxic bisretinoid compounds (See Quazi and Molday, PNAS, vol 111, no 13, pg. 5024-5029, doi: 10.1073/pnas.1400780111). Dysfunctional ABCA4 may result in accumulation of bisretinoid compounds which can damage cells. Common mutations in the ABC4 gene include c.5461- 101T>C and c.4539_2001G>A (See, e g., Dulla, et al., Investigative Opthalmology & Visual Science, July 2018, Vol., 59, 5315; and Garanto, Et. AL, Genes (Basel), 2019 Jun 14; 10(6):452, doi: 10.3390/genesl0060452).
[0097] In embodiments, a cargo conjugate includes a TM that modulates target gene expression and/or target protein activity related to one or more target genes and/or target proteins associated with STGD, such as STGD1, STGD2, and STDG3. In embodiments, the target gene is ABCA4. In embodiments, the TM is an ASO. In embodiments, the ASO is a PMO. Other diseases
[0098] A number of other genes and proteins associated with other diseases of the eye are known and can be readily identified through, for example, a literature search. The cargo conjugates may include a TM that targets such genes or gene products.
Therapeutic Moieties
[0099] The cargo conjugates provided herein comprises an ocular delivery construct and a therapeutic moiety or therapeutic agent. The therapeutic moiety may be any suitable therapeutic moiety for treating a disease of the eye. As used herein, the terms “therapeutic moiety” or “therapeutic agent” refer to any molecule (e.g., polypeptide, small molecule, oligonucleotide, gene editing machinery) that is designed to have and/or has prophylactic or other biological activity. In embodiments, the therapeutic moiety selectively binds to a target molecule associated with an ocular disease. In embodiments, the target molecule is a macromolecule implicated in a disease or pathology of the eye. In embodiments, the target molecule is a polypeptide or protein. In embodiments, the target molecule is an oligonucleotide. In embodiments, the oligonucleotide target comprises DNA. In embodiments, the oligonucleotide target comprises genomic DNA. In embodiments, the oligonucleotide target comprises RNA. In embodiments, the oligonucleotide target comprises mRNA. In embodiments, the target molecule is associated with an ocular disease. In embodiments, selective binding of the therapeutic moiety with the target molecule is useful for the treatment of a disease, pathology or other abnormal state or condition of the eye. In embodiments, selective binding of the therapeutic moiety to the target molecule upregulates expression or activity of the target molecule. In embodiments, selective binding of the therapeutic moiety to the target molecule downregulates expression or activity of the target molecule.
[0100] In embodiments, the therapeutic moiety comprises a therapeutic oligonucleotide. In embodiments, the therapeutic moiety comprises a polypeptide. In embodiments, the therapeutic moiety comprises a small molecule. In embodiments, the therapeutic moiety includes one or more components of gene editing machinery.
Therapeutic Oligonucleotides
[0101] In embodiments, the therapeutic moiety comprises a therapeutic oligonucleotide. In embodiments, the therapeutic oligonucleotide comprises an antisense oligonucleotide (ASO). In embodiments, the therapeutic oligonucleotide comprises siRNA, RNAi, microRNA, antagomir, an aptamer, a ribozyme, an immunostimulatory oligonucleotide, a decoy oligonucleotide, a supermir, a miRNA mimic, a miRNA inhibitor, or a combination thereof (See, for example, Chery, J., “RNA therapeutics: RNAi and antisense mechanisms and clinical applications,” Postdoc J, July 2016, 4(7):35-50, and Zhu, et al., “RNA-based therapeutics: an overview and prospectus,: Cell Death & Disease, 23 July 2022, 12(644) (doi: 10.1038/s41419-022-05075-2).
[0102] In embodiments, therapeutic oligonucleotides are provided that include from about 5 to about 100 nucleic acids in length. In embodiments, the therapeutic oligonucleotide is from about 5 to about 50, about 8 to about 40, about 10 to about 30, about 15 to about 30, or about 20 to about 30 nucleotides in length. In embodiments, the therapeutic oligonucleotide includes one or more modified nucleosides, one or more modified internucleoside linkages, one or more conjugate groups, or combinations thereof.
Antisense Oligonucleotides (ASO)
[0103] In embodiments, the therapeutic oligonucleotide is an antisense oligonucleotide (ASO) directed to a target gene or a target transcript associated with a disease of the eye. The ASO may be directed to and bind to a target nucleotide sequence located within a target gene or a target transcript. In embodiments, the target nucleotide sequence is within a target gene and/or target transcript associated with an eye disease.
[0104] The term “antisense oligonucleotide” refers to an oligonucleotide that is at least partially complementary to a target sequence within a target polynucleotide. An antisense oligonucleotide (ASO) is a single stranded molecule that contains DNA, RNA, or combinations or modifications thereof that are at least partially complementary to a chosen sequence, e.g., a target nucleotide sequence within a target gene or target transcript.
[0105] An ASO may modulate one or more aspects of gene expression and/or protein function via hybridization of the ASO with a target nucleotide sequence. The ASO may modulate one or more aspects of gene expression and/or protein junction through various mechanisms. For example, hybridization of an ASO to a target nucleotide sequence of a target transcript may modulate splicing of a target transcript such as, for example, via exon skipping; exon inclusion; alternative splicing; prevent polyadenylation of the target transcript; increase the target transcript stability; induce target transcript degradation; prevent translation of the target transcript; or any combination thereof. ASOs have been demonstrated to be effective and targeted inhibitors of protein synthesis, and, consequently, can be used to modulate gene expression of a targeted gene. [0106] In embodiments, the ASO hybridizes with a target sequence of a target gene or target transcript having sequence from about 5 to about 50 nucleotides in length, which can also be referred to as the length of the ASO. In embodiments, the ASO is from about 5 to about 50, about 8 to about 40, about 10 to about 30, about 15 to about 30, or about 20 to about 30 nucleotides in length. In embodiments, the ASO is at least about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15, and up to about 16, about 17, about 18, about 19, about 20, about 21 , about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31 , about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, or about 50 nucleotides in length. In embodiments, the ASO is about 15 nucleotides in length. In embodiments, the ASO is about 16 nucleotides in length. In embodiments, the ASO is about 17 nucleotides in length. In embodiments, the ASO is about 18 nucleotides in length. In embodiments, the ASO is about 19 nucleotides in length. In embodiments, the ASO is about 20 nucleotides in length. In embodiments, the ASO is about 21 nucleotides in length. In embodiments, the ASO is about 22 nucleotides in length. In embodiments, the ASO is about 23 nucleotides in length. In embodiments, the ASO is about 24 nucleotides in length. In embodiments, the ASO is about 25 nucleotides in length. In embodiments, the ASO is about 26 nucleotides in length. In embodiments, the ASO is about 27 nucleotides in length. In embodiments, the ASO is about 28 nucleotides in length. In embodiments, the ASO is about 29 nucleotides in length. In embodiments, the ASO is about 30 nucleotides in length.
[0107] In embodiments, the ASO may be less than about 100 percent complementary to a target nucleotide sequence. As used herein, the term “percent complementarity” refers to the number of nucleobases of an ASO that have nucleobase complementarity with a corresponding nucleobase of target nucleotide sequence by the total length (number of nucleobases) of the ASO. One skilled in the art recognizes that the inclusion of mismatches is possible without eliminating the activity of the antisense compound. In embodiments, the ASOs contain no more than about 15%, no more than about 10%, no more than 5%, or no mismatches. In embodiments, the ASOs are at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, or 100% complementary to a target nucleic acid. Percent complementarity of an oligonucleotide is calculated by dividing the number of complementary nucleobases by the total number of nucleobases of the oligonucleotide. Percent complementarity of a region of an oligonucleotide is calculated by dividing the number of complementary nucleobases in the region by the total number of nucleobases region.
[0108] In embodiments, incorporation of nucleotide affinity modifications allows for a greater number of mismatches compared to an unmodified compound. Similarly, certain oligonucleotide sequences may be more tolerant to mismatches than other oligonucleotide sequences. One of ordinary skill in the art is capable of determining an appropriate number of mismatches between a an ASO and a target nucleotide sequence, such as by determining melting temperature (Tm). Tm or change in Tm (ATm) can be calculated by techniques that are familiar to one of ordinary skill in the art. For example, techniques described in Freier et al. (Nucleic Acids Research, 1997, 25, 22: 4429-4443) allow one of ordinary skill in the art to evaluate nucleotide modifications for their ability to increase the melting temperature of an RNA:DNA duplex.
Therapeutic oligonucleotides design
[0109] Design of a therapeutic oligonucleotides (e g., an ASO) will depend upon the target gene. Targeting a therapeutic oligonucleotide to a particular target nucleotide sequence can be a multistep process. The process usually begins with the identification of gene of interest. The transcript of the gene of interest is analyzed and a target nucleotide sequence is identified. In embodiments, the target gene is a gene associated with a disease of the eye.
[0110] One of skill in the art will be able to design, synthesize, and screen therapeutic oligonucleotides of different nucleobase sequences to identify a sequence that results in antisense activity. For example, a therapeutic oligonucleotide can be designed that inhibits expression of a target gene. Methods for designing, synthesizing, and screening therapeutic nucleotides for antisense activity against a preselected target nucleic acid and/or target gene can be found, for example in "Antisense Drug Technology, Principles, Strategies, and Applications" Edited by Stanley T. Crooke, CRC Press, Boca Raton, Florida, which is incorporated by reference in its entirety for any purpose.
[0111] The efficacy of a therapeutic oligonucleotide (e.g., an ASO) may be assessed by evaluating the antisense activity effected by their administration. As used herein, the term "antisense activity" refers to any detectable and/or measurable activity attributable to the hybridization of a therapeutic oligonucleotide to its target nucleotide sequence. Such detection and/or measuring may be direct or indirect. In embodiments, antisense activity is assessed by detecting and or measuring the amount of target protein in a cell or population of cells before and after administration of the therapeutic oligonucleotide to the cell or population of cells. In embodiments, antisense activity is assessed by detecting and/or measuring the amount of target transcript in a cell or population of cells.
Therapeutic Oligonucleotides Structure
[0112] Therapeutic oligonucleotides comprise nucleosides linked through intemucleoside linkages. Nucleosides include a pentose sugar (e.g., ribose or deoxyribose) and a nitrogenous base (nucleobase or simply base) covalently attached to sugar. The naturally occurring (traditional) bases found in DNA and/or RNA are adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U). The naturally occurring (traditional) sugars found in DNA and/or RNA deoxyribose (DNA) and ribose (RNA). A naturally occurring (traditional) nucleoside linkage is a phosphodiester bond. In embodiments, the therapeutic oligonucleotides may have all natural sugars, natural bases, and natural intemucleoside linkages.
[0113] Chemically modified nucleosides are routinely used for incorporation into therapeutic oligonucleotides to enhance one or more properties, such as nuclease resistance, pharmacokinetics, or affinity for a target gene or target transcript. Non-limiting examples of nucleosides are provided in FIG. 2 and in Khvorova et al. Nature Biotechnology (2017) 35: 238-248, which is incorporated by reference herein in its entirety. In embodiments, a therapeutic oligonucleotide has one or more modified nucleosides. In embodiments, a therapeutic oligonucleotide has one or more modified sugars. In embodiments, a therapeutic oligonucleotide has one or more modified bases. In embodiments, a therapeutic oligonucleotide has one or more modified intemucleoside linkages.
[0114] In general, a nucleobase is any group that contains one or more atoms or groups of atoms capable of hydrogen bonding to a base of another nucleic acid. In addition to natural nucleobases, many modified nucleobases or nucleobase mimetics known to those skilled in the art are amenable with the compounds described herein. The terms modified nucleobase and nucleobase mimetic can overlap, but generally a modified nucleobase refers to a nucleobase that is fairly similar in structure to the parent nucleobase, such as for example a 7-deaza purine, a 5-methyl cytosine, or a G-clamp, whereas a nucleobase mimetic generally includes more complicated structures, such as for example a tricyclic phenoxazine nucleobase mimetic. Methods for preparation of the above noted modified nucleobases are well known to those skilled in the art.
[0115] In embodiments, a therapeutic oligonucleotide includes one or more nucleosides having a modified sugar moiety. In embodiments, the furanosyl sugar ring of a natural nucleoside can be modified. A furanosyl sugar ring may be modified in any suitable manner, including, but not limited to, addition of a substituent group, bridging of two non-geminal ring atoms to form a bicyclic nucleic acid (BNA) and substitution of an atom or group such as -S-, -N(R)- or -C(R1 )(R2) for the ring oxygen at the 4'-position. Modified sugar moieties are well known and can be used to alter, typically increase, the affinity of the antisense compound for its target and/or increase nuclease resistance. A representative list of modified sugars includes, but is not limited to, non- bicyclic substituted sugars, especially non-bicyclic 2'-substituted sugars having a 2'-F, 2 -OCH3 or a 2'-O(CH2)2-OCH3 substituent group, and 4-thio modified sugars. Sugars can also be replaced with a sugar mimetic group, for example, a morpholino ring, a methylenemorpholine ring, among others.
[0116] In embodiments, a therapeutic oligonucleotide may include one or more bicyclic modified sugars (SNA's), such as, for example, LNA (4*-(CH2)-O-2' bridge), 2'-thio-LNA (4'-(CH2)-S-2' bridge), 2'-amino-LNA (4'-(CH2)-NR-2' bridge), ENA (4'-(CH2)2-O-2' bridge), 4'-(CH2)3-2' bridged BNA, ^-(CIECHfCIB))-^ bridged BNA" cEt (4'-(CH(CH3>0-2' bridge), and cMOE BNAs (4'-(CH(CH2OCH3)-O-2' bridge).
[0117] In embodiments, a therapeutic oligonucleotide may include one or more locked nucleic acids" (LN As) in which the 2'-hydroxyl group of the ribosyl sugar ring is linked to the 4' carbon atom of the sugar ring thereby forming a 2'-C,4'-C-oxymethylene linkage to form the bicyclic sugar moiety. The synthesis and preparation of the LNA monomers adenine, cytosine, guanine, 5- methyl-cytosine, thymine and uracil, along with their oligomerization, and nucleic acid recognition properties have been described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). LNAs and preparation thereof are also described in WO 98/39352 and WO 99/14226.
[0118] Intemucleoside linking groups link the nucleosides or otherwise modified monomer units of an oligonucleotide together. The two main classes of intemucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus containing intemucleoside linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidate, phosphorodiamidate, and phosphorothioates. Representative non-phosphorus containing intemucleoside linking groups include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-O-C(O)-S-), thionocarbamate (-O-C(OXNH)-S-); siloxane (-O-Si(H)2-O-); and N.N'-dimethylhydrazine (- CH2-N(CH3)-N(CH3)-). Therapeutic oligonucleotides having non-phosphorus intemucleoside linking groups are referred to as oligonucleosides. Modified intemucleoside linkages, compared to natural phosphodiester linkages, can be used to alter, typically increase, nuclease resistance of the therapeutic oligonucleotides. Intemucleoside linkages having a chiral atom can be prepared racemic, chiral, or as a mixture. Representative chiral intemucleoside linkages include, but are not limited to, alkylphosphonates and phosphorothi oates. Methods of preparation of phosphorous- containing and non-phosphorous-containing linkages are well known to those skilled in the art.
[0119] In embodiments, a phosphate group can be linked to the 2', 3' or 5' (or 6', for a 6 membered ring, such as a methylenemorpholine ring) hydroxyl moiety of the sugar (or sugar mimetic). In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. Within oligonucleotides, the phosphate groups are commonly referred to as forming the intemucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3' to 5* phosphodiester linkage. In embodiments, the oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO) comprising a backbone of methylenemorpholine rings linked through phosphorodiamidate intemucleoside linkages.
[0120] PMOs are uncharged nucleic acid analogs bind to target nucleic acid through base paring. PMOs that bind to mRNA may block interaction of proteins to the mRNA through steric blockade (See, e.g., Nan and Zhang, Front. Microbiol. 20 April 2019 (doi.org/10.3389/fmicb .2018.00750)). As uncharged, or net neutral charged, oligonucleotides, PMOs are particularly effective for intracellular delivery with ocular delivery construct.
[0121] The therapeutic oligonucleotides may contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations that may be defined, in terms of absolute stereochemistry, as (R) or (S); a or 0; or as (D) or (L). Included in the antisense compounds provided herein are all such possible isomers, as well as their racemic and optically pure forms.
[0122] In embodiments, therapeutic oligonucleotides are modified by covalent attachment of one or more conjugate groups. In general, conjugate groups modify one or more properties of the attached therapeutic oligonucleotides including but not limited to pharmacodynamic, pharmacokinetic, binding, absorption, cellular distribution, cellular uptake, chaige and clearance. Conjugate groups are routinely used in the chemical arts and are linked directly or via an optional linking moiety or linking group to a parent compound such as a therapeutic oligonucleotides. Conjugate groups include without limitation, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins and dyes. In embodiments, the conjugate group is a polyethylene glycol (PEG), and the PEG is conjugated to either the therapeutic oligonucleotide, a linker, an EP, or the cyclic peptide.
Gene-Editing Machinery
[0123] In embodiments, the therapeutic moiety comprises one or more component of gene-editing machineiy. As used herein, “gene-editing machinery” refers to protein, nucleic acids, or combinations thereof, which may be used to edit a genome. Non-limiting examples of gene-editing machinery include guide RNAs (gRNAs), nucleases, nuclease inhibitors, and combinations and complexes thereof.
[0124] The gene editing machineiy may be used to repair a mutated gene or to introduce a mutation into a gene. The gene may be a gene associated with a disease of the eye.
[0125] In embodiments, a linker conjugates the ocular delivery construct to the one or more components of gene-editing machinery. Any linker described in this disclosure or that is known to a person of skill in the art may be utilized. gRNA
[0126] Tn embodiments, the therapeutic moiety includes a guide RNA (gRNA). A gRNA targets a genomic loci in a prokaryotic or eukaryotic cell.
[0127] In embodiments, the gRNA is a single-molecule guide RNA (sgRNA). A sgRNA includes a spacer sequence and a scaffold sequence. A spacer sequence is a short nucleic acid sequence used to target a nuclease (e.g., a Cas9 nuclease) to a specific nucleotide region of interest (e.g., a genomic DNA sequence to be cleaved). In embodiments, the spacer may be about 17-24 bases in length, such as about 20 bases in length.
[0128] In embodiments, the spacer targets a site that immediately precedes a 5' protospacer adjacent motif (PAM). The PAM sequence may be selected based on the desired nuclease. For example, the PAM sequence may be any one of the PAM sequences shown in Table 1 below, wherein N refers to any nucleic acid, R refers to A or G, Y refers to C or T, W refers to A or T, and V refers to A or C or G. Table 1. Nucleases and PAM sequences
Figure imgf000039_0001
[0129] Tn embodiments, a spacer may target a sequence of a mammalian gene, such as a human gene. In embodiments, the spacer may target a mutant gene. In embodiments, the spacer may target a coding sequence. In embodiments, the spacer may target an exonic sequence. In embodiments, the spacer may target a polyadenylation site (PS). In embodiments, the spacer may target a sequence element of a PS. In embodiments, the spacer may target a polyadenylation signal (PAS), an intervening sequence (IS), a cleavage site (CS), a downstream element (DES), or a portion or combination thereof. In embodiments, a spacer may target a splicing element (SE) or a cis-splicing regulatory element (SRE).
[0130] The scaffold sequence is the sequence within the sgRNA that is responsible for nuclease (e.g., Cas9) binding. The scaffold sequence does not include the spacer/targeting sequence. In embodiments, the scaffold may be about 10 to about 150 nucleotides in length, or about 50 to about 100 nucleotides in length.
[0131] In embodiments, the gRNA is single guide RNA molecule comprising the spacer and the scaffold. In embodiments, the gRNA comprises two molecules that hybridize to form the gRNA. An example of a gRNA that includes two molecules is a gRNA comprising a crRNA and tracrRNA. In embodiments, the gRNA or one or more components thereof may further include a poly(A) tail.
[0132] In embodiments, a compound that includes a CPP is conjugated to a nucleic acid that includes a gRNA or a component thereof. In embodiments, the nucleic acid includes about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11 , about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 gRNAs or components thereof. In embodiments, the gRNAs recognize the same target. In embodiments, the gRNAs recognize different targets.
[0133] In embodiments, a compound that includes a CPP is conjugated to a nucleic acid designed to express the gRNA within a cell. The nucleic acid may include a promoter sequence to drive expression of the gRNA.
Nuclease
[0134] In embodiments, the therapeutic moiety includes a nuclease. In embodiments, the nuclease is a Type II, Type V-A, Type V-B, Type VC, Type V-U, Type VI-B nuclease. In embodiments, the nuclease is a transcription, activator-like effector nuclease (TALEN), a meganuclease, or a zinc-finger nuclease or a modified form or variant thereof. In embodiments, the nuclease is a Cas9, Casl2a (Cpfl), Casl2b, Casl2c, Tnp-B like, Casl3a (C2c2), Casl3b, or Casl4 nuclease or a modified form or variant thereof. For example, in embodiments, the nuclease is a Cas9 nuclease or a Cpfl nuclease
[0135] In embodiments, the ocular delivery construct is conjugated to a nucleic acid encoding a nuclease. In embodiments, the nucleic acid encoding a nuclease includes a sequence encoding a promoter, wherein the promoter drives expression of the nuclease. gRNA and Nuclease Combinations
[0136] Tn embodiments, the therapeutic moiety includes a ribonucleoprotein (RNP) that includes a gRNA and a nuclease. A RNP is a complex of a gRNA bound to a nuclease. The gRNA, the nuclease, or both may be covalently attached to an ocular delivery construct to form a cargo conjugate having an RNP therapeutic moiety.
[0137] In embodiments, a composition that includes: (a) a cargo conjugate comprising an ocular delivery' construct conjugated to a gRNA and (b) a nuclease is delivered to a cell. In embodiments, a composition that includes: (a) a cargo conjugate comprising an ocular delivery construct conjugated to a nuclease and (b) an gRNA is delivered to a cell. In embodiments, a composition that includes: (a) a first cargo conjugate comprising a first delivery construct conjugated to a gRNA and (b) a second cargo conjugate comprising a second delivery construct conjugated to a nuclease is delivered to a cell. In embodiments, the first delivery construct and the second delivery construct are the same. In embodiments, the first delivery construct and the second delivery construct are different. [0138] In embodiments, a cargo conjugate comprises an ocular delivery construct conjugated to a nucleic acid encoding a gRNA and/or a nuclease. In embodiments, the nucleic acid encoding a nuclease and a gRNA includes a sequence encoding a promoter, wherein the promoter drives expression of the nuclease and the gRNA. In embodiments, the nucleic acid encoding a nuclease and a gRNA includes two promoters, wherein a first promoter controls expression of the nuclease and a second promoter controls expression of the gRNA. In embodiments, the nucleic acid encoding a gRNA and a nuclease encodes from about 1 to about 20 gRNAs, or from about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, or about 19, and up to about 20 gRNAs. In embodiments, the gRNAs recognize different targets. In embodiments, the gRNAs recognize the same target.
Nuclease Inhibitors
[0139] In embodiments, the therapeutic moiety includes a nuclease inhibitor. A limitation of gene editing is potential off-target editing. The delivery of a nuclease inhibitor may limit off-target editing. In embodiments, the nuclease inhibitor is a polypeptide, polynucleotide, or small molecule.
Therapeutic polypeptides
[0140] Tn embodiments, the therapeutic moiety includes a therapeutic polypeptide. In embodiments, the therapeutic polypeptide includes a peptide inhibitor. In embodiments, the peptide inhibitor inhibits a protein associated with a disease of the eye. In embodiments, the therapeutic polypeptide includes a peptide replacement therapy to functionally replace an aberrantly expressed protein associated with a disease of the eye.
[0141] In embodiments, the therapeutic moiety includes a protein or a fragment thereof. In embodiments, the therapeutic moiety includes an RNA binding protein or an RNA binding fragment thereof. In embodiments, the therapeutic moiety includes an enzyme. In embodiments, the therapeutic moiety includes an RNA-cleaving enzyme or an active fragment thereof.
Antibodies
[0142] In embodiments, the therapeutic moiety includes an antibody or an antigen-binding fragment. Antibodies and antigen-binding fragments can be derived from any suitable source, including human, mouse, camelid (e.g., camel, alpaca, llama), rat, ungulates, or non-human primates (e.g., monkey, rhesus macaque).
[0143] The term “antibody” includes intact polyclonal or monoclonal antibodies and antigenbinding fragments thereof. For example, a native immunoglobulin molecule includes two heavy chain polypeptides and two light chain polypeptides. Each of the heavy chain polypeptides associate with a light chain polypeptide by virtue of interchain disulfide bonds between the heavy and light chain polypeptides to form two heterodimeric proteins or polypeptides (i.e., a protein that includes two heterologous polypeptide chains). The two heterodimeric proteins then associate by virtue of additional interchain disulfide bonds between the heavy chain polypeptides to form an immunoglobulin protein or polypeptide.
[0144] In embodiments, the therapeutic moiety is an antigen-binding fragment that binds to a target protein associated with a disease of the eye. An antibody may modulate the activity of the target protein to which it binds. In embodiments, the therapeutic moiety is an antigen-binding fragment that binds to a target transcript of a protein (Ye et al., PNAS (2008), 105(l):82-87; and Jung et al., (RNA (2014), 20(6): 805-814). In embodiments, an antigen-binding fragment that binds to a target protein includes 1, 2, 3, 4, 5, or all 6 CDRs of a variable heavy chain (VH) and/or a variable light chain (VL) sequence from an antibody that specifically binds to the target protein. In embodiments, the antigen binding fragment includes 1, 2, or 3 of the CDRs of a camelid single domain antibody such as the VHH region. In embodiments, the antigen-binding fragment that binds to a target protein is a portion of a full-length antibody, such as Fab, F(ab’)2, Fab’, Fv fragments, minibodies, diabodies, single domain antibody (dAb), single-chain variable fragments (scFv), multispecific antibodies formed from antibody fragments, or any other modified configuration of the immunoglobulin molecule that includes an antigen-binding site or fragment of the required specificity.
[0145] In embodiments, the therapeutic moiety includes a bispecific antibody. Bispecific antibodies (BsAbs) are antibodies that can simultaneously bind two separate and unique antigens (or different epitopes of the same antigen). In embodiments, the therapeutic moiety includes a bispecific antibody that can simultaneously bind to a target protein associated with an eye disease and another target protein. Non-limiting examples include scFv (single-chain variable fragment), BsDb (bispecific diabody), scBsDb (single-chain bispecific diabody), scBsTaFv (single-chain bispecific tandem variable domain), DNL-(Fab)3 (dock-and-lock trivalent Fab), sdAb (singledomain antibody), and BssdAb (bispecific single-domain antibody).
[0146] BsAbs with an Fc region are useful for carrying out Fc mediated effector functions such as antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. They have the half-life of normal IgG. On the other hand, BsAbs without the Fc region (bispecific fragments) rely solely on their antigen-binding capacity for carrying out therapeutic activity. Due to their smaller size, these fragments have better solid-tumor penetration rates. BsAb fragments do not require glycosylation, and they may be produced in bacterial cells. The size, valency, flexibility and half-life of BsAbs to suit the application.
[0147] In embodiments, the therapeutic moiety includes a “diabody.” The term diabody refers to a bispecific antigen-binding antibody fragment in which VH and VL domains are expressed in a single polypeptide chain using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen-binding sites (see, e g., Holliger et al.. Proc. Natl. Acad. Sci. USA 90:6444-48 (1993) and Poljak et al., Structure 2:1121- 23 (1994)). Diabodies may be designed to bind to two distinct antigens and are bi-specific antigen binding constructs.
[0148] In embodiments, the therapeutic moiety includes a “nanobody” or a “single domain antibody” (which can also be referred to herein as sdAbs or VHH). Single domain antibody refers to an antigen-binding fragment that includes a single monomeric variable antibody domain comprising one variable domain (VH) of a heavy-chain antibody. In embodiments, the variable chain is the VHH of a cam elid single chain antibody.
[0149] In embodiments, the therapeutic moiety includes a minibody.
[0150] In embodiments, the therapeutic moiety is an antibody mimetic. Antibody mimetics are compounds that, like antibodies, can specifically bind antigens, but that are not structurally related to antibodies. They are usually artificial peptides or proteins with a molar mass of about 3 to 20 kD (compared to the molar mass of antibodies at -150 kDa.). Examples of antibody mimetics include affibody molecules affilins, affimers, affitins, alphabodies anticalins, avimers, DARPins, fynomers Kunitz domain peptides and monobodies.
Other Peptides
[0151] In embodiments, the therapeutic moiety includes a peptide. In embodiments, the peptide acts as an agonist, increasing the activity of a target protein. In embodiments, the peptide acts as an antagonist, decreasing the activity of a target protein. In embodiments, the peptide is configured to inhibit protein-protein interaction (PPI). Protein-protein interactions (PPIs) are important in many biochemical processes, including transcription of nucleic acid and various post-translational modifications of translated proteins. PPIs can be experimentally determined by biophysical techniques such as X-ray crystallography, NMR spectroscopy, surface plasma resonance (SPR), bio-layer interferometry (BLI), isothermal titration calorimetry (ITC), radio-ligand binding, spectrophotometric assays and fluorescence spectroscopy. Peptides that inhibit protein-protein interaction can be referred to as peptide inhibitors.
[0152] In embodiments, the therapeutic moiety includes a peptide inhibitor. In embodiments, the peptide inhibitor includes from about 5 to about 100 amino acids, from about 5 to about 50 amino acids; from about 15 to about 30 amino acids; or from about 20 to about 40 amino acids. In embodiments, the peptide inhibitor includes one or more chemical modifications, for example, to reduce proteolytic degradation and/or to improve in vivo half-life. In embodiments, the peptide inhibitor includes one or more synthetic amino acids and/or a backbone modification. In embodiments, the peptide inhibitor has an a-helical structure.
[0153] In embodiments, the peptide inhibitor is configured to disrupt one or more function of a protein associated with a disease of the eye. In embodiments, the peptide inhibitor is configured to disrupt formation of protein complexes. In embodiments, binding of the peptide inhibitor to the protein blocks dimer formation.
Small Molecules
[0154] In embodiments, the therapeutic moiety includes a small molecule for treating a di sease of the eye. In embodiments, the small molecule does not readily gain access to an intracellular compartment of a cell of the eye when delivered by itself (not conjugated to an ocular delivery construct).
Ocular Delivery Constructs
[0155] A cargo conjugate includes an ocular delivery construct. An ocular delivery construct may be used to transport a cargo (e.g., a therapeutic moiety) to a structure of the eye, an eye tissue, and/or a cell-type of the eye. In embodiments, an ocular delivery construct may be used to transport a cargo across a cell membrane, for example, to deliver the cargo to the cytosol or nucleus of a cell in the eye. An ocular delivery construct can comprise a cyclic cell penetrating peptide (cCPP); a cCPP and a linker; a cCPP and an exocyclic peptide (EP); or an endosomal escape vehicle (EEV) which comprises a cCPP, an EP, and a linker.
[0156] The configuration of the ocular delivery construct depends at least in part on the components of the ocular delivery construct. Two or more components that are coupled, conjugated, or linked are a part of the same compound. In embodiments, the ocular delivery construct comprises a cCPP, and the cCPP is conjugated to the cargo to from a cargo conjugate. In embodiments, the ocular delivery construct comprises a cCPP and a linker, and the cCCP is coupled to the linker and the linker is conjugated to the cargo to form a cargo conjugate. In embodiments, the ocular delivery construct comprises a cCPP and an EP, and the cCPP is coupled to the EP and the EP is conjugated to the cargo to from the cargo conjugate; the cCPP is coupled to the EP and the cCPP is conjugated to the cargo to form the cargo conjugate; or both the cCPP and the EP are conjugated to the cargo to form the cargo conjugate. In embodiments, the ocular delivery construct comprises an EEV, and the EP is coupled to the cCPP, the cCPP is coupled to the linker, and the linker is conjugated to the cargo, the cCPP is coupled to the EP, the EP is coupled to the linker, and the linker is conjugated to the cargo; the cCPP is coupled to the linker, the EP is coupled to the linker, and the EP is conjugated to the cargo, the EP is coupled to the linker, the cCPP is coupled to the linker, and the cCPP is conjugated to the cargo; or the EP is coupled to the linker, the cCPP is coupled to the linker, and the linker is conjugated to the cargo to from a cargo conjugate.
Cell Penetrating Peptides (CPP)
[0157] The ocular delivery constructs comprise at least one cell penetrating peptide (CPP). The cell penetrating peptide can be a cyclic cell penetrating peptide (cCPP) In embodiments, the ocular delivery constructs comprise one cell penetrating peptide (cCPP). In embodiments, the ocular delivery constructs comprise two cell penetrating peptides (cCPP). In embodiments, the cCPP is capable of penetrating a cell membrane. In embodiments, the cCPP can deliver the cargo to the cytosol of the cell. The cCPP can deliver the cargo to a cellular location where a target gene, target transcript, and/or target protein is located. To conjugate the cCPP to a cargo, an EP, and/or a linker, at least one bond or lone pair of electrons on the cCPP can be replaced.
[0158] The total number of amino acid residues in the cCPP is in the range of from 6 to 20 amino acid residues, e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues, inclusive of all ranges and subranges therebetween. The cCPP can comprise 6 to 13 amino acid residues. The cCPP can comprise 6 to 10 amino acids. By way of example, cCPP comprising 6-10 amino acid residues can have a structure according to any of Formula I-A to I-E:
Figure imgf000046_0001
, wherein AAi, AA2, AA3, AA4, AAs, AAe, AA?, AAg, AAg, and AA10 are amino acid residues.
[0159] The cCPP can comprise 6 to 8 amino acids. The cCPP can comprise 8 amino acids.
[0160] Each amino acid in the cCPP may be a natural or non-natural amino acid. Abbreviations used herein for some natural and non-natural amino acids are shown in Table 2.
[0161 J As used herein, the term "amino acid" refers to compounds having an amino group and a carboxylic acid group. Most amino acids (except for glycine) also have a side chain. As used herein, “amino acid side chain” or "side chain" refers to the characterizing substituent bound to the a-carbon of the amino acid.
[0162] An “a-amino acid” i s an amino acid in which the amino group is attached to the first (alpha) carbon adjacent to the carboxylic acid group, such that the carbon atom of the carbonyl is separated from the nitrogen atom of the amino group by one carbon atom. A “b-amino acid” (also called “beta-amino acid,” and “P-amino acid”) is an analog of an a -amino acid in which the amino group is attached to the second (beta) carbon, rather than the alpha-carbon, such that the carbon atom of the carbonyl is separated from the nitrogen atom of the amino group by two carbon atoms. Examples of b-amino acids include but are not limited to b-alanine and b-homophenylalanine. [0163] An “uncharged” amino acid is an amino acid that does not have a charge at a physiological pH (between 5.0 and 8.0). It is noted that histidine can exist in neutral or positively charged forms at physiological pH.
[0164] A side chain that does not comprise an aryl or heteroaryl group, can be referred to herein as a “non-aiyl” side chain. In embodiments, the side chain that does not comprise an aryl or heteroaryl group can be uncharged and is referred to herein as an uncharged, non-aryl side chain. Amino acids with uncharged non-aryl amino side chains include, but are not limited to, histidine, threonine; serine; leucine; isoleucine, valine; neopentylglycine, alanine; homoalanine, homoserine; 3-(4-thiazolyl)-alanine; 3-(4-furanyl)-alanine; 3-(4-thienyl)-alanine; and b-amino acid derivatives thereof.
[0165] The term “non-natural amino acid” refers to an organic compound that is a congener of a natural amino acid in that it has a structure similar to a natural amino acid so that it mimics the structure and reactivity of a natural amino acid. The non-natural amino acid can be a modified amino acid, and/or amino acid analog, that is not one of the 20 common naturally occurring amino acids or the rare natural amino acids selenocysteine or pyrrolysine. Non-natural amino acids can also be a D-isomer of a natural amino acid. Examples of suitable amino acids include, but are not limited to, alanine, allosoleucine, arginine, citrulline, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, napthylalanine, phenylalanine, proline, pyroglutamic acid, serine, threonine, tryptophan, tyrosine, valine, a derivative thereof, or combinations thereof.
Table 2. Amino Acid Abbreviations
Figure imgf000047_0001
Figure imgf000048_0001
[0166] In embodiments, the cCPP can comprise 2 contiguous amino acids with hydrophobic side chains. In embodiments, the cCPP can comprise 3 contiguous amino acids with hydrophobic side chains. The hydrophobicity of amino acid residues can be measured and/or calculated using a variety of techniques. In embodiments, the hydrophobicity of an amino acid residue can be determined by calculating its consensus value on the consensus scale of D. Eisenberg et al., using the method described in D. Eisenberg et al., “Hydrophobic Moments and Protein Structure,” Faraday Symp. Chem. Soc. 1982, 17, 109-120 (e.g., D. Eisenberg et al.). A hydrophobic amino acid is an amino acid that has a hydrophobic side chain.
[0167] In embodiments, one or two amino acids in the cCPP can have no side chain. In embodiments, all amino acids in the cCPP have a side chain. As used herein, when no side chain is present, the amino acid has two hydrogen atoms on the carbon atom(s) (e.g., -CH?) linking the amine and carboxylic acid of the amino acid residue. The amino acid having no side chain can be glycine or beta-alanine.
[0168] The cCPP can comprise from 6 to 20, from 6 to 10, or from 6 to 8 amino acid residues, wherein: (i) at least two amino acids can, independently, be glycine, b-alanine, serine, histidine or 4-aminobutyric acid; (ii) at least two amino acids can have a side chain comprising an aryl or heteroaryl group; and (iii) at least two amino acid has a side chain comprising a guanidine group, or a protonated form thereof. In embodiments, (i) two amino acids can, independently, be glycine, b-alanine, serine, histidine or 4-aminobutyric acid; (ii) two or three amino acids can have a side chain comprising an aryl or heteroaryl group; and (iii) two amino acid has a side chain comprising a guanidine group, or a protonated form thereof.
[0169] In embodiments, one amino acid of the cCPP can be glycine, b-alanine, serine, histidine, or 4-aminobutyric acid. In embodiments, two amino acids can be, independently, glycine, b- alanine, serine, histidine, or 4-aminobutyric acid. In embodiments, three amino acids can be glycine, b-alanine, serine, histidine, or 4-aminobutyric acid.
[0170] In embodiments, one amino acid of the cCPP can have a side chain comprising an aryl or heteroaryl group. In embodiments, two amino acids of the cCPP can have a side chain comprising an aryl or heteroaryl group. In embodiments, three amino acids of the cCPP can have a side chain comprising an aryl or heteroaryl group.
[0171] In embodiments, one amino acid of the cCPP can have a side chain that does not comprise an aryl or heteroaryl group, referred to herein as a “non-aryl” side chain. In embodiments, the side chain that does not comprise an aryl or heteroaryl group can be uncharged and is referred to herein as an uncharged, non-aryl side chain. In embodiments, two amino acids of the CPP (e g., cCPP) can have an uncharged, non-aryl side chain. In embodiments, three amino acids of the CPP (e.g., cCPP) can have an uncharged, non-aryl side chain. Amino acids with uncharged non-aryl amino side chains include, but are not limited to, histidine; threonine; serine; leucine; isoleucine; valine; neopentylglycine; alanine; homoalanine; homoserine; 3-(4-thiazolyl)-alanine, 3-(4-furanyl)- alanine; and 3-(4-thienyl)-alanine.
[0172] In embodiments, one amino acid of the cCPP has a side chain comprising a guanidine group, or a protonated form thereof. In embodiments, two amino acids of the cCPP can have a side chain comprising a guanidine group, or a protonated form thereof. In embodiments, three amino acids of the cCPP can have a side chain comprising a guanidine group, or a protonated form thereof. In embodiments, four amino acids of the cCPP can have a side chain comprising a guanidine group, or a protonated form thereof.
[0173] In embodiments, the cCPP can comprise 4 to 20 amino acids, such as 6 to 20 amino acids, wherein: (i) at least one amino acid has a side chain comprising a guanidine group, or a protonated form thereof; (ii) at least one amino acid has no side chain or a side chain comprising
Figure imgf000050_0001
Figure imgf000050_0003
, or a protonated form thereof; and (iii) at least two amino acids independently have a side chain comprising an aryl or heteroaryl group.
[0174] In embodiments, at least two amino acids can have no side chain or a side chain comprising
Figure imgf000050_0002
, or a protonated form thereof. As used herein, when no side chain is present, the amino acid has two hydrogen atoms on the carbon atom(s) (e g., -CH2-) linking the amine and carboxylic acid.
[0175] In embodiments, the amino acid having no side chain can be glycine or P-alanine.
[0176] In embodiments, the cCPP can comprise from 6 to 20 amino acid residues, wherein: (i) at least two amino acids can be glycine, p-alanine, or 4-aminobutyric acid residues; (ii) at least two amino acids can have a side chain comprising an aryl or heteroaryl group; and (iii) at least two amino acids can have a side chain comprising a guanidine group,
Figure imgf000051_0001
Figure imgf000051_0002
, or a protonated form thereof.
[0177] In embodiments, the cCPP can comprise from 6 to 20 amino acid residues, wherein: (i) 2 amino acids can independently be glycine, P-alanine, or 4-aminobutyric acid residues; (ii) 2 or 3 amino acids can have a side chain comprising an aryl or heteroaryl group; and (iii) 2 amino acids have a side chain comprising a guanidine group,
Figure imgf000051_0003
Figure imgf000051_0004
, or a protonated form thereof.
[0178] In embodiments, the cCPP can comprise from 6 to 20 amino acid residues, wherein: (i) at least three amino acids can independently be glycine, P-alanine, or 4-aminobutyric acid residues; (ii) at least one amino acid can have a side chain comprising an aryl or heteroaryl group; and (iii) at least one amino acid can have a side chain comprising a guanidine group,
Figure imgf000051_0005
Figure imgf000051_0006
, or a protonated form thereof.
Glycine and Related Amino Acid Residues
[0179] In embodiments, the cCPP can comprise (i) 1, 2, 3, 4, 5, or 6 glycine, P-alanine, 4- aminobutyric acid residues, or combinations thereof. In embodiments, the cCPP can comprise (i) 2 glycine, P-alanine, 4-aminobutyric acid residues, or combinations thereof. In embodiments, the cCPP can comprise (i) 3 glycine, P-alanine, 4-aminobutyric acid residues, or combinations thereof. In embodiments, the cCPP can comprise (i) 4 glycine, p-alanine, 4-aminobutyric acid residues, or combinations thereof. In embodiments, the cCPP can comprise (i) 5 glycine, P-alanine, 4- aminobutyric acid residues, or combinations thereof. In embodiments, the cCPP can comprise (i) 6 glycine, P-alanine, 4-aminobutyric acid residues, or combinations thereof. In embodiments, the cCPP can comprise (i) 3, 4, or 5 glycine, P-alanine, 4-aminobutyric acid residues, or combinations thereof. In embodiments, the cCPP can comprise (i) 3 or 4 glycine, P-alanine, 4-aminobutyric acid residues, or combinations thereof.
[0180] In embodiments, the cCPP can comprise (i) 1, 2, 3, 4, 5, or 6 glycine residues. The cCPP can comprise (i) 2 glycine residues. In embodiments, the cCPP can comprise (i) 3 glycine residues. In embodiments, the cCPP can comprise (i) 4 glycine residues. In embodiments, the cCPP can comprise (i) 5 glycine residues. In embodiments, the cCPP can comprise (i) 6 glycine residues. In embodiments, the cCPP can comprise (i) 3, 4, or 5 glycine residues. In embodiments, the cCPP can comprise (i) 3 or 4 glycine residues. In embodiments, the cCPP can comprise (i) 2 or 3 glycine residues. In embodiments, the cCPP can comprise (i) 1 or 2 glycine residues.
[0181] In embodiments, the cCPP can comprise (i) 3, 4, 5, or 6 glycine, p-alanine, 4-aminobutyric acid residues, or combinations thereof. In embodiments, the cCPP can comprise (i) 3 glycine, p- alanine, 4-aminobutyric acid residues, or combinations thereof. In embodiments, the cCPP can comprise (i) 4 glycine, P-alanine, 4-aminobutyric acid residues, or combinations thereof. In embodiments, the cCPP can comprise (i) 5 glycine, P-alanine, 4-aminobutyric acid residues, or combinations thereof. In embodiments, the cCPP can comprise (i) 6 glycine, P-alanine, 4- aminobutyric acid residues, or combinations thereof. In embodiments, the cCPP can comprise (i) 3, 4, or 5 glycine, P-alanine, 4-aminobutyric acid residues, or combinations thereof. In embodiments, the cCPP can comprise (i) 3 or 4 glycine, P-alanine, 4-aminobutyric acid residues, or combinations thereof.
[0182] In embodiments, the cCPP can comprise at least three glycine residues. In embodiments, the cCPP can comprise (i) 3, 4, 5, or 6 glycine residues. In embodiments, the cCPP can comprise (i) 3 glycine residues. In embodiments, the cCPP can comprise (i) 4 glycine residues. In embodiments, the cCPP can comprise (i) 5 glycine residues. In embodiments, the cCPP can comprise (i) 6 glycine residues. In embodiments, the cCPP can comprise (i) 3, 4, or 5 glycine residues. The cCPP can comprise (i) 3 or 4 glycine residues.
[0183] In embodiments, none of the glycine, P-alanine, or 4-aminobutyric acid residues in the cCPP are contiguous. In embodiments, two or three glycine, P-alanine, 4-or aminobutyric acid residues can be contiguous. In embodiments, two glycine, P-alanine, or 4-aminobutyric acid residues can be contiguous.
[0184] In embodiments, none of the glycine residues in the cCPP are contiguous. For example, each glycine residues in the cCPP can be separated by an amino acid residue that is not glycine.
[0185] In embodiments, two or more of the glycine residues in the cCPP are contiguous. In embodiments, two or three glycine residues are contiguous. In embodiments, two glycine residues are contiguous
Amino Acid Side Chains with an Aryl or heteroaryl Group
[0186] In embodiments, the cCPP can comprise (ii) 2, 3, 4, 5 or 6 amino acid residues independently having a side chain comprising an aryl or heteroaryl group. In embodiments, the cCPP can comprise (ii) 2 amino acid residues independently having a side chain comprising an aryl or heteroaryl group. In embodiments, the cCPP can comprise (ii) 3 amino acid residues independently having a side chain comprising an aryl or heteroaryl group. In embodiments, the cCPP can comprise (ii) 2, 3, or 4 amino acid residues independently having a side chain comprising an aryl or heteroaryl group. In embodiments, the cCPP can comprise (ii) 2 or 3 amino acid residues independently having a side chain comprising an aryl or heteroaiyl group.
[0187] In embodiments, the cCPP can comprise (ii) 2, or 3amino acid residues independently having a side chain comprising an aryl group. In embodiments, the cCPP can comprise (ii) 2 amino acid residues independently having a side chain comprising an aryl group. In embodiments, the cCPP can comprise (ii) 3 amino acid residues independently having a side chain comprising an aryl group. In embodiments, the cCPP can comprise (ii) 2 or 3 amino acid residues independently having a side chain comprising an aryl group.
[0188] The aryl group can be a 6- to 14-membered aryl. Aryl can be phenyl, naphthyl or anthracenyl, each of which is optionally substituted. Aryl can be phenyl or naphthyl, each of which is optionally substituted. The heteroaryl group can be a 6- to 14-membered heteroaryl having 1, 2, or 3 heteroatoms selected from N, O, and S. Heteroaryl can be pyridyl, quinolyl, or isoquinolyl.
[0189] The amino acid residue having a side chain comprising an aryl or heteroaryl group can each independently be bis(homonaphthylalanine); homonaphthylalanine; naphthylalanine; phenylglycine; bis(homophenylalanine); homophenylalanine, phenylalanine, tryptophan, or tyrosine, each of which is optionally substituted with one or more substituents. The amino acid having a side chain comprising an aryl or heteroaryl group can each independently be selected from: naphthylalanine; homophenylalanine and phenylalanine.
[0190] The amino acid residue having a side chain comprising an aryl or heteroaryl group can each be independently a residue of phenylalanine, naphthylalanine, phenyl glycine, homophenylalanine, homonaphthylalanine, bis(homophenylalanine), bis-(homonaphthylalanine), tryptophan, or tyrosine, each of which is optionally substituted with one or more substituents. The amino acid residue having a side chain comprising an aryl group can each independently be a residue of tyrosine, phenylalanine; 1 -naphthylalanine, 2-naphthylalanine; tryptophan; phenylglycine; homophenylalanine; or P-homophenylalanine;. The amino acid residue having a side chain comprising an aryl group can each independently be a residue of phenylalanine, 2- naphthylalanine, homophenylalanine, P-homophenylalanine or homonaphthylalanine, each of which is optionally substituted with one or more substituents. The amino acid residue having a side chain comprising an aryl group can each be independently a residue of phenylalanine, 2- naphthylalanine, homophenylalanine, or P-homophenylalanine, each of which is optionally substituted with one or more substituents. The amino acid residue having a side chain comprising an aryl group can each be independently a residue of phenylalanine or 2-naphthylalanine, each of which is optionally substituted with one or more substituents. At least one amino acid residue having a side chain comprising an aryl group can be a residue of phenylalanine. Two amino acid residues having a side chain comprising an aryl group can be residues of phenylalanine. Each amino acid residue having a side chain comprising an aryl group can be a residue of phenylalanine.
[0191] In embodiments, none of the amino acids having the side chain comprising the aryl or heteroaryl group are contiguous. In embodiments, two amino acids having the side chain comprising the aryl or heteroaryl group can be contiguous. In embodiments, two contiguous amino acids can have opposite stereochemistry. In embodiments, the two contiguous amino acids can have the same stereochemistry. In embodiments, three amino acids having the side chain comprising the aryl or heteroaryl group can be contiguous. In embodiments, three contiguous amino acids can have the same stereochemistry. In embodiments, three contiguous amino acids can have alternating stereochemistry.
[0192] The amino acid residues comprising aryl or heteroaryl groups can be L-amino acids. The amino acid residues comprising aryl or heteroaryl groups can be D-amino acids. The amino acid residues comprising aryl or heteroaryl groups can be a mixture of D- and L-amino acids. [0193] The optional substituent can be any atom or group which does not significantly reduce (e.g., by more than 50%) the cytosolic delivery efficiency of the cCPP, e.g., compared to an otherwise identical sequence which does not have the substituent. The optional substituent can be a hydrophobic substituent or a hydrophilic substituent. The optional substituent can be a hydrophobic substituent. The substituent can increase the solvent-accessible surface area (as defined herein) of the hydrophobic amino acid. The substituent can be halogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, acyl, alkylcarbamoyl, alkylcarboxamidyl, alkoxycarbonyl, alkylthio, or arylthio. The substituent can be halogen.
Amino Acid Residues Beninga Side Chain Comprising a Guanidine Group, Guanidine Replacement Group, or Protonated Form Thereof
[0194] As used herein, guanidine refers to the structure:
Figure imgf000055_0001
[0195] As used herein, a protonated form of guanidine refers to the structure:
Figure imgf000055_0002
[0196] Guanidine replacement groups refer to functional groups on the side chain of amino acids that will be positively charged at or above physiological pH or those that can recapitulate the hydrogen bond donating and accepting activity of guanidinium groups.
[0197] The guanidine replacement groups may facilitate cell penetration and delivery of a therapeutic agent while reducing toxicity associated with guanidine groups or protonated forms thereof. The cCPP can comprise at least one amino acid having a side chain comprising a guanidine or guanidinium replacement group. The cCPP can comprise two amino acids having a side chain comprising a guanidine or guanidinium replacement group. The cCPP can comprise three amino acids having a side chain comprising a guanidine or guanidinium replacement group. The cCPP can comprise four amino acids having a side chain comprising a guanidine or guanidinium replacement group. The cCPP can comprise five amino acids having a side chain comprising a guanidine or guanidinium replacement group. The cCPP can comprise six amino acids having a side chain comprising a guanidine or guanidinium replacement group.
[0198] The guanidine or guanidinium group can be an isostere of guanidine or guanidinium. The guanidine or guanidinium replacement group can be less basic than guanidine.
H
[0199] As used herein, a guanidine replacement group refers to
Figure imgf000056_0001
Figure imgf000056_0002
, or a protonated form thereof.
[0200] In embodiments, a cCPP comprising from 6 to 20 amino acids residues is provided, wherein: (i) at least two amino acids have a side chain comprising a guanidine group, or a protonated form thereof; (ii) at least two amino acids have no side chain or a side chain comprising
Figure imgf000056_0003
, or a protonated form thereof; and (iii) at least two amino acids residues independently have a side chain comprising an aryl or heteroaryl group.
[0201] In embodiments, at least two amino acids residues can have no side chain or a side chain comprising
Figure imgf000056_0006
protonated form thereof. As used herein, when no side chain is present, the amino acid residue have two hydrogen atoms on the carbon atom(s) (e g., -CH2-) linking the amine and carboxylic acid. Glycine or b-alanine are examples of amino acids with no side chain.
[0202] In embodiments, the cCPP can comprise at least one amino acid having a side chain comprising one of the following moieties:
Figure imgf000056_0004
Figure imgf000056_0005
, or a protonated form thereof. [0203] In embodiments, the cCPP can comprise at least two amino acids each independently having one of the following moieties
Figure imgf000057_0011
, or a protonated form thereof. In embodiments, at least two amino acids can
Figure imgf000057_0002
have a side chain comprising the same moiety selected from:
Figure imgf000057_0003
Figure imgf000057_0004
, or a protonated form thereof. In embodiments, at least one amino acid can have a side chain comprising
Figure imgf000057_0005
or a protonated form thereof
In embodiments, at least two amino acids can have a side chain comprising
Figure imgf000057_0001
, or a protonated form thereof. In embodiments, one, two, three, or four amino acids can have a side chain comprising , or a protonated form thereof. In embodiments, one amino acid can
Figure imgf000057_0006
have a side chain comprising , or a protonated form thereof. In embodiments, two
Figure imgf000057_0007
amino acids can have a side chain comprising
Figure imgf000057_0008
, or a protonated form thereof.
Figure imgf000057_0010
, or a protonated form thereof, can be attached to the terminus of the amino acid side chain,
Figure imgf000057_0009
can be attached to the terminus of the amino acid side chain. [0204] In embodiments, the cCPP can comprise (iii) 2, 3, 4, 5 or 6 amino acid residues independently having a side chain comprising a guanidine group, guanidine replacement group, or a protonated form thereof. In embodiments, the cCPP can comprise (iii) 2 amino acid residues independently having a side chain comprising a guanidine group, guanidine replacement group, or a protonated form thereof. In embodiments, the cCPP can comprise (iii) 3 amino acid residues independently having a side chain comprising a guanidine group, guanidine replacement group, or a protonated form thereof. In embodiments, the cCPP can comprise (iii) 4 amino acid residues independently having a side chain comprising a guanidine group, guanidine replacement group, or a protonated form thereof. In embodiments, the cCPP can comprise (iii) 5 amino acid residues independently having a side chain comprising a guanidine group, guanidine replacement group, or a protonated form thereof. In embodiments, the cCPP can comprise (iii) 6 amino acid residues independently having a side chain comprising a guanidine group, guanidine replacement group, or a protonated form thereof. In embodiments, the cCPP can comprise (iii) 2, 3, 4, or 5 amino acid residues independently having a side chain comprising a guanidine group, guanidine replacement group, or a protonated form thereof. In embodiments, the cCPP can comprise (iii) 2, 3, or 4 amino acid residues independently having a side chain comprising a guanidine group, guanidine replacement group, or a protonated form thereof. In embodiments, the cCPP can comprise (iii) 2 or 3 amino acid residues independently having a side chain comprising a guanidine group, guanidine replacement group, or a protonated form thereof.
[0205] In embodiments, the amino acid residues can independently have a side chain comprising the guanidine group, guanidine replacement group, or the protonated form thereof that are not contiguous. In embodiments, two amino acid residues can independently have the side chain comprising the guanidine group, guanidine replacement group, or the protonated form thereof can be contiguous. In embodiments, three amino acid residues can independently have the side chain comprising the guanidine group, guanidine replacement group, or the protonated form thereof can be contiguous. In embodiments, four amino acid residues can independently have the side chain comprising the guanidine group, guanidine replacement group, or the protonated form thereof can be contiguous. In embodiments, the contiguous amino acid residues can have the same stereochemistry. In embodiments, the contiguous amino acids can have alternating stereochemistry. [0206] In embodiments, the amino acid residues independently having the side chain comprising the guanidine group, guanidine replacement group, or the protonated form thereof, can be L-amino acids. In embodiments, the amino acid residues independently having the side chain comprising the guanidine group, guanidine replacement group, or the protonated form thereof, can be D-amino acids. In embodiments, the amino acid residues independently having the side chain comprising the guanidine group, guanidine replacement group, or the protonated form thereof, can be a mixture of L- or D-amino acids.
[0207] In embodiments, each amino acid residue having the side chain comprising the guanidine group, or the protonated form thereof, can independently be a residue of arginine, homoarginine, 2-amino-3 -propionic acid, 2-amino-4-guanidinobutyric acid or a protonated form thereof. In embodiments, each amino acid residue having the side chain comprising the guanidine group, or the protonated form thereof, can independently be a residue of arginine or a protonated form thereof.
[0208] In embodiments, each amino acid having the side chain comprising a guanidine replacement group, or protonated form thereof, can independently be
Figure imgf000059_0002
Figure imgf000059_0001
or a protonated form thereof. Those skilled in the art will appreciate that the N- and/or C-termini of the above non-natural aromatic hydrophobic amino acids, upon incorporation into the peptides, form amide bonds.
[0209] In embodiments, the cCPP can comprise a first amino acid having a side chain comprising an aryl or heteroaryl group and a second amino acid having a side chain comprising an aryl or heteroaryl group, wherein an N-terminus of a first glycine forms a peptide bond with the first amino acid having the side chain comprising the aryl or heteroaryl group, and a C-terminus of the first glycine forms a peptide bond with the second amino acid having the side chain comprising the aryl or heteroaryl group. Although by convention, the term “first amino acid” often refers to the N-terminal amino acid of a peptide sequence, as used herein “first amino acid” is used to distinguish the referent amino acid from another amino acid (e.g., a “second amino acid”) in the cCPP such that the term “first amino acid” may or may not refer to an amino acid located at the N-terminus of the peptide sequence. [0210] In embodiments, the cCPP can comprise an N-terminus of a second glycine forms a peptide bond with an amino acid having a side chain comprising an aryl or heteroaryl group, and a C- terminus of the second glycine forms a peptide bond with an amino acid having a side chain comprising a guanidine group, or a protonated form thereof.
[0211] In embodiments, the cCPP can comprise a first amino acid having a side chain comprising a guanidine group, or a protonated form thereof, and a second amino acid having a side chain comprising a guanidine group, or a protonated form thereof, wherein an N-terminus of a third glycine forms a peptide bond with a first amino acid having a side chain comprising a guanidine group, or a protonated form thereof, and a C-terminus of the third glycine forms a peptide bond with a second amino acid having a side chain comprising a guanidine group, or a protonated form thereof.
[0212] In embodiments, the cCPP can comprise a residue of asparagine, aspartic acid, glutamine, glutamic acid, or homoglutamine. In embodiments, the cCPP can comprise a residue of asparagine. In embodiments, the cCPP can comprise a residue of glutamine.
[0213] In embodiments, the cCPP can comprise a residue of tyrosine; phenylalanine; 1- naphthylalanine, 2-naphthylalanine; tryptophan; homophenylalanine; or p-homophenylalanine.
[0214] While not wishing to be bound by theory, it is believed that the chirality of the amino acids in the cCPPs may impact cytosolic uptake efficiency. In embodiments, the cCPP can comprise at least one D amino acid. In embodiments, the cCPP can comprise one to fifteen D amino acids. In embodiments, the cCPP can comprise one to ten D amino acids. In embodiments, the cCPP can comprise 1, 2, 3, 4, 5, 6, 7 or 8 D amino acids. In embodiments, the cCPP can comprise at least one L amino acid. In embodiments, the cCPP can comprise one to fifteen L amino acids. In embodiments, the cCPP can comprise one to ten L amino acids. In embodiments, the cCPP can comprise 1, 2, 3, 4, 5, 6, 7 or 8 L amino acids. In embodiments, the cCPP can comprise 2, 3, 4, 5, 6, 7, or 8 contiguous amino acids having alternating D and L chirality. In embodiments, the cCPP can comprise three contiguous amino acids having the same chirality. In embodiments, the cCPP can comprise two contiguous amino acids having the same chirality. In embodiments, at least two of the amino acids can have the opposite chirality. In embodiments, the at least two amino acids having the opposite chirality can be adjacent to each other. In embodiments, at least three amino acids can have alternating stereochemistry relative to each other. In embodiments, the at least three amino acids having the alternating chirality relative to each other can be adjacent to each other. In embodiments, at least four amino acids have alternating stereochemistry relative to each other. In embodiments, the at least four amino acids having the alternating chirality relative to each other can be adjacent to each other. In embodiments, at least two of the amino acids can have the same chirality. In embodiments, at least two amino acids having the same chirality can be adjacent to each other. In embodiments, at least two amino acids have the same chirality and at least two amino acids have the opposite chirality. In embodiments, the at least two amino acids having the opposite chirality can be adjacent to the at least two amino acids having the same chirality. Accordingly, adjacent amino acids in the cCPP can have any of the following sequences: D-L; L- D; D-L-L-D; L-D-D-L; L-D-L-L-D; D-L-D-D-L; D-L-L-D-L; or L-D-D-L-D. In embodiments, the amino acid residues that form the cCPP can all be L-amino acids. In embodiments, the amino acid residues that form the cCPP can all be D-amino acids.
[0215] In embodiments, at least two of the amino acids can have a different chirality. In embodiments, at least two amino acids having a different chirality can be adjacent to each other. In embodiments, at least three amino acids can have different chirality relative to an adjacent amino acid. In embodiments, at least four amino acids can have different chirality relative to an adjacent amino acid. In embodiments, at least two amino acids have the same chirality and at least two amino acids have a different chirality. In embodiments, one or more amino acid residues that form the cCPP can be achiral. In embodiments, the cCPP can comprise a motif of 3, 4, or 5 amino acids, wherein two amino acids having the same chirality can be separated by an achiral amino acid. The cCPPs can comprise the following sequences: D/L-X-D/L; D/L-X-D/L-X; D/L-X-D/L-X-D/L; D- X-D; D-X-D-X; D-X-D-X-D; L-X-L; L-X-L-X; or L-X-L-X-L, wherein DZL indicates that the amino acid can be a D or an L amino acid and X is an achiral amino acid. The achiral amino acid can be glycine.
[0216] In embodiments, an amino acid having a side chain comprising:
Figure imgf000061_0001
protonated form thereof, can be adjacent to an amino acid having a side chain comprising an aryl or
H heteroaryl group. In embodiments, an amino acid having a side chain comprising:
Figure imgf000061_0002
Figure imgf000062_0001
, or a protonated form thereof, can be adjacent to at least one amino acid having a side chain comprising a guanidine or protonated form thereof. In embodiments, an amino acid having a side chain comprising a guanidine or protonated form thereof can be adjacent to an amino acid having a side chain comprising an aryl or heteroaryl group. In embodiments, two amino acids having a side chain comprising:
Figure imgf000062_0002
protonated forms there, can be adjacent to each other. In embodiments, two amino acids having a side chain comprising a guanidine or protonated form thereof are adjacent to each other. In embodiments, a cCPP can comprise at least two contiguous amino acids having a side chain can comprise an aryl or heteroaryl group and at least two non-adjacent amino acids having a side chain comprising:
Figure imgf000062_0004
Figure imgf000062_0003
or a protonated form thereof. In embodiments, a cCPP can comprise at least two contiguous amino acids having a side chain comprising an aryl or heteroaryl group and at least two non-adjacent amino acids having a side chain comprising , or a protonated form
Figure imgf000062_0005
thereof. In embodiments, the adjacent amino acids can have the same chirality. In embodiments, the adjacent amino acids can have the opposite chirality. Other combinations of amino acids can have any arrangement of D and L amino acids, e.g., any of the sequences described in the preceding paragraph.
[0217] In embodiments, at least two amino acids having a side chain comprising:
Figure imgf000062_0006
protonated form thereof, are alternating with at least two amino acids having a side chain comprising a guanidine group or protonated form thereof. [0218] In embodiments, the cCPP can comprise the structure of Formula (A):
Formula (A)
Figure imgf000063_0001
, or a protonated form thereof, wherein:
Ri, Ra, Rs, R», RS, Re, and R7 are independently H or an amino acid side chain;
AAsc is an amino acid side chain; and q is 1, 2, 3 or 4.
[0219] The cCPP of the general Formula (A) can have any configuration and/or amino acid side chain as described in the published PCT application NO. US2020/066459 (WO2021127650A1) or US Patent No. 11,225,506.
[0220] In embodiments, AAsc can be
Figure imgf000063_0002
, wherein t can be an integer from
0 to 5. AAsc can be , wherein t can be 0 or an integer from 1 to 5. t can be 1 to 5. T is
Figure imgf000063_0003
2 or 3. t can be 2. t can be 3. In embodiments, AAsc can be conjugated to a linker.
[0221] In embodiments, the cCPP are of the general Formula (A) or a protonated form thereof, wherein:
Ri, R2, and Rs are each independently H or an aryl or heteroaiyl side chain of an amino acid; at least two of Ri, Ri, and Rs is an aryl or heteroaryl side chain of an amino acid;
R4, RS, Re, R7 are independently H or an amino acid side chain; at least two of Ri, Rs, Re, R7 is H or a side chain of 3-guanidino-2-aminopropionic acid, 4-guanidino-2-aminobutanoic acid, arginine, homoarginine, N-methylarginine, N,N- dimethylarginine, 2,3 -diaminopropionic acid, 2,4-diaminobutanoic acid, lysine, N-methyllysine, N,N-dimethyllysine, N-ethyllysine, N,N,N-trimethyllysine, 4-guanidinophenylalanine, citrulline, serine, histidine, N,N-dimethyllysine, 0-homoarginine, 3-(l-piperidinyl)alanine;
AAsc is an amino acid side chain; and q is 1, 2, 3 or 4.
[0222] In embodiments the cCPP is of Formula (A), where at least one of R*, Rs, Re, R7 are independently an uncharged, non-aromatic side chain of an amino acid. In embodiments, at least two of Ri, Rs, Re, R7 are, independently, H or a side chain of serine, histidine, or citrulline.
[0223] In embodiments, compounds are provided that include a cyclic peptide having from 6 to 12 amino acids, wherein at least two amino acids of the cyclic peptide are charged amino acids; at least two amino acids of the cyclic peptide are aromatic hydrophobic amino acids; and at least two amino acids of the cyclic peptide are uncharged, non-aromatic amino acids. In embodiments, at least two charged amino acids of the cyclic peptide are arginine. In embodiments, at least two aromatic, hydrophobic amino acids of the cyclic peptide are phenylalanine, naphtha alanine (3- Naphth-2-yl-alanine), or a combination thereof. In embodiments, at least two uncharged, non- aromatic amino acids of the cyclic peptide are citrulline, glycine or a combination thereof. In embodiments, the compound is a cyclic peptide having from 6 to 12 amino acids wherein two amino acids of the cyclic peptide are arginine; at least two amino acids are aromatic, hydrophobic amino acids selected from phenylalanine, naphtha alanine, homophenylalanine and combinations thereof; and at least two amino acids are uncharged, non-aromatic amino acids selected from citrulline, serine, histidine, glycine, and combinations thereof. In embodiments, the compound is a cyclic peptide having from 6 to 12 amino acids wherein two amino acids of the cyclic peptide are arginine; two or three amino acids are, independently, aromatic, hydrophobic amino acids selected from phenylalanine, 2 -naphthylalanine, 0-homophenylalanine; and two amino acids are independently, uncharged, non-aromatic amino acids selected from citrulline, serine, histidine, and glycine.
[0224] The cCPP can comprise the structure of Formula (I):
Figure imgf000065_0001
or a protonated form thereof, wherein:
Ri. Rz, and Rs can each independently be H or an amino acid residue having a side chain comprising an aryl or heteroaryl group; at least two of Ri, Rz, and Rs is an aryl or heteroaiyl side chain of an amino acid;
R* and R6 are independently H or an amino acid side chain;
AAsc is an amino acid side chain; q is 1, 2, 3 or 4; and each m is independently an integer 0, 1, 2, or 3.
[0225] A cCPP of Formula (A) may be of Formula (I)
[0226] In embodiments, the cCPP are of Formula (I) or (A), where Ri, Rz, and R3 can each independently be H; -alkylene-ary 1; or -alkylene -heteroaryl. Ri, Rz, and Rs can each independently be H, -Ci-3alkylene-aryl or -Cisalkylene-heteroaryl. Ri, Rz, and Rs can each independently be H or -alkylene-ary] . Ri, Rz, and Rs can each independently be H or -Ci-salkylene-aryl. Ci-salkylene can be methylene. Aryl can be a 6- to 14-membered aryl. Heteroaryl can be a 6- to 14-membered heteroaryl having one or more heteroatoms selected from N, O, and S. Aryl can be selected from phenyl, naphthyl, or anthracenyl. Aryl can be phenyl or naphthyl. Aryl can be phenyl. Heteroaiyl can be pyridyl, quinolyl, and isoquinolyl. Ri, Rz, and Rs can each independently be H, -Ci- salkylene-Ph or -C1-a3lkylene-Naphthyl. Ri, Rz, and Rs can each independently be H, -CHzPh, or -CHzNaphthyl. Ri, R2, and Rs can each independently be H or -CHzPh. [0227] In embodiments, the cCPP are of Formula (I) or (A), where Ri, R2, and R3 can each independently be the side chain of tyrosine, phenylalanine; 1 -naphthylalanine; 2-naphthylalanine; tryptophan; 3-benzothienylalanine, 4-phenylphenylalanine; 3,4-difluorophenylalanine; 4- trifluoromethylphenylalanine; 2,3,4,5,6-pentafluorophenylalanine; homophenylalanine; P- homophenylalanine; 4-tert-butyl-phenylalanine; 4-pyridinylalanine; 3-pyridinylalanine; 4- methylphenylalanine; 4-fluorophenylalanine; 4-chlorophenylalanine; or 3-(9-anthryl)-alanine.
[0228] In embodiments, the cCPP are of Formula (I) or (A), where Ri can be the side chain of tyrosine. Ri can be the side chain of phenylalanine. Ri can be the side chain of 1 -naphthylalanine. Ri can be the side chain of 2-naphthylalanine. Ri can be the side chain of tryptophan. Ri can be the side chain of homophenylalanine. Ri can be H.
[0229] In embodiments, the cCPP are of Formula (I) or (A), where Ra can be the side chain of tyrosine. Ra can be the side chain of phenylalanine. Ra can be the side chain of 1 -naphthylalanine. Ra can be the side chain of 2-naphthylalanine. Ra can be the side chain of tryptophan. Ra can be the side chain of homophenylalanine. Ra can be H.
[0230] In embodiments, the cCPP are of Formula (I) or (A), where R7 can be the side chain of tyrosine. Rj can be the side chain of phenylalanine. Rs can be the side chain of 1 -naphthylalanine. Rs can be the side chain of 2-naphthylalanine. Ra can be the side chain of tryptophan. Ra can be the side chain of homophenylalanine. Ra can be H.
[0231] In embodiments, the cCPP are of Formula (J) or (A), where R4 can be H, or a side chain of arginine, citrulline, serine or histidine. Rt can be H. R4 can be a side chain of arginine. R4 can be a side chain of citrulline. R4 can be a side chain of serine. R4 can be a side chain of histidine.
[0232] In embodiments, the cCPP are of Formula (A), where Rs can be H, or a side chain of arginine, citrulline, serine or histidine. Rs can be H. Rs can be a side chain of arginine. Rs can be a side chain of citrulline. Rs can be a side chain of serine. Rs can be a side chain of histidine.
[0233] In embodiments, the cCPP are of Formula (I) or (A), where Re can be H, or a side chain of arginine, citrulline, serine or histidine. Re can be H. Re can be a side chain of arginine. Re can be a side chain of citrulline. Re can be a side chain of serine. Re can be a side chain of histidine.
[0234] In embodiments, the cCPP are of Formula (A), where R7 can be H, or a side chain of arginine, citrulline, serine or histidine. R7 can be H. R7 can be a side chain of arginine. R7 can be a side chain of citrulline. R7 can be a side chain of serine. R7 can be a side chain of histidine. [0235] In embodiments, the cCPP are of Formula (I) or (A), where one, two, or three of Ri, R2, R3, R4, Rs, Re, and R7 can be H. One of Ri, R2, Rs, R4, Rs, Re, and R7 can be H. Two of Ri, R2, Rs, R4, Rs, Re, and R7 can be H. Three of Ri, R2, Rs, Rs, Re, and R7 can be H. At least one of Ri, R2, Rs, R4, Rs, Re, and R7 can be H.
[0236] In embodiments, the cCPP are of Formula (I) or (A), where one of Ri, R2, or Rs, is H.
[0237] In embodiments, the cCPP are of Formula (I) or (A), where at least one of R», Rs, Re, and R7 can be H or a side chain of arginine, citrulline, serine, or histidine. At least one of R4, Rs, Re, and R7 is H. At least one of R4, Rs, Re, and R7 can be side chain of arginine. At least one of R4, Rs, Re, and R7 can be side chain of citrulline. At least one of R4. Rs, Re, and R7 can be side chain of serine. At least one of R4, Rs, Re, and R7can be side chain of histidine. One of R4, Rs, Re, and R7 can be H or a side chain of arginine, citrulline, serine, or histidine. One of R4, Rs, Re, and R7 is H. One of R4, Rs, Re, and R7 can be side chain of arginine. One of R4, Rs, Re, and R7 can be side chain of citrulline. One of R4, Rs, Re, and R7 can be side chain of serine. One of R4, Rs, Re, and R7 can be side chain of histidine.
[0238] In embodiments, the cCPP are of Formula (I) or (A), where two of R4, Rs, Re, and R7 can be H or a side chain of arginine, citrulline, serine, or histidine. Two of Rt, Rs, Re, and R7 can be H. Two of R4, Rs, Re, and R7 can be side chain of arginine. Two of R4, Rs, Re, and R7 can be side chain of citrulline. Two of R-4, Rs, Re, and R7 can be side chain of serine. Two of R», Rs, Re, and R7 can be side chain of histidine.
[0239] In embodiments, the cCPP are of Formula (A), where three of Rt, Rs, Re, and R7 can be H or a side chain arginine, citrulline, serine or histidine. Three of Ri, Rs, Re, and R7 can be H. Three of R4, Rs, Re, and R7 can be side chain of arginine. Three of R4, Rs, Re, and R7 can be side chain of citrulline. Three of R4, Rs, Re, and R7 can be side chain of serine. Three of R4, Rs, Re, and R7 can be side chain of histidine.
[0240] In embodiments, the cCPP are of Formula (A), where AAsc can be a side chain of a residue of asparagine, glutamine, or homoglutamine. AAsc can be a side chain of a residue of glutamine. The cCPP can further comprise a linker conjugated to the AAsc, e g., the residue of asparagine, glutamine, or homoglutamine. Hence, the cCPP can further comprise a linker conjugated to the asparagine, glutamine, or homoglutamine residue. The cCPP can further comprise a linker conjugated to the glutamine residue. [0241] In embodiments, the cCPP are of Formula (A), where q can be 1, 2, or 3. q can 1 or 2. q can be 1. q can be 2. q can be 3. q can be 4.
[0242] In embodiments, the cCPP are of Formula (A), where m can be an integer from 1 to 3. m can be 1 or 2. m can be 0. m can be 1. m can be 2. m can be 3.
[0243] The cCPP of Formula (A) or (I) can comprise the structure of Formula (I-a) or Formula (I- b):
Formula (I-a):
Figure imgf000068_0001
or protonated form thereof, wherein AAsc, Ri, R2, R7, R4, andm are as defined herein relative to Formula (A) and/or Formula (I).
[0244] The cCPP of Formula (A) or (I) can comprise the structures of Formulae (1-1), (1-2), (1-3), (1-4), (1-5), (1-6) or (1-7): Formulae (7-1), (1-2), (1-3), (7-4), (1-5), (1-6) or (1-7)
Figure imgf000069_0001
protonated form thereof, wherein AAsc and m are as defined herein relative to Formula (A) and/or Formula (F).
[0245] The cCPP can comprise one of the following sequences: FfΦRrRr, FGFGRGR; GfFGrGr, FfфGRGR; FfFGRGR; FfфGrGr; FGFGRRR; or FGFRRRR. The cCPP can have one of the following sequences: FfΦDRrRrQ, FGFGRGRQ; GfFGrGrQ, FfΦ GRGRQ; FfFGRGRQ; FfΦGrGrQ; FGFGRRRQ; orFGFRRRRQ.
[0246] The cCPP of Formula (A) or Formula (I) can have the structure of Formula (I-c):
Formula (I-c):
Figure imgf000070_0001
wherein R4, R6, q, m and AAscare as defined herein.
[0247] The cCPP of Formula (IA) or Formula (I) can have the structure of Formula (I-d):
Formula (1-d)
Figure imgf000071_0001
protonated form thereof, wherein R4, Rs, q, m, and AAscare as defined herein.
[0248] The cCPP of Formula (A) or Formula (I) can have the structure of Formula (I-e):
Formula (I-e):
Figure imgf000071_0002
, or a protonated form thereof,
[0249] wherein; Ri, R4, Re, q, m, and AAscare as defined herein. In embodiments, Ra is H. The cCPP of Formula (A) can be selected from :
Figure imgf000071_0003
[0250] The cCPP of Formula (A) can be selected from:
Figure imgf000072_0003
[0251] The cCPP of Formula (A) can be selected from:
Figure imgf000072_0002
[0252]
[0253] In embodiments, the cCPP is selected from:
Figure imgf000072_0001
Where <I> = L-naphthylalanine; <|) = D-naphthylalanine; 62 = L-norleucine [0254] In embodiments, the cCPP can be of the Formula (II)
Formula (II):
Figure imgf000073_0001
wherein:
R1, R2, and R3 can each independently be H or an amino acid residue having a side chain comprising an aryl or heteroaryl group; at least two of Ri, R7, and R3 is an aryl or heteroaryl side chain of an amino acid;
R4, R5, R6, R7 are independently H or an amino acid side chain; at least two of R4, Rs, Re, R7 are independently a side chain of arginine,
AAsc is an amino acid side chain; nx is 0 or 1; and q is 1, 2, 3 or 4.
[0255] In embodiments, the cCPP can be of the Formula (HI)
Formula (111):
Figure imgf000074_0001
wherein:
Ri, R2, and R3 can each independently be H or an amino acid residue having a side chain comprising an aryl or heteroaryl group; at least two of R1, R2, and R3 is an aryl or heteroaryl side chain of an amino acid;
R4, and R6 are independently H or an amino acid side chain;
AAscis an amino acid side chain; nx is 0 or 1; each m is independently an integer 0, 1, 2, or 3; and q is 1, 2, 3 or 4.
[0256] In embodiments, the cCPP is of Formula (II) or (HI) where at least one of Ri, Rz, or R3 are, independently, H. In embodiments, the cCPP is of Formula (II) or Formula (III) where at least one of Ri, Rz, and R3 are, independently, an amino acid residue having a side chain comprising an aryl or heteroaryl group. In embodiments, the amino acid residue having a side chain comprising an aryl or heteroaryl group is phenylalanine, beta homophenylalanine, or 3-(2-naphthyl)-alanine. In embodiments, the cCPP is of Formula (II) where at least two of R4, Re are each independently an amino acid residue having a side chain comprising a charged group. In embodiments, the amino acid residue having a side chain comprising a charged group is arginine. In embodiments, the cCPP is of Formula (II) or (HI) where q is 1.
[0257] In embodiments, the cCPP is of Formula (II) where nx is 1 and where Rs and R7 are a side chain of arginine. In embodiments, the cCPP is of Formula (II) where nx is 1, wherein the aryl or heteroaryl group is phenylalanine, beta homophenylalanine, or 3-(2-naphthyl)-alanine, and where at least two of RA, RS, Re and R7 are the side chain of arginine. In embodiments, the cCPP is of Formula (II) where nx is 1 , where Ri, Rs, Re and R7 are the side chain of arginine. In embodiments, the cCPP is of Formula (II) where nx is 1, where Rs and R7 are the side chain of arginine, and RA and Re are H.In embodiments, the cCPP is of Formula (II) or (HI) where at least one of RA, RS (if present), Re, or R7 (if present) are H or the amino acid side chain of serine or histidine. In embodiments the cCPP is of Formula (II) or (HI) where at least two of RA, RS (if present), Re, or R7 (if present) are, independently, H or the amino acid side chain of serine or histidine. In embodiments the cCPP is of Formula (II) where at least three of RA, RS, Re, or R7 are, independently, H or the amino acid side chain of serine or histidine. In embodiments the cCPP is of Formula (II) where at least four of RA, RS, Re, or R7 are, independently, H or the amino acid side chain of serine or histidine.
[0258] In embodiments of the cCPP of Formula (II): at least two of Ri, R2, and Rs are independently a side chain of phenylalanine, betahomophenylalanine, or naphthylalanine; at least two of RA, Rs, Re, or R7 are independently a side chain of arginine, at least two of RA, RS, Re, or R7 are independently H or a side chain of arginine, serine or histidine; AAsc is an amino acid side chain; n.K is 0 or 1; and q is 1. It is understood that nx is 1 when Ri is a side chain of betahomophenylalanine.
[0259] In embodiments of the cCPP of Formula (III): at least two of Ri, R2, and Rs are independently a side chain of phenylalanine, or naphthylalanine; RA and Re are each independently H or a side chain of arginine, serine or histidine; AAsc is an amino acid side chain; nx is 0 or 1; and q is 1.
[0260] In embodiments, the cCPP is of Formula (II) or (III), where two of RA, RS (if present), Re, or R7 (if present) are independently a side chain of serine. In embodiments, the cCPP is of Formula (II) or (HI), where two of RA, RS (if present), Re, or R7 (if present) are independently a side chain of histidine. In embodiments, the cCPP is of Formula (II) or (III), where two of RA, RS (if present), Re, or R7 (if present) are independently, H. [0261] In embodiments, the CPP is of Formula (II) wherein: at least two of Ri, R2, and Ri are independently a side chain of phenylalanine, betahomophenylaline, or naphthylalanine; at least two of R4, Rs, Rd, or R7 are independently a side chain of arginine; at least two of R4, Rs, Rs, or R7 are independently H or a side chain of an uncharged non-aryl amino acid selected from histidine, threonine, serine, leucine, isoleucine, valine, neopentylglycine, alanine, homoalanine, homoserine, 3-(4-thiazolyl)-alanine, 3-(4-furanyl)-alanine, and 3-(4-thienyl)-alanine; AAsc is an amino acid side chain; nx is 0 or 1; and q is 1. It is understood that nx is 1 when Ri is a side chain of betahomophenylalanine.
[0262] In embodiments, the CPP is of Formula (III) wherein: at least two of Ri, R2, and Ri are independently a side chain of phenylalanine, naphthylalanine, orbetahomophenylanine; R4 and R7 are independently H or a side chain of an uncharged non-aryl amino acid selected from histidine, threonine, serine, leucine, isoleucine, valine, neopentylglycine, alanine, homoalanine, homoserine, 3-(4-thiazolyl)-alanine, 3-(4-furanyl)-alanine, and 3-(4-thienyl)-alanine; AAsc is an amino acid side chain; nx is 0 or 1; and q is 1. It is understood that nx is 1 when Ri is a side chain of betahomophenylal anine.
[0263] In embodiments, the CPP is of Formula (II) wherein: at least two of Ri, R2, and Ra are independently a side chain of phenylalanine, betahomophenylalanine, or naphthylalanine; at least two of R4, Rs, Rs, or R7 are independently a side chain of arginine; at least two of R4, Rs, Rs, or R7 are independently a side chain of serine or histidine; AAsc is an amino acid side chain, nx is 0 or 1; and q is 1. It is understood that nx is 1 when Ri is a side chain of betahomophenylalanine.
[0264] In embodiments, the CPP is of Formula (DI) wherein, at least two of Ri, R2, and Ra are independently a side chain of phenylalanine, betahomophenylaline, or naphthylalanine; Rt and Rs are independently a side chain of serine or histidine; AAsc is an amino acid side chain; nx is 0 or 1; and q is 1. It is understood that nx is 1 when Ri is a side chain of betahomophenylalanine.
[0265] In embodiments, the CPP is of the general Formula (II) or (DI), wherein at least one of Ri, R2, or Ri is H. In embodiments, the CPP is of the general Formula (II) or (III), wherein at least one of Ri, R2, or Rais a side chain of phenylalanine. In embodiments, the CPP is of the general Formula (II) or (III), wherein at least two of Ri, R2, or Raare a side chain of phenylalanine. In embodiments, the CPP is of the general Formula (D) or (ID), wherein at least one of Ri, R2, or Ra is a side chain of naphthylalanine. [0266] In embodiments, the CPP is of the general Formula (II), wherein at least two of R4, Rs, Re, or R7 are independently a side chain of serine or histidine. In embodiments, the CPP is of the general Formula (III), wherein R4, and Re are independently H, or a side chain of serine or histidine. In embodiments, the CPP is of the general Formula (III), wherein R4, and Re are the side chain of serine. In embodiments, the CPP is of the general Formula (IH), wherein R4, and Re are the side chain of histidine. In embodiments, the CPP is of the general Formula (III), wherein R4, and Re are H.
[0267] In embodiments, the CPP is of the general Formula (II) or (III), wherein at least one of R4, Rs (if present), Re, R7 (if present) are independently an uncharged, non-aryl side chain of an amino acid. In embodiments, the CPP is of the general Formula (II) or (III), wherein at least two of R4, Rs (if present), Re, or R7 (if present) are independently side chains of an uncharged non-aryl amino acid (e g., histidine, threonine, serine, leucine, isoleucine, valine, neopentylglycine, alanine, homoalanine, homoserine, 3-(4-thiazolyl)-alanine, 3-(4-furanyl)-alanine, and 3-(4-thienyl)- alanine). In embodiments, the CPP is of the general Formula (II) or (III), wherein at least two of R4, Rs (if present), Rs, or R7 (if present) are independently side chains of an uncharged non-aryl amino acid selected from histidine, threonine, serine, leucine, isoleucine, valine, neopentylglycine, alanine, homoalanine, homoserine, 3-(4-thiazolyl)-alanine, 3-(4-furanyl)-alanine, and 3-(4- thienyl)-alanine. In embodiments, the CPP is of the general Formula (II) or (III), wherein at least two of R4, Rs (if present), Rs, or R7 (if present) are independently side chains of an uncharged non- aryl amino acid selected from histidine, and serine.
[0268] In embodiments, the CPP is of the general Formula (II), wherein at least one of R4, Rs, Re, R7 is, independently, H. In embodiments, the CPP is of the general Formula (II), wherein two of R4, Rs, Re, R7 are, independently, H. In embodiments, the CPP is of the general Formula (III), wherein at least one of R4 or Re is, independently, H. In embodiments, the CPP is of the general Formula (III), wherein two of R4 and Re are H.
[0269] In embodiments, compounds are provided that include a cyclic peptide having from 6 to 12 amino acids, wherein at least two amino acids of the cyclic peptide are charged amino acids, at least two amino acids of the cyclic peptide are aryl or heteroaryl hydrophobic amino acids and at least two amino acids of the cyclic peptide are uncharged, non-aryl amino acids. In embodiments, at least two charged amino acids of the cyclic peptide are arginine. In embodiments, at least two aryl or heteroaryl, hydrophobic amino acids of the cyclic peptide are phenylalanine, naphthylalanine (3-naphth-2-yl-alanine), betahomophenylalanine, or a combination thereof. In embodiments, at least two uncharged, non-aryl amino acids of the cyclic peptide are glycine. In embodiments, two of the uncharged amino acids are serine, histidine or a combination thereof.
[0270] In embodiments, the CPP) may comprise one of the following sequences: FGFGHGH; FGFSHSH; FGFGHGHQ; or FGFSHSHQ. In embodiments, the cCPP can comprise one of the following sequences: βhF-FΦSRSR, phF-Fd>GRGR, bhF-f-d>GrGr; bhF-fd>SRSR; or FfOSrSr. The cCPP can comprise one of the following sequences: βhF-Ffl>SRSRQ, βhF-F0GRGRQ, bhF- fDGrGrQ; bhF-fΦSRSRQ; or FfCbSrSrQ. In embodiments, the cCPP can comprise one of the following sequences: FfFSRSR; FGFSRSR; βhF-f-Nal-SRSR; FfFSRSRQ; FGFSRSRQ; or βhF- f-Nal-SRSRQ.
[0271] The cCPP of F ormula (AV) or F ormula (II) can have the structure of F ormula (II- A) or (II- B):
Formula (III-A)
Figure imgf000078_0001
Formula (1I1-B)
Figure imgf000079_0001
wherein AAsc is as defined herein.
[0272] The cCPP can comprise one of the following sequences: phF-FOSRSR, phF-FCDGRGR, bhF-f-ΦGrGr; bhF-fd>SRSR; or FfiDSrSr. The cCPP can comprise one of the following sequences: phF-FΦ>SRSRQ, phF-FΦDGRGRQ,bhF-f<hGrGrQ; bhF-fOSRSRQ, or Ffф SrSrQ.
[0273] In embodiments, the cCPP can comprise one of the following sequences: FfFSRSR; FGFSRSR; βhF-f-Nal-SRSR; FflFSRSRQ; FGFSRSRQ; or βhF-f-Nal-SRSRQ.
[0274] In embodiments, AAsc can be wherein t can be an integer
Figure imgf000079_0003
from 0 to 5. AAsc can be wherein t can be 0 or an integer from 1 to 5. t can be 1 to 5.
Figure imgf000079_0002
T is 2 or 3. t can be 2. t can be 3. In embodiments, AAsc can be conjugated to a linker.
[0275] The cCPP can be selected from:
Figure imgf000079_0004
Exocyclic Peptides
[0276] In embodiments, the ocular delivery construct comprises a cyclic cell penetrating peptide (cCPP) and an exocyclic peptide (EP); or an endosomal escape vehicle (EEV) that comprises a cCPP, an EP, and one or more linkers.
[0277] The EP can comprise a sequence of a nuclear localization signal (NLS). The exocyclic peptide (EP) can comprise from 2 to 10 amino acid residues e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues, inclusive of all ranges and values therebetween. The EP can comprise from 6 to 9 amino acid residues. The EP can comprise from 4 to 8 amino acid residues.
[0278] Each amino acid in the exocyclic peptide may be a natural or non-natural amino acid. The term “non-natural amino acid” refers to an organic compound that is a congener of a natural amino acid in that it has a structure similar to a natural amino acid so that it mimics the structure and reactivity of a natural amino acid. The non-natural amino acid can be a modified amino acid, and/or amino acid analog, that is not one of the 20 common naturally occurring amino acids or the rare natural amino acids selenocysteine or pyrrolysine. Non-natural amino acids can also be the D- isomer of the natural amino acids. Examples of suitable amino acids include, but are not limited to, alanine, allosoleucine, arginine, citrulline, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, napthylalanine, phenylalanine, proline, pyroglutamic acid, serine, threonine, tryptophan, tyrosine, valine, a derivative thereof, or combinations thereof. These, and others amino acids, are listed in Table 2 along with their abbreviations used herein. For example, the amino acids can be A, G, P, K, R, V, F, H, Nal, or citrulline.
[0279] The EP can comprise at least one positively charged amino acid residue, e g., at least one lysine residue and/or at least one amine acid residue comprising a side chain comprising a guanidine group, or a protonated form thereof. The EP can comprise 1 or 2 amino acid residues comprising a side chain comprising a guanidine group, or a protonated form thereof. The amino acid residue comprising a side chain comprising a guanidine group can be an arginine residue. Protonated forms can mean salt thereof throughout the disclosure.
[0280] The EP can comprise at least two, at least three or at least four or more lysine residues. The
EP can comprise 2, 3, or 4 lysine residues. The amino group on the side chain of each lysine residue can be substituted with a protecting group, including, for example, trifluoroacetyl (-COCF3), allyl oxycarbonyl (Alloc), l-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde), or (4,4- dimethyl-2,6-dioxocyclohex-l-ylidene-3)-methylbutyl (ivDde) group. The amino group on the side chain of each lysine residue can be substituted with a trifluoroacetyl (-COCF3) group. The protecting group can be included to enable amide conjugation. The protecting group can be removed after the EP is conjugated to a cCPP.
[0281] The EP can comprise at least 2 amino acid residues with a hydrophobic side chain. The amino acid residue with a hydrophobic side chain can be selected from valine, proline, alanine, leucine, isoleucine, and methionine. The amino acid residue with a hydrophobic side chain can be valine or proline.
[0282] The EP can comprise at least one positively charged amino acid residue, e.g., at least one lysine residue and/or at least one arginine residue. The EP can comprise at least two, at least three or at least four or more lysine residues and/or arginine residues.
[0283] The EP can comprise KK, KR, RR, HH, UK, HR, RH, KKK, KGK, KBK, KBR, KRK, KRR, RKK, RRR KKH, KHK, HKK, HRR HRH, HHR HBH, HHH, HHHH, KHKK, KKHK, KKKH, KHKH, HKHK, KKKK, KKRK, KRKK, KRRK, RKKR RRRR, KGKK, KKGK, HBHBH, HBKBH, RRRRR KKKKK, KKKRK, RKKKK, KRKKK, KKRKK, KKKKR KBKBK, RKKKKG, KRKKKG, KKRKKG, KKKKRG, RKKKKB, KRKKKB, KKRKKB, KKKKRB, KKKRKV, RRRRRR, HHHHHH, RHRHRH, HRHRHR KRKRKR, RKRKRK, RBRBRB, KBKBKB, PKKKRKV, PGKKRKV, PKGKRKV, PKKGRKV, PKKKGKV, PKKKRGV or PKKKRKG, wherein B is beta-alanine. The amino acids in the EP can have D or L stereochemistry.
[0284] The EP can comprise KK, KR RR, KKK, KGK, KBK, KBR, KRK, KRR, RKK, RRR, KKKK, KKRK, KRKK, KRRK, RKKR, RRRR KGKK, KKGK, KKKKK, KKKRK, KBKBK, KKKRKV, PKKKRKV, PGKKRKV, PKGKRKV, PKKGRKV, PKKKGKV, PKKKRGV or PKKKRKG. The EP can comprise PKKKRKV, RR, RRR RHR RBR RBRBR RBHBR or HBRBH, wherein B is beta-alanine. The EP can comprise PKKKRKV. The amino acids in the EP can have D or L stereochemistry.
[0285] The EP can consist of KK, KR RR KKK, KGK, KBK, KBR KRK, KRR RKK, RRR KKKK, KKRK, KRKK, KRRK, RKKR, RRRR KGKK, KKGK, KKKKK, KKKRK, KBKBK, KKKRKV, PKKKRKV, PGKKRKV, PKGKRKV, PKKGRKV, PKKKGKV, PKKKRGV or PKKKRKG. The EP can consist of PKKKRKV, RR RRR, RHR RBR RBRBR RBHBR or HBRBH, wherein B is beta-alanine. The EP can consist of PKKKRKV. The amino acids in the EP can have D or L stereochemistry.
[0286] The EP can comprise an amino acid sequence identified in the art as a nuclear localization sequence (NLS). The EP can consist of an amino acid sequence identified in the art as a nuclear localization sequence (NLS). The EP can comprise an NLS comprising the amino acid sequence PKKKRKV. The EP can consist of an NLS comprising the amino acid sequence PKKKRKV. The EP can comprise an NLS comprising an amino acid sequence selected from NLSKRPAAIKKAGQAKKKK, PAAKRVKLD, RQRRNELKRSF,
RMRKFKNKGKDTAELRRRRVEVSVELR, KAKKDEQILKRRNV, VSRKRPRP, PPKKARED, PQPKKKPL, SALIKKKKKMAP, DRLRR, PKQKKRK, RKLKKKIKKL, REKKKFLKRR KRKGDEVDGVDEVAKKKSKK and RKCLQAGMNLEARKTKK. The EP can consist of aann NLS comprising an amino acid sequence selected from NLSKRPAAIKKAGQAKKKK, PAAKRVKLD, RQRRNELKRSF,
RMRKFKNKGKDTAELRRRRVEVSVELR, KAKKDEQILKRRNV, VSRKRPRP, PPKKARED, PQPKKKPL, SALIKKKKKMAP, DRLRR, PKQKKRK, RKLKKKIKKL, REKKKFLKRR KRKGDEVDGVDEVAKKKSKK and RKCLQAGMNLEARKTKK
[0287] All exocyclic sequences can also contain an N-terminal acetyl group. Hence, for example, the EP can have the structure: Ac-PKKKRKV.
Endosomal Escape Vehicles (EEVs)
[0288] In embodiments, a cargo conjugate comprising a cargo and an ocular delivery construct is provided. In embodiments, the ocular delivery construct comprises an endosomal escape vehicle (EEV). The EEV can comprise at least one cell penetrating peptide (CPP), for example, a cyclic cell penetrating peptide (cCPP), which is conjugated to an exocyclic peptide (EP). In embodiments, the EEV comprises one cCPP. In embodiments, the EEV comprises one or more linkers.
[0289] The configuration of the cargo conjugate comprising an EEV may vaiy. In embodiments, the EP can be coupled to the cargo. In embodiments, the EP can be coupled to the cCPP. In embodiments, the EP can be coupled to the cargo and the cCPP. In embodiments, the EP can be coupled to the linker. In embodiments, the cCPP can be coupled to the cargo. In embodiments, the cCPP can be coupled to the EP. In embodiments, the cCPP can be coupled to the cargo and the EP. In embodiments, the cCPP can be coupled to the linker. In embodiments, the cargo can be coupled to the EP. In embodiments, the cargo can be coupled to the cCPP. In embodiments, the cargo can be coupled to the cCPP and the EP. In embodiments, the cargo can be coupled to the linker. In embodiments, the cCPP and the EP can be coupled to the linker and the cargo can be coupled to the cCPP or the EP. In embodiments, the cCPP and the cargo can be coupled to the linker and the EP can be coupled to the cCPP or the cargo. In embodiments, the EP and the cargo can be coupled to the linker and the cCPP can be coupled to the EP or the cargo. In embodiments, the cCPP, the EP, and the cargo can be coupled to the linker.
[0290] Coupling between the EP, cargo, cCPP, linker, or combinations thereof, may be non- covalent or covalent. In embodiments, the EP can be attached through a peptide bond to the N- terminus of the cCPP. In embodiments, the EP can be attached through a peptide bond to the C- terminus of the cCPP. In embodiments, EP can be attached to the cCPP through a side chain of an amino acid in the cCPP. In embodiments, the EP can be attached to the cCPP through a side chain of a lysine which can be conjugated to the side chain of a glutamine in the cCPP. In embodiments when the cargo is a therapeutic oligonucleotide, the EP can be conjugated to the 5' or 3'. In embodiments, the EP can be conjugated to an amino group of the linker. In embodiments, the EP can be coupled to a linker via the C-terminus of the EP and a cCPP through a side chain on the cCPP and/or EP. For example, an EP may comprise a terminal lysine which can then be coupled to a cCPP containing a glutamine through an amide bond. When the EP contains a terminal lysine, and the side chain of the lysine can be used to conjugate the EP to the cCPP and the C- or N- terminus may be attached to the linker or the cargo.
Linker
[0291] One or more linkers (L) may be used to link the components of a delivery construct and/or the cargo conjugates. A linker is a moiety that covalently couples two or more components of a delivery construct and/or a cargo construct; includes one or more functional group that can be used to conjugate one or more components to a linker; or both. In embodiments, the functional group on the linker may be reacted with a functional group on a component (e.g., an EP, cCPP, or cargo) to form a reaction product and covalently couple to component to the linker and link the component to one or more other components covalently attached to the linker.
[0292] The delivery constructs and/or cargo conjugates of the disclosure can include one or more linkers. The linker can link a cargo to the cCPP. The linker can link an EP to the cCPP. The linker can link the cCPP to the EP and the cargo. The linker can be attached to the side chain of an amino acid of the cCPP, and the cargo can be attached at a suitable position on the linker. [0293] The linker can be any appropriate moiety which can conjugate a cCPP to one or more additional components, e.g., an exocyclic peptide (EP) and/or a cargo. Prior to conjugation (e.g., to the cCPP and/or one or more additional components), the linker has two or more functional groups, each of which is independently capable of forming a covalent bond (e.g., to the cCPP and/or one or more additional components). If the cargo is an oligonucleotide, the linker can be covalently bound to the 5' end of the cargo or the 3' end of the oligonucleotide cargo. The linker can be covalently bound to the 5' end of the oligonucleotide cargo. The linker can be covalently bound to the 3* end of the oligonucleotide cargo. If the cargo is a peptide, the linker can be covalently bound to the N-terminus or the C-terminus of the peptide cargo. The linker can be covalently bound to the backbone (e g., somewhere in the middle and not at a terminus or termini) of the oligonucleotide or peptide cargo. The linker can be any appropriate moiety that conjugates a cCPP described herein to a therapeutic moiety such as an oligonucleotide, peptide or small molecule.
[0294] While not wishing to be bound by theory, the linkers can be chemically functionalized to influence efficacy and tolerability of the resultant construct, for example to increase efficacy. In embodiments, efficacy is increased without decreasing tolerability. For example, the linker can comprise one or more PEG (polyethylene glycol) regions and/or the linker can vary in length. The linker length (e g., between the EP and cCPP) can influence efficacy and tolerability. In some instances, as linker length decreases, efficacy increases and tolerability decreases; and as linker length increases, efficacy decreases and tolerability increases. In embodiments, as the length of the linker increases (e.g., between a cargo and EEV), the tolerability increases.
[0295] The linker can comprise hydrocarbon linker.
[0296] The linker can comprise a cleavage site. The cleavage site can be a disulfide, or caspasecleavage site (e.g, Val-Cit-PABC).
[0297] The linker can comprise: (i) one or more D or L amino acids, each of which is optionally substituted; (ii) optionally substituted alkylene; (iii) optionally substituted alkenylene; (iv) optionally substituted alkynylene; (v) optionally substituted carbocyclyl; (vi) optionally substituted heterocyclyl; (vii) one or more -(R1’J-R2)z”- subunits, wherein each of R1 and R2, at each instance, are independently selected from alkylene, alkenylene, alkynylene, carbocyclyl, and heterocyclyl, each J is independently C, NR3, -NR3C(O)-, S, and O, wherein R3 is independently selected from H, alkyl, alkenyl, alkynyl, carbocyclyl, and heterocyclyl, each of which is optionally substituted, and z” is an integer from 1 to 50; (viii) -(RI-J)z”- or -(J-R^z”-, wherein each of R1, at each instance, is independently alkylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl, each J is independently C, NR3, -NR3C(O)-, S, or O, wherein R3 is H, alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl, each of which is optionally substituted, and z” is an integer from 1 to 50; or (ix) the linker can comprise one or more of (i) through (x). [0298] The linker can comprise one or more D or L amino acids and/or -(R^J-R^z”-, wherein each of R1 and R2, at each instance, are independently alkylene, each J is independently C. NR3, - NR3C(O)-, S, and O, wherein R4 is independently selected from H and alkyl, and z” is an integer from 1 to 50; or combinations thereof.
[0299] The linker can comprise a -(OCH2CH2)Z- (e g., as a spacer), wherein z' is an integer from 1 to 60, e g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 29, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60. “-(OCHiCFbjz can also be referred to as polyethylene glycol (PEG).
[0300] The linker can comprise one or more amino acids. The linker can comprise a peptide. The linker can comprise a -(OCHaCHjjz-, wherein z' is an integer from 1 to 60, and a peptide . The peptide can comprise from 2 to 10 amino acids. The linker can further comprise a functional group (FG) capable of reacting through click chemistry. FG can be an azide or alkyne, and a triazole is formed when the ASO is conjugated to the linker. The linker can further comprise a carboxylic acid functional group capable of reacting with an amine to form an amide when an ASO is conjugated to the linker.
[0301] The linker can comprise (i) a 0 alanine residue and lysine residue; (ii) -(J-R'jz”; or (iii) a combination thereof. Each R1 can independently be alkylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl, each J is independently C, NR3, -NR3C(O)-, S, or O, wherein R3 is H, alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl, each of which is optionally substituted, and z” can be an integer from 1 to 50. Each R1 can be alkylene and each J can be O.
[0302] The linker can comprise (i) residues of 0-alanine, glycine, lysine, 4-aminobutyric acid, 5- aminopentanoic acid, 6-aminohexanoic acid or combinations thereof; and (ii) -(R^Jjz”- or -(J- R’)z”. Each R1 can independently be alkylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl, each J is independently C, NR3, -NR3C(O)-, S, or O, wherein R3 is H, alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl, each of which is optionally substituted, and z” can be an integer from 1 to 50. Each R1 can be alkylene and each J can be O. The linker can comprise glycine, beta-alanine, 4-aminobutyric acid, 5-aminopentanoic acid, 6-aminohexanoic acid, or a combination thereof.
[0303] The linker can be a trivalent linker. The linker can have the structure:
Figure imgf000086_0001
Figure imgf000086_0002
, wherein Ai, Bi, and Ci, can independently be a hydrocarbon linker (e.g., NRH-(CHz)n-COOH), a PEG linker (e.g., NRH-(CHzO)n-COOH, wherein R is H, methyl or ethyl) or one or more amino acid residue, and Z is independently a protecting group. The linker can also incoiporate a cleavage site, including a disulfide [NH2- (CH2O)n-S-S-(CH2O)n-COOH], or caspase-cleavage site (Val-Cit-PABC).
[0304] The hydrocarbon can be a residue of glycine or beta-alanine.
[0305] The linker can be bivalent and link the cCPP to an AC. The linker can be bivalent and link the cCPP to an exocyclic peptide (EP).
[0306] The linker can be trivalent and link the cCPP to an AC and to an EP.
[0307] The linker can be a bivalent or trivalent C1-C50 alkylene, wherein 1-25 methylene groups are optionally and independently replaced by -N(H)-, -N(CI-C4 alkyl)-, -N(cycloalkyl)-, -O-, - C(O)-, -C(O)O-, -S-, -S(O)-, -S(O)2-, -S(O)2N(CI-C4 alkyl)-, -S(O)2N(cycloalkyl)-, -N(H)C(O)-, -N(CI-C4 alkyl)C(O)-, -N(cycloalkyl)C(O)-, -C(O)N(H)-, -C(O)N(Ci-C4 alkyl), - C(O)N(cycloalkyl), aryl, heterocyclyl, heteroaiyl, cycloalkyl, or cycloalkenyl. The linker can be a bivalent or trivalent C1-C50 alkylene, wherein 1-25 methylene groups are optionally and independently replaced by -N(H)-, -O-, -C(O)N(H)-, or a combination thereof.
[0308] The cargo can be coupled to the glutamic acid of the cyclic peptide, which converts the glutamic acid to glutamine. The linker (L) can couple the cargo to the glutamine/glutamic acid of the cyclic peptide. In embodiments, a linker (L) is covalently bound to the backbone of the cargo.
[0309] The linker can have the structure:
Figure imgf000087_0001
, wherein: each AA is independently an amino acid residue; * is the point of attachment to the AAsc, and AAsc is side chain of an amino acid residue of the cCPP; x is an integer from 0-10; y is an integer from 1-5; and z Is an integer from 1-10. x can be an integer from 0-5. x can be an integer from 0-3. X can be 1. x can be 0. y can be an integer from 2-4. y can be 4. z can be an integer from 1-5. z can be an integer from 1-3. z can be 1. Each AA can independently be selected from glycine, P-alanine, 4-aminobutyric acid, 5-aminopentanoic acid, and 6-aminohexanoic acid.
[0310] The cCPP can be attached to the cargo through a linker (“L”). The linker can be conjugated to the cargo through a bonding group (“M”).
[0311] The linker can have the structure:
Figure imgf000087_0002
, wherein: x is an integer from 0-10; y is an integer from 1-5; z is an integer from 1-10, each AA is independently an amino acid residue; * is the point of attachment to the AAsc, and AAsc is side chain of an amino acid residue of the cCPP; and M is a bonding group defined herein.
[0312] In embodiments where a linker has a terminal NH that is conjugated to an exocyclic peptide (EP), one of ordinary skill in the art would understand that the terminal NH of the linker may be a part of the EP. For example, the terminal NH of the linker may be a part of an amide bond formed between the reaction of the carboxylic acid of the C-terminal amino acid of the EP with an amine of the linker. As such, the terminal NH of the linker may be covalently bonded to the carbonyl of the C-terminal amino acid of the EP.
[0313] As one of ordinary skill in the art would understand, when an amino acid side chain of the cCPP is covalently attached to the linker, the stated side chain includes a covalent bond between an atom of the side chain and the linker. For example, in embodiments when the linker is covalently attached to the side chain of glutamine, the glutamine side chain includes a covalent bond between the nitrogen of the side chain of glutamine and the linker.
[0314] The linker can have the structure:
Figure imgf000088_0001
wherein: y is an integer from 1-5; z' is an integer from 1-60; A is the point of attachment to the EP; * is the point of attachment to the AAsc, and AAsc is a side chain of an amino acid residue of the cCPP; and M is a bonding group defined herein.
[0315] The linker can have the structure:
[0316]
Figure imgf000088_0002
wherein: x' is an integer from 1-60; y is an integer from 1-5; z' is an integer from 1-60; * is the point of attachment to the AAsc, and AAsc is a side chain of an amino acid residue of the cCPP; and M is a bonding group defined herein. The terminal NH of the linker may be conjugated to an EP via the terminal C of the EP.
[0317] The linker can have the structure:
Figure imgf000088_0003
wherein: x' is an integer from 1-60; y is an integer from 1 -5; and z' is an integer from 1-60; * is the point of attachment to the AAsc, and AAsc is a side chain of an amino acid residue of the cCPP. The terminal NH of the linker may be conjugated to an EP.
(0318] x' can be an integer from 0-60, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 29, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60, inclusive of all ranges and subranges therebetween, x' can Be an integer from 5-15. x' can Be an integer from 9-13. x' can be an integer from 1-5. x' can be 1.
[0319] y can be an integer from 1-5, e.g., 1, 2, 3, 4, or 5, inclusive of all ranges and subranges therebetween, y can be an integer from 2-5. y can be an integer from 3-5. y can be 3 or 4. y can be 4 or 5. y can be 3. y can be 4. y can be 5.
[0320] z can be an integer from 1-10, e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, inclusive of all ranges and subranges therebetween.
[0321] z' can be an integer from 1-60, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 29, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60, inclusive of all ranges and subranges therebetween, z' can Be an integer from 5-15. z' can Be an integer from 9-13. z' can be 11.
[0322] As discussed above, the linker or M (wherein M is part of the linker) can be covalently bound to cargo at any suitable location on the cargo. The linker or M (wherein M is part of the linker) can be covalently bound to the 3' end of oligonucleotide cargo or the 5' end of an oligonucleotide cargo. The linker or M (wherein M is part of the linker) can be covalently bound to the N-terminus or the C-terminus of a peptide cargo. The linker or M (wherein M is part of the linker) can be covalently bound to the backbone of an oligonucleotide or a peptide cargo.
[0323] The linker can be bound to the side chain of aspartic acid, glutamic acid, glutamine, asparagine, or lysine, or a modified side chain of glutamine or asparagine (e.g., a reduced side chain having an amino group), on the cCPP. The linker can be bound to the side chain of lysine on the cCPP.
[0324] The linker can have a structure:
Figure imgf000089_0001
wherein:
M is a group that conjugates L to an AC;
AAsis a side chain or terminus of an amino acid on the cCPP; each AAx is independently an amino acid residue; o is an integer from 0 to 10; and p is an integer from 0 to 5.
[0325] The linker can have a structure:
Figure imgf000090_0001
wherein:
M is a group that conjugates L to a cargo (e.g., an ASO);
AAsis a side chain or terminus of an amino acid on the cCPP; each AAx is independently an amino acid residue; o is an integer from 0 to 10; and p is an integer from 0 to 5.
[0326] The linker can have a structure:
Figure imgf000090_0002
wherein:
M is a group that conjugates L to a cargo (e.g., an ASO);
*is the point of attachment of the linker to an AAscof a cCPP; and z' is an integer from 0 to 60.
[0327] M can comprise an alkylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl, each of which is optionally substituted. M can comprise or be selected from:
Figure imgf000090_0003
Figure imgf000091_0001
, wherein
R is alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl.
[0328] M can comprise or be selected from:
Figure imgf000091_0002
wherein: B is a nucleobase of an oligonucleotide cargo (e g., the 3' base of an oligonucleotide cargo); and R10 is alkylene, cycloalkyl, or wherein a is 0 to 10.
Figure imgf000091_0003
Figure imgf000092_0001
Figure imgf000093_0001
wherein o is an integer from 0 to 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10); s' is an integer from 0 to 10 (e g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10); c' is an integer from 0 to 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10); and B is a nucleobase of an oligonucleotide cargo (e.g., the 3' base of an oligonucleotide cargo).
[0330] M can be
Figure imgf000093_0002
and a is O to 10. M can be
Figure imgf000093_0004
[0331] M ean be a heterobifunctional crosslinker, e g.,
Figure imgf000093_0003
which is disclosed in Williams et al. Curr. Protoc Nucleic Acid Chem. 2010, 42, 4.41.1-4.41.20, incorporated herein by reference its entirety.
[0332] M ean be -C(O)-. [0333] M can be or comprise
Figure imgf000094_0005
can be or comprise wherein
Figure imgf000094_0001
f is 0 to 10. M can be
Figure imgf000094_0003
. M can be or comprise
Figure imgf000094_0002
wherein B is a nucleobase of an oligonucleotide cargo (e.g., the 3' base of an oligonucleotide cargo such as a PMO). [0334] AAs can be a side chain or terminus of an amino acid on the cCPP. Non-limiting examples of AAS include aspartic acid, glutamic acid, glutamine, asparagine, or lysine, or a modified side chain of glutamine or asparagine (e.g., a reduced side chain having an amino group). AAs can be an AAsc as defined herein.
[0335] Each AAx is independently a natural or non-natural amino acid. One or more AAx can be a natural amino acid. One or more AAx can be a non-natural amino acid. One or more AAx can be a p-amino acid. The p-amino acid can be p-alanine.
[0336] o can be an integer from 0 to 10, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. o can be 0, 1, 2, or
3. o can be 0. o can be 1. o can be 2. o can be 3.
[0337] p can be 0 to 5, e.g., 0, 1, 2, 3, 4, or 5. p can be 0. p can be 1. p can be 2. p can be 3. p can be 4. p can be 5.
[0338] The linker can have the structure:
Figure imgf000094_0004
wherein M, AAs. each -(R1-J-R2)z”-, o and z” are defined herein; r can be 0 or 1.
[0339] In embodiments, r is 0. In embodiments, r is 1 [0340] The linker can have the structure:
Figure imgf000095_0001
wherein each of M, AA$, o, p, q, r and z” can be as defined herein.
[0341] z" can be an integer from 1 to 50, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50, inclusive of all ranges And values therebetween, z " can Be an integer from 5-20. z " can be an integer from 10-15.
[0342] The linker can have the structure:
Figure imgf000095_0003
wherein:
M, AAs and o are as defined herein.
[0343] Other non-limiting examples of suitable linkers include:
Figure imgf000095_0002
O
Figure imgf000096_0001
Figure imgf000097_0001
wherein M and AA$ are as defined herein.
[0344] Provided herein is a compound comprising a cCPP and an AC that is complementary to a target in a pre-mRNA sequence further comprising L, wherein the linker is conjugated to the AC through a bonding group (M), wherein
Figure imgf000097_0002
[0345] Provided herein is a compound comprising a cCPP and an antisense compound (AC), for example, an antisense oligonucleotide, that is complementary to a target in a pre-mRNA sequence, wherein the compound further comprises L, wherein the linker is conjugated to the AC through a bonding group (M), wherein M comprises or is selected from:
Figure imgf000097_0003
wherein t' is 0 to 10 wherein each R is independently an alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl, wherein R1 is
Figure imgf000098_0001
, and t' is 2.
[0346] M can
Figure imgf000098_0002
, wherein t' is O to 10. M ean be
Figure imgf000098_0003
[0347] The linker can have the structure:
Figure imgf000098_0004
wherein AAs is as defined herein, and m' is 0-10. [0348] The linker can be of the formula.
Figure imgf000098_0005
wherein z' is defined herein. [0349] The linker can be of the formula:
Figure imgf000099_0001
, wherein
“base” is a nucleobase of an oligonucleotide cargo (e g., the 3' base of an oligonucleotide cargo such as PMO); and z' is defined herein.
[0350] The linker can be of the formula:
[0351] wherein
Figure imgf000099_0002
base” is a nucleobase of an oligonucleotide cargo (e.g., the 3' base of an oligonucleotide cargo such as PMO); and z' is defined herein. The linker can be of the formula:
Figure imgf000099_0003
, wherein “base” is a nucleobase of an oligonucleotide cargo (e.g., the 3' base of an oligonucleotide cargo such as PMO). [0352] The linker can be of the formula:
Figure imgf000100_0001
“base” is a nucleobase of an oligonucleotide cargo (e g., the 3' base of an oligonucleotide cargo such as PMO).
[0353] The linker can be of the formula:
[0354]
Figure imgf000100_0003
wherein: “base” is a nucleobase of an oligonucleotide cargo (e.g., the 3' base of an oligonucleotide cargo such as a PMO), and z' is defined herein.
[0355] The linker can comprise:
Figure imgf000100_0002
wherein:
“base” a nucleobase of an oligonucleotide cargo (e g., the 3' base of an oligonucleotide cargo) base is a nucleobase of an oligonucleotide cargo.
[0356] The linker can comprise:
Figure imgf000101_0001
Figure imgf000101_0002
wherein: “base” is a nucleobase of an oligonucleotide cargo (e g., the 3' base of an oligonucleotide cargo); and z' is defined herein.
[0357] The linker can be covalently bound to a cargo at any suitable location on the oligonucleotide cargo. The linker is covalently bound to the 3' end of cargo or the 5' end of an oligonucleotide cargo. The linker can be covalently bound to the backbone of an oligonucleotide cargo.
[0358] The linker can be bound to the side chain of aspartic acid, glutamic acid, glutamine, asparagine, or lysine, or a modified side chain of glutamine or asparagine (e.g., a reduced side chain having an amino group), on the cCPP. The linker can be bound to the side chain of lysine on the cCPP.
Delivery Constructs and Cargo Conjugates
[0359] The components of an ocular cargo conjugate can be conjugated to a linker defined herein.
An ocular delivery construct can be linked to a cargo to from a cargo conjugate. The cargo can be linked to the ocular delivery construct through a linker. The cargo can be conjugated to the linker using any conjugation reaction disclosed herein to form a bonding group (M).
[0001] In embodiments, a cargo is directly conjugated to the cCPP of an ocular delivery' construct to form a cargo conjugate. In embodiments, at least one atom of the cCPP can be replaced by a cargo or at least one lone pair can form a bond to a cargo. In embodiments, at least one atom of an amino acid side chain of the cCPP is replaced by a cargo or at least one lone pair of the atom forms a bond to a cargo. In embodiments, a hydrogen atom on the NHj group of the carboxamide side chain of a cCPP can be replaced by a bond to the cargo. In embodiments, a hydrogen atom on the NH? group of the carboxamide of a glutamine side chain of the cCPP can be replaced by a bond to the cargo.
[0360] In embodiments, a cargo is linked to an ocular delivery construct through a linker to form a cargo conjugate. In embodiments, the ocular delivery construct comprises a cCPP and a linker. In embodiments, the AAscof a cCPP is conjugated to a linker and the cargo is conjugated to the linker thereby forming a cargo conjugate.
[0361] The cargo can be any therapeutic moiety such as a peptide, oligonucleotide, or small molecule. The cargo can be a peptide sequence or a non-peptidyl therapeutic agent. The cargo can be a therapeutic moiety that is an antibody or an antigen binding fragment thereof, including, but not limited to an scFv or nanobody.
[0362] The cCPP can be conjugated to a linker defined herein. The linker can be conjugated to an AAsc of the cCPP as defined herein.
[0363] The linker can comprise a -(OCIfcCEbjz- subunit (e.g., as a spacer), wherein z' is an integer from 1 to 60, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 29, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60. “-(OCHzCHa),' is also referred to as PEG. In embodiments, z' is 11. In embodiments, z' is 1.
[0364] The linker can comprise a -(OCEbCHk)/- subunit (e.g., as a spacer), wherein z' is an integer from 1 to 23. e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23. “-(OCH2CH2)Z' is also referred to as PEG. In embodiments, z' is 11. In embodiments, z" is 1. [0365] An endosomal escape vehicle (EEV) can comprise a cyclic cell penetrating peptide (cCPP), an exocyclic peptide (EP) and linker, and can be conjugated to a cargo to form an EEV-conjugate (cargo conjugate) comprising the structure of Formula (Z):
[0366] EEVs comprising a cyclic cell penetrating peptide (cCPP), linker and exocyclic peptide (EP) are provided. A cargo construct comprising an EEV can comprise the structure of Formula (X):
Formula (X):
Figure imgf000103_0001
wherein: EP is an exocyclic peptide; cargo is a therapeutic moiety as defined herein; cCPP is a cCPP; and x', y, z', and M are defined herein. In embodiments, cCPP is a cCPP of Formula (I). In embodiments, cCPP is a cCPP of Formula (II).
[0367] A cargo construct comprising an EEV can comprise the structure of Formula (B ):
Formula (B):
Figure imgf000104_0001
or a protonated form thereof, wherein:
Ri, Rz, and R3 are each independently H or an aryl or heteroaryl side chain of an amino acid;
Rt and R6 are independently H or an amino acid side chain; peptide is an exocyclic peptide as defined herein;
M is a bonding group as defined herein; each m is independently an integer from 0-3; cargo is a therapeutic moiety as defined herein; n is an integer from 0-2; x' is an integer from 1 -20; y is an integer from 1-5; q is 1-4; and z' is an integer from 1-20.
[0368] In embodiments the cargo conjugate comprises the structure of Formula (B) or a protonated form thereof, wherein: Ri, R2, and Rs are each independently H or an aryl or heteroaryl side chain of an amino acid; at least two of Ri, R2, and Rs are an aryl or heteroaryl side chain of an amino acid;
R4 and R6 are independently H or an amino acid side chain; peptide is an exocydic peptide (EP); each m is independently an integer from 0-3; cargo is a therapeutic moiety as defined herein; n is an integer from 0-2; x' is an integer from 0-20; y is an integer from 1-5; q is 1-4; and z' is an integer from 0-20.
[0369] In embodiments the cargo conjugate comprises the structure of Formula (B) or a protonated form thereof wherein:
Ri, R2, and Rs can each independently be H or an amino acid residue having a side chain comprising an aryl group,
R4 and Rs are independently H or an amino acid side chain; peptide is an exocydic peptide as defined herein;
M is a bonding group as defined herein; cargo is a therapeutic moiety as defined herein; each m is independently an integer from 0-3; n is an integer from 0-2; x' is an integer from 1 -20; y is an integer from 1-5; q is an integer from 1-4; and z' is an integer from 1-20.
[0370] A cargo conjugate comprising an EEV can comprise the structure of Formula (B-l) or
(B-2):
Figure imgf000106_0001
 Formula (B-2):
Figure imgf000107_0001
, or a protonated form thereof, wherein
Ri, R2, and Rs are each independently H or an aryl or heteroaryl side chain of an amino acid;
R4 is H or an amino acid side chain;
EP is an exocyclic peptide as defined herein; each m is independently an integer from 0-3; cargo is a therapeutic moiety as defined herein; x' is an integer from 1-20; y is an integer from 1-5, and z' is an integer from 1-20.
[0371] A cargo conjugate comprising an EEV can comprise the structure of Formula (C): Formula (C):
Figure imgf000108_0001
or a protonated form thereof, wherein EP, R1, R2, R3, R4, and m are as defined above in Formula (B); cargo is a therapeutic moiety as defined herein; AA can be an amino acid as defined herein; n can be an integer from 0-2, x can be an integer from 0-10; y can be an integer from 1-5; and z can be an integer from 1-10.
[0372] A cargo construct comprising an EEV can comprise the structure of Formula the structure of Formula (D):
Formula (D):
Figure imgf000109_0001
or a protonated form thereof, wherein:
RI, Rz, and R3 can each independently be H or an amino acid residue having a side chain comprising an aryl or heteroaryl group; at least two of Ri, Ra, and Ra is an aryl or heteroaryl side chain of an amino acid;
R4, and Rs are independently H or an amino acid side chain;
AAscis an amino acid side chain; nx is 0 or 1; cargo is a therapeutic moiety as defined herein;
M is a bonding group as defined herein. q is 1, 2, 3 or 4; each m is independently an integer from 0-3; n is an integer from 0-2; x' is an integer from 1-20; y is an integer from 1-5; q is an integer from 1-4; and z' is an integer from 1-20. [0373] In embodiments, cCPP is a cCPP of Formula (X), (B), (B-l), (B-2), (C), or (D) wherein x' is 1. In embodiments, cCPP is a cCPP of Formula (X), (B), (B-l), (B-2), (C), or (D) wherein y is 1. In embodiments, cCPP is a cCPP of Formula (X), (B), (B-l ), (B-2), (C), or (D) wherein z' is 2. In embodiments, cCPP is a cCPP of Formula (X), (B), (B-l), (B-2), (C), or (D) wherein z' is 11. In embodiments, cCPP is a cCPP of Formula (X), (B), (B-l), (B-2), (C), or (D) wherein M comprises
Figure imgf000110_0003
embodiments, cCPP is a cCPP of Formula (B), (C), or (D) wherein M is or comprises
Figure imgf000110_0001
, wherein t' is 0 to 10. Tn embodiments, cCPP is a cCPP of
Formula (B), (C), or (D) wherein M is or comprises
Figure imgf000110_0002
Delivery constructs before conjugation to a cargo
[0374] Delivery constructs having a structure prior to conjugation to a cargo are provided. The delivery constructs can include a cCPP, a cCPP and a linker, or an EEV (cCPP, EP, and linker). Prior to conjugation to the cargo, the linker can include a reactive group. The reactive group can react with a reactive group on a cargo to form at least a portion of a bonding group M as described herein. Examples of reactive groups include an azide (Ns) and a carboxylic acid. In embodiments, where the delivery construct prior to conjugation to a cargo includes an azide, click chemistry may be used to conjugate the delivery construct to a cargo. In embodiments, an azide reactive group may be incorporated in a delivery construct as an amino acid derivative, for example, azidolysine (K(N3)). In embodiments, where the delivery construct prior to conjugation to a cargo includes a carboxylic acid, amid bond forming chemistry may be used to conjugate the delivery construct to a cargo.
[0375] A delivery construct prior to conjugation to a cargo can be selected from cyclo[Ff-Nal-RrRrQ]-PEGz-OH; and cyclo[Ff-Nal-RrRrQ]-PEGz-K(N3)-NH2, wherein x' and z' are, independently, an integer from 0 to 12.
[0376] A delivery construct prior to conjugation to a cargo can be selected from Ac-PKKKRKV-PEGx-K(cyclo[GfFGrGrQ])-PEGz-OH; and Ac-PKKKRKV-PEGx-K(cyclo[GfFGrGrQ])-PEGz-K(N3)-NH2 wherein x' and z' are, independently, an integer from 0 to 12.
[0377] A delivery construct prior to conjugation to a cargo can be selected from Ac-PKKKRKV-PEGx-K(cyclo[FfFGRGRQ])-PEGz'-OH; and Ac-PKKKRKV-PEGx'-K(cyclo[FfFGRGRQ])-PEGz’-K(N3)-NH2 wherein x' and z' are, independently, an integer from 0 to 12.
[0378J A delivery construct prior to conjugation to a cargo can be selected from Ac-PKKKRKV-PEGx,-K(cyclo[Ff-Nal-GrGrQ])-PEGz-OH; and Ac-PKKKRKV- PEGx-K(cyclo[Ff-Nal-GrGrQ])-PEGz-K(N3)-NH2 wherein x' and z' are, independently, an integer from 0 to 12.
[0379] A delivery construct prior to conjugation to a cargo can be selected from Ac-PKKKRKV-PEGx -K(cyclo[FGFGRGRQ])-PEGz-OH; and Ac-PKKKRKV-PEGx'-K(cyclo[FGFGRGRQ])-PEGz-K(N3)-NH2 wherein x' and z' are, independently, an integer from 0 to 12.
[0380] A delivery construct prior to conjugation to a cargo can be selected from Ac-KKKRK-PEGx-K(cyclo(FGFGRGRQ))-PEGz'-OH ; and Ac-KKKRK-PEGx-K(cyclo(FGFGRGRQ))-PEGz’-K(N3)-NH2 wherein x' and z' are, independently, an integer from 0 to 12.
[0381] A delivery construct prior to conjugation to a cargo can be selected from Ac-PKKKRKV-PEGx-K(cyclo[FGFRRRRQ])-PEGz-OH; and Ac-PKKKRKV-PEGx--K(cyclo[FGFRRRRQ])-PEGz’- K(N3)-NH2 wherein x' and z' are, independently, an integer from 0 to 12.
[0382] A delivery construct prior to conjugation to a cargo can be selected from Ac-PKKKRKV-PEGx-K(cyclo[FF<PGRGRQ])-PEGz-OH; and Ac-PKKKRKV-PEGx-K(cyclo[FF<PGRGRQ])-PEG/-K(N3)-NH 2 wherein x' and z' are, independently, an integer from 0 to 12.
[0383] A delivery construct prior to conjugation to a cargo can be selected from Ac-PKKKRKV-PEGx-K(cyclo[βhF-F0SRSRQ])-PEGz -OH; and Ac-PKKKRKV-PEGx-K(cyclo[βhF-FOSRSRQ])-PEGz- K(N3)-NH2 wherein x' and z' are, independently, an integer from 0 to 12. [0384] A delivery construct prior to conjugation to a cargo can be selected from Ac-PKKKRKV-PEGx-K(cyclo[βhF--Φ GRGRQ])-PEGz-OH; and Ac-PKKKRKV-PEGx’-K(cyclo[βhF-F0GRGRQ])-PEG2- K(N3)-NH2, wherein x' and z' are, independently, an integer from 0 to 12.
[0385] A delivery construct prior to conjugation to a cargo can be selected from
Ac-PKKKRKV-PEGx’-K(cyclo[bhF-f-Φ>GrGrQ])-PEG/-OH; and
Ac-PKKKRKV-PEGx-K(cyclo[bhF-f-ΦGrGrQ])-PEGz'- K(N3)-NH2, wherein x' and z' are, independently, an integer from 0 to 12.
[0386] A delivery construct prior to conjugation to a cargo can be selected from Ac-PKKKRKV-PEGx-K(cyclo[bhF-f-ΦDGrGrQ])-PEGz-OH, and Ac-PKKKRKV-PEGx-K(cyclo[bhF-f-ΦSRSRQ])-PEGz'- K(N3)-NH2, wherein x' and z' are, independently, an integer from 0 to 12.
[0387] K(N3) is azidolysine; and a terminal OH indicates a terminal carboxylic acid. In embodiments, PEG2 is miniPEGi or miniPEG. In embodiments, x' is 0. In embodiments, x' is 2. In embodiments, x' is 4. In embodiments, x' is 8. In embodiments, x' is 12. In embodiments, z' is 0. In embodiments, z' is 2. In embodiments, z' is 4. In embodiments, z' is 8. In embodiments, z' is 12.
[0388] A delivery construct prior to conjugation to a cargo can be selected from cyclo[Ff-Nal-RrRrQ]-PEGi2-OH; cyclo[Ff-Nal-RrRrQ]-PEGi2-K(N3)-NH2;
Ac-PKKKRKV-K(cyclo[Ff-Nal-GrGrQ])-PEGi2-K(N3)-NH2;
Ac-PKKKRKV-K(cyclo[Ff-Nal-GrGrQ])-miniPEG2-K(N3)-NH2;
Ac-PKKKRKV- PEG2-K(cyclo[Ff-Nal-GrGrQ]>PEGi2-OH; and
Ac-PKKKRKV-miniPEG2-K(cyclo[FF0GRGRQ])-PEG2-K(N3)-NH2.
[0389] A delivery construct prior to conjugation to a cargo can be selected from
Ac-PKKKRKV- PEG2-K(cyclo[GfFGrGrQ])-PEG2-K(N3)-NH2;
Ac-PKKKRKV- PEG2-K(cyclo[FfFGRGRQ])-PEG2-K(N3)-NH2;
Ac-PKKKRKV- PEG2-K(cyclo[FGFGRGRQ])-PEGi2-OH;
Ac-PKKKRKV- PEG2-K(cyclo[FGFRRRRQ])-PEGi2-OH;
Ac-PKKKRKV-miniPEG2-K(cyclo[FGFGRGRQ])-PEGi2-K(N3)-Ml2;
Ac-PKKKRKV-miniPEG2-K(cyclo[FGFGRGRQ])-miniPEG>-K(N3)-NH2; and Ac-PKKKRKV-miniPEG2-K(cyclo[FfFGRGRQ])-miniPECh-K(N3)-NH2.
[0390] A delivery construct prior to conjugation to a cargo can be: Ac-KKKRK-miniPEG2-K(cyclo(FGFGRGRQ))-miniPEG2-K(N3)-NH2.
[0391] A delivery construct prior to conjugation to a cargo can be selected from Ac-PKKKRKV-miniPEG2-K(cyclo[βhF-f-ΦI SRSRQ])-PEGi2-OH; and Ac-PKKKRKV-miniPEG2-K(cyclo[βhF-FΦI>GRGRQ])-PEGi2-OH. Ac-PKKKRKV-miniPEG2-K(cyclo[bhF-f-Φ>GrGrQ])-PEGi2-OH; and Ac-PKKKRKV-miniPEG2-K(cyclo[bhF-f<DSRSRQ])-PEGi2-OH.
[0392] It is understood that a terminal OH indicates a terminal carboxylic acid and KflSb) is azidolysine. In embodiments, PEG2 is miniPEGz or miniPEG.
[0393] In embodiments, a delivery construct having an azide group that can be used in a click chemistry reaction to conjugate a cargo to the delivery construct can be selected from cyclo[Ff-Nal-RrRrQ]-PEGz’-K(N3)-NH2 Ac-PKKKRKV-PEG x'-K(cy cl o[GfFGrGrQ])-PEGz'-K(N3)-NH2; Ac-PKKKRKV-PEG x-'K(cyclo[FfFGRGRQ])-PEG z-K(N3)-NH2; Ac-PKKKRK V-PEG x-K(cyclo[Ff-Nal-GrGrQ])-PEG z’-K(N3)-NH2; Ac-PKKKRKV-PEG x-K(cyclo[FGFGRGRQ])-PEGz-K(N3)-NH2; Ac-PKKKRKV-PEG x-K(cyclo[FGFRRRRQ])-PEGz-K(N3>NH2; Ac-PKKKRKV-K(cyclo[Ff-Nal-G-r-G-rQ])- PEG /-K(N3)-NH2;
Ac-PKKKRK V-K(cyclo[Ff-Nal-G-r-G-rQ])- PEG z-K(N3)-NI-I2, Ac-PKKKRKV-PEG x-K(cyclo[FGFGRGRQ])-PEGz'-K(N3)-NH2; Ac-PKKKRKV-PEG x’-K(cyclo[FfFGRGRQ])-PEGz-K(N3)-NH2; Ac-KKKRK-PEGx-K(cyclo[FGFGRGRQ])-PEGz-K(N3)-NH2; Ac-PKKKRK V-PEG x-K(cy cl o[FFO>GRGRQ])-PEGz'-K(N3)-NH2; Ac-PKKKRKV-PEG x-K(cyclo[0hF-FΦDSRSRQ])-PEG z-K(N3)-NH2; Ac-PKKKRKV-PEG x'-K(cyclo[phF-F^GRGRQ])-PEGz-K(N3)-NH2; and Ac-PKKKRKV-PEG x-K(cyclo[FGFGRGRQ])-PEGz’-K(N3)-NH2; wherein K(Ns) is azidolysine; and x' and z' are, independently, an integer from 0 to 12. In embodiments, x' is 0. In embodiments, x' is 2. In embodiments, x' is 4. In embodiments, x' is 8. In embodiments, x' is 12. In embodiments, z' is 0. In embodiments, z' is 2. In embodiments, z' is 4. In embodiments, z' is 8. In embodiments, z' is 12. In embodiments, PEGt* is miniPEG. In embodiments, PEG?/ is miniPEG or miniPEG.
[0394] In embodiments, a delivery construct having an azide group that can be used in a click chemistry reaction to conjugate a cargo to the delivery construct can be cyclo[Ff-Nal-RrRrQ]- PEG2-K(N3)-NHz. In embodiments, the delivery construct can be cyclo[Ff-Nal-RrRrQ]-PEGi?- K(N3)-NH2.
[0395] In embodiments, a delivery construct having an azide group that can be used in a click chemistry reaction to conjugate a cargo to the delivery construct can be Ac-PKKKRKV-PEG- K(cyclo[GfFGrGrQ])-PEG2-K(N3)-NH? where each PEG2 may, independently, be miniPEG2. In embodiments, the delivery construct can be Ac-PKKKRKV-PEG-K(cyclo[GfFGGrQ])-PEGi?- K(N3)-NH2 where PEG2 may be miniPEG2.
[0396] In embodiments, a delivery construct having an azide group that can be used in a clickchemistry reaction to conjugate a cargo to the delivery construct can be Ac-PKKKRKV-PEG- K(cyclo[FfFGRGRQ])-PEG-K(N3)-NH? where each PEG2 may, independently, be miniPEG2. In embodiments, the delivery construct can be Ac-PKKKRKV-PEG-K(cyclo[FfFGRGRQ])-PEGi?- K(N3)-NH2 where PEG2 may be miniPEG2.
[0397] In embodiments, the delivery construct can be Ac-PKKKRKV-PEG-K(cyclo[Ff-Nal- GrGrQ])-PEG- K(N3)-NH? where each PEG? may, independently, be miniPEG?. In embodiments, the delivery construct can be Ac-PKKKRKV-PEG-K(cyclo[Ff-Nal-GrGQ])-PEGi?-K(N3)-NH? where PEG? may be miniPEG2.
[0398] In embodiments, the delivery ccoonnssttrruucctt ccaann bbee Ac-PKKKRKV-PEG2- K(cyclo[FGFGRGRQ])-PEG2 K(N3)-NH2 where each PEG? may, independently, be miniPEG2. In embodiments, the delivery construct can be Ac-PKKKRKV-PEG2-K(cyclo[FGFGRGRQ])-PEGi2 K(N3)-NH? where PEG2 may be miniPEG2.
[0399] In embodiments, the delivery construct can be Ac-PKKKRKV-PEG- K(cyclo[FGFRRRRQ])-PEG K(N3)-NH? where each PEG2 may, independently, be miniPEG2. In embodiments, the delivery construct can be Ac-PKKKRKV-PEG-K(cyclo[FGFRRRRQ])-PEGi? K(N3>NH2 where PEG may be miniPEG.
[0400] In embodiments, the delivery construct can be Ac-PKKKRKV-K(cyclo[Ff-Nal-G-r-G- rQ])-PEG2 K(N3)-NH? where each PEG may, independently, be miniPEG2. In embodiments, the delivery construct can be Ac-PKKKRKV-K(cyclo[Ff-Nal-G-r-G-rQ])-PEGi2 K(Ns)-NH2 where PEG may be miniPEG.
[0401] In embodiments, the delivery construct can be Ac-PKKKRKV-K(cyclo[Ff-Nal-G-r-G- rQ])-PEG K(N3)-NH2 where each PEG? may, independently, be miniPEG. In embodiments, the delivery construct can be Ac-PKKKRKV-K(cyclo[f-Nal-G-r-G-rQ])-PEGi2 K(N3)-NH? where PEG may be miniPEG.
[0402] In embodiments, tthhee delivery construct can be Ac-PKKKRKV-PEG-
K(cyclo[FGFGRGRQ])- PEG-K(N3)-NH2 where each PEG may, independently, be miniPEG. In embodiments, the delivery construct can be Ac-PKKKRKV-PEG-K(cyclo[FGFGRGRQ])- PEGi2-K(N3)-NH2 where PEG2 may be miniPEG2.
[0403] In embodiments, the delivery construct ccaann bbee Ac-PKKKRKV-PEGz- K(cyclo[FfFGRGRQ])- PEG-K(N3)-NH2 where each PEG2 may, independently, be miniPEG. In embodiments, the delivery construct can be Ac-PKKKRKV-PEG-K(cyclo[FfFGRGRQ])- PEGi2-K(N3)-NH2 where PEG2 may be miniPEGz.
[0404] In embodiments, the delivery construct can be Ac-KKKRK-PEG-K(cyclo(F GF GRGRQ)- PEG-K(N3)-NH2 where each PEG2 may, independently, be miniPEG2. In embodiments, the delivery construct can be Ac-KKKRK-PEG-K(cyclo(FGFGRGRQ))-PEGi2-K(N3)-NH2 where PEG2 may be miniPEG?.
[0405] In embodiments, the delivery construct can be Ac-PKKKRKV-PEG- K(cyclo[FFΦ GRGRQ])- PEG-K(N3)-NH? where each PEG? may, independently, be miniPEG. In embodiments, the delivery' construct can be Ac-PKKKRKV-PEG2-K(cy'clo[FFC>GRGRQ])- PEGi?-K(N3)-NH2 where PEG may be miniPEG.
[0406] In embodiments, the delivery construct can be Ac-PKKKRKV-PEG-K(cyclo[phF- F0SRSRQ])-PEG-K(N3)-NH2 where each PEG may, independently, be miniPEG. In embodiments, the delivery construct can be Ac-PKKKRKV-PEG2-K(cyclo[piiF-FΦI’SRSRQ])- PEG12-K(N3)-NH2 where PEG may be miniPEG.
[0407] In embodiments, the delivery construct can be Ac-PKKKRKV-PEG?-K(cyclo[βhF- FΦ GRGRQ])-PEG-K(N3)-NH2 where each PEG may, independently, be miniPEG. In embodiments, the delivery construct can be Ac-PKKKRKV-PEG2-K(cyclo[phF-FΦ GRGRQ])- PEGi2-K(N3)-NH2 where PEG2 may be miniPEG. [0408] In embodiments, the delivery construct ccaann bbee Ac-PKKKRKV-PEG2- K(cyclo[FGFGRGRQ])- PEG2-K(N3)-NH2 where each PEG2 may, independently, be miniPEGz. In embodiments, the delivery construct can be Ac-PKKKRKV-PEGz-K(cyclo[FGFGRGRQ])- PEG12-K(N3)-NH2 where PEGz may be mini PEG.
[0409] In embodiments, a delivery construct having an azide group that can be used in a click chemistry reaction to conjugate a cargo to the delivery construct can be selected from
Figure imgf000116_0001
wherein OH indicates a terminal carboxylic acid; and x' and z' are, independently, an integer from 0 to 12. In embodiments, x' is 0. In embodiments, x' is 2. In embodiments, x' is 4. In embodiments, x' is 8. In embodiments, x' is 12. In embodiments, z' is 0. In embodiments, z' is 2. In embodiments, z' is 4. In embodiments, z' is 8. In embodiments, z' is 12. In embodiments, PEG/ is miniPEGz. In embodiments, PEG/ is miniPEG.
[0410] In embodiments, a delivery construct having an azide group that can be used in a click chemistry reaction to conjugate a cargo to the delivery construct can be cyclo[Ff-Nal-RrRrQ]- PEG2-OH. In embodiments, the delivery construct can be cyclo[Ff-Nal-RrRrQ]-PEGi2-OH.
[0411] In embodiments, a delivery construct having an azide group that can be used in a click chemistry reaction to conjugate a cargo to the delivery construct can be Ac-PKKKRKV-PEG- K(cyclo[GfFGGQ])-PEG-OH where each PEG may, independently, be miniPEG. In embodiments, the delivery construct can be Ac-PKKKRKV-PEG-K(cyclo[GfFGGrQ])-PEGi2- OH where PEG may be miniPEGz.
[0412] In embodiments, a delivery construct having an azide group that can be used in a click chemistry reaction to conjugate a cargo to the delivery construct can be Ac-PKKKRKV-PEG- K(cyclo[FfFGRGRQ])-PEG2-OH where each PEGz may, independently, be miniPEGz. In embodiments, the delivery construct can be Ac-PKKKRKV-PEG-K(cyclo[FfFGRGRQ])-PEGi2- OH where PEGz may be miniPEGz.
[0413] In embodiments, the delivery construct can be Ac-PKKKRKV-PEG-K(cyclo[Ff-Nal- GrGrQ])-PEG-OH where each PEG may. independently, be miniPEGz. In embodiments, the delivery construct can be Ac-PKKKRKV-PEG-K(cyclo[Ff-Nal-GrGrQ])-PEGi2-OH where PEGz may be miniPEG.
[0414] In embodiments, tthhee delivery ccoonnssttrruucctt ccaann bbee Ac-PKKKRKV-PEG- K(cyclo[FGFGRGRQ])-PEGz-OH where each PEGz may, independently, be miniPEG. In embodiments, the delivery construct can be Ac-PKKKRKV-PEGz-K(cyclo[FGFGRGRQ])- PEG12-OH where PEG may be mini PEG?.
[0415] In embodiments, the delivery ccoonnssttrruucctt ccaann bbee Ac-PKKKRKV-PEGz- K(cyclo[FGFRRRRQ])-PEG-OH where each PEG may, independently, be miniPEG. In embodiments, the delivery construct can be Ac-PKKKRKV-PEG-K(cyclo[FGFRRRRQ])- PEGiz-OH where PEG may be miniPEG.
[0416] In embodiments, the delivery construct can be Ac-PKKKRKV-K(cyclo[Ff-Nal-G-r-G- rQ])-PEGz-OH where each PEG may, independently, be miniPEGz. In embodiments, the delivery construct can be Ac-PKKKRKV-K(cyclo[Ff-Nal-G-r-G-rQ])-PEGi2-OH where PEG may be miniPEGz.
[0417] In embodiments, the delivery construct can be Ac-PKKKRKV-K(cyclo[Ff-Nal-G-r-G- rQ])-PEG K(N3)-NHZ where each PEG may, independently, be miniPEGz. In embodiments, the delivery construct can be Ac-PKKKRKV-K(cyclo[Ff-Nal-G-r-G-rQ])-PEGiz-OH where PEG may be miniPEGz.
[0418] In embodiments, tthhee delivery ccoonnssttrruucctt ccaann bbee Ac-PKKKRKV-PEG- K(cyclo[FGFGRGRQ])-PEG2-OH where each PEG may, independently, be miniPEG. In embodiments, the delivery construct can be Ac-PKKKRKV-PEGz-K(cyclo[FGFGRGRQ])- PEG12-OH where PEG may be miniPEG. [0419] In embodiments, tthhee delivery construct can be Ac-PKKKRKV-PEG2-
K(cyclo[FfFGRGRQ])-PEG2-OH where each PEG2 may, independently, be miniPEG2. In embodiments, the delivery construct can be Ac-PKKKRKV-PEG2-K(cyclo[FfFGRGRQ])- PEG12-OH where PEG2 may be miniPEG2.
[0420] In embodiments, the delivery construct can be Ac-KKKRK-PEG2-K(cyclo(FGFGRGRQ)- PEG2-OH where each PEG2 may, independently, be miniPEG2. In embodiments, the delivery construct can be Ac-KKKRK-PEG2-K(cyclo[FGFGRGRQ])-PEGi?-OH where PEG2 may be miniPEG2.
[0421] In embodiments, the delivery construct can be Ac-PKKKRKV-PEG2-
K(cyclo[FFd>GRGRQ])-PEG2-OH where each PEG2 may, independently, be miniPEG2. In embodiments, the delivery construct can be Ac-PKKKRKV-PEG2.-K(cyclo[FFOGRGRQ])- PEG12-OH where PEG2 may be miniPEG2.
[0422] In embodiments, the delivery construct can be Ac-PKKKRKV-PEG2-K(cyclo[βhF- FOSRSRQD-PEG2-OH where eachPEG2 may, independently, be miniPEG2. In embodiments, the delivery construct can be Ac-PKKKRKV-PEG2-K(cyclo[βhF-FΦDSRSRQ])-PEGi2-OH where PEG2 may be miniPEG2.
[0423] In embodiments, the delivery construct can be Ac-PKKKRKV-PEG2-K(cyclo[phF- FΦGRGRQ])-PEG2-OH where each PEG2 may, independently, be miniPEG2. In embodiments, the delivery construct can be Ac-PKKKRKV-PEG2-K(cyclo[phF-F<DGRGRQ])-PEGi?-OH where PEG2 may be miniPEG2.
[0424] In embodiments, tthhee delivery construct can be Ac-PKKKRKV-PEG2-
K(cyclo[FGFGRGRQ])-PEG2-OH where each PEG2 may, independently, be miniPEG2. In embodiments, the deliveiy construct can be Ac-PKKKRKV-PEG2-K(cyclo[FGFGRGRQ])- PEG12-OH where PEG2 may be miniPEG2.
Methods of Making
[0425] The cargo conjugates and the components of the cargo conjugates can be prepared in a variety of ways known to one skilled in the art of organic synthesis or variations thereon as appreciated by those skilled in the art. The compounds can be prepared from readily available starting materials. Reaction conditions can vary with the particular reactants or solvents used, but such conditions can be determined by one skilled in the art. [0426] Variations on the compounds described herein include the addition, subtraction, or movement of the various constituents as described for each compound. Similarly, when one or more chiral centers are present in a molecule, the chirality of the molecule can be changed. Additionally, compound synthesis can involve the protection and deprotection of various chemical groups. The use of protection and deprotection, and the selection of appropriate protecting groups can be determined by one skilled in the art. The chemistry of protecting groups can be found, for example, in Wuts and Greene, Protective Groups in Organic Synthesis, 4th Ed., Wiley & Sons, 2006, which is incorporated herein by reference in its entirety.
[0427] The starting materials and reagents used in preparing the disclosed compounds and compositions are either available from commercial suppliers such as Aldrich Chemical Co., (Milwaukee, WI), Acros Organics (Morris Plains, NJ), Fisher Scientific (Pittsburgh, PA), Sigma (St. Louis, MO), Pfizer (New York, NY), GlaxoSmithKline (Raleigh, NC), Merck (Whitehouse Station, NJ), Johnson & Johnson (New Brunswick, NJ), Aventis (Bridgewater, NJ), AstraZeneca (Wilmington, DE), Novartis (Basel, Switzerland), Wyeth (Madison, NJ), Bristol-Myers-Squibb (New York, NY), Roche (Basel, Switzerland), Lilly (Indianapolis, IN), Abbott (Abbott Park, IL), Schering Plough (Kenilworth, NJ), or Boehringer Ingelheim (Ingelheim, Germany), or are prepared by methods known to those skilled in the art following procedures set forth in references such as Fieser and Fieser’s Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991), Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and Suppiementals (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991); March’s Advanced Organic Chemistry, (John Wiley and Sons, 4th Edition); and Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989). Other materials, such as the pharmaceutical carriers can be obtained from commercial sources.
[0428] Reactions to produce the compounds described herein can be carried out in solvents, which can be selected by one of skill in the art of organic synthesis. Solvents can be substantially nonreactive with the starting materials (reactants), the intermediates, or products under the conditions at which the reactions are carried out, e.g., temperature and pressure. Reactions can be carried out in one solvent or a mixture of more than one solvent. Product or intermediate formation can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e g., ‘H or 13C) infrared spectroscopy, spectrophotometry (e g., UV-visible), or mass spectrometry, or by chromatography such as high-performance liquid chromatography (HPLC) or thin layer chromatography.
[0429| In embodiments, portions of the cargo constructs can be prepared by solid phase peptide synthesis wherein the amino acid a-N-terminus is protected by an acid or base protecting group. Such protecting groups should have the properties of being stable to the conditions of peptide linkage formation while being readily removable without destruction of the growing peptide chain or racemization of any of the chiral centers contained therein. Suitable protecting groups are 9- fluorenylmethyloxycarbonyl (Fmoc), t-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz), biphenylisopropyloxycarbonyl, t-amyloxycarbonyl, isobomyloxycarbonyl, a,a-dimethyl-3,5- dimethoxybenzyloxycarbonyl, o-nitrophenylsulfenyl, 2-cyano-t-butyloxycarbonyl, and the like. The 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group can be used for the synthesis of the disclosed compounds. Other side chain protecting groups are, for side chain amino groups like lysine and arginine, 2,2,5,7,8-pentamethylchroman-6-sulfonyl (pmc), nitro, p-toluenesulfonyl, 4- methoxybenzene- sulfonyl, Cbz, Boc, and adamantyloxycarbonyl, for tyrosine, benzyl, o- bromobenzyloxy-carbonyl, 2,6-dichlorobenzyl, isopropyl, t-butyl (t-Bu), cyclohexyl, cyclopenyl and acetyl (Ac); for serine, t-butyl, benzyl and tetrahydropyranyl; for histidine, trityl, benzyl, Cbz, p-toluenesulfonyl and 2,4-dinitrophenyl; for tryptophan, formyl; for asparticacid and glutamic acid, benzyl and t-butyl and for cysteine, triphenylmethyl (trityl).
[0430] In the solid phase peptide synthesis method, the a-C-terminal amino acid is attached to a suitable solid support or resin. Suitable solid supports useful for the above synthesis are those materials which are inert to the reagents and reaction conditions of the stepwise condensationdeprotection reactions, as well as being insoluble in the media used. Solid supports for synthesis of a-C-terminal carboxy peptides is 4-hydroxymethylphenoxymethyl-copoly(styrene-l% divinylbenzene) or 4-(2',4*-dimethoxyphenyl-Fmoc-aminomethyl)phenoxyacetamidoethyl resin available from Applied Biosystems (Foster City. Calif.). The a-C-terminal amino acid is coupled to the resin by means of N,N'-dicyclohexylcarbodiimide (DCC), N.N1 -diisopropylcarbodiimide (DIC) or O-benzotriazol-l-yl-N,N,N',N'-tetramethyluroniumhexafluorophosphate (HBTU), with or without 4-dimethylaminopyridine (DMAP), 1 -hydroxybenzotriazole (HOBT), benzotriazol- 1- yloxy-tris(dimethylamino)phosphoniumhexafluorophosphate (BOP) oorr bis(2-oxo-3- oxazolidinyl)phosphine chloride (BOPCI), mediated coupling for from about 1 to about 24 hours at a temperature of between 10°C and 50°C in a solvent such as dichloromethane or DMF. When the solid support is 4-(2',4l-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy-acetamidoethyl resin, the Fmoc group is cleaved with a secondary amine, for example, piperidine, prior to coupling with the a-C-terminal amino acid as described above. One method for coupling to the deprotected 4 (2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy-acetamidoethyl resin is O-benzotriazol-1- yl-N,N,N',N'-tetramethyluroniumhexafluorophosphate (HBTU, 1 equiv.) and 1- hydroxybenzotriazole (HOBT, 1 equiv.) in DMF. The coupling of successive protected amino acids can be carried out in an automatic polypeptide synthesizer. In one example, the a-N-terminus in the amino acids of the growing peptide chain are protected with Fmoc. The removal of the Fmoc protecting group from the a-N-terminal side of the growing peptide is accomplished by treatment with a secondary amine, for example, piperidine. Each protected amino acid is then introduced in about 3 -fold molar excess, and the coupling can be carried out in DMF. The coupling agent can be O-benzotriazol-l-yl-N,N,N',N,-tetramethyluroniumhexafluorophosphate (HBTU, 1 equiv.) and 1- hydroxybenzotriazole (HOBT, 1 equiv.). At the end of the solid phase synthesis, the polypeptide is removed from the resin and deprotected, either successively or in a single operation. Removal of the polypeptide and deprotection can be accomplished in a single operation by treating the resinbound polypeptide with a cleavage reagent comprising thianisole, water, ethanedithiol and trifluoroacetic acid. In cases wherein the a-C-terminal of the polypeptide is an alkylamide, the resin is cleaved by aminolysis with an alkylamine. Alternatively, the peptide can be removed by transesterification, e.g. with methanol, followed by aminolysis or by direct transamidation. The protected peptide can be purified at this point or taken to the next step directly. The removal of the side chain protecting groups can be accomplished using the cleavage cocktail described above. The fully deprotected peptide can be purified by a sequence of chromatographic steps employing any or all of the following types: ion exchange on a weakly basic resin (acetate form); hydrophobic adsorption chromatography on underivitized polystyrene-divinylbenzene (for example, Amberlite XAD); silica gel adsorption chromatography; ion exchange chromatography on carboxymethylcellulose; partition chromatography, e.g. on Sephadex G-25, LH-20 or countercurrent distribution; high performance liquid chromatography (HPLC), especially reversephase HPLC on octyl- or octadecylsilyl-silica bonded phase column packing.
[0431] Polymers, such as PEG groups, can be attached to an oligonucleotide, such as an ASO, a cCCP, EP, or ocular delivery construct under any suitable conditions. Any means known in the art can be used, including via acylation, reductive alkylation, Michael addition, thiol alkylation or other chemoselective conjugation/ligation methods through a reactive group on the PEG moiety (e g., an aldehyde, amino, ester, thiol, a-haloacetyl, maleimido or hydrazino group) to a reactive group on the ASO a cCCP, an EEV, or a compound comprising an EEV (e.g., an aldehyde, amino, ester, thiol, a-haloacetyl, maleimido or hydrazino group). Activating groups which can be used to link the water soluble polymer to one or more proteins include without limitation sulfone, maleimide, sulfhydryl, thiol, triflate, tresylate, azidirine, oxirane, 5-pyridyl, and alpha-halogenated acyl group (e.g., a-iodo acetic acid, a-bromoacetic acid, a-chloroacetic acid). If attached to the ASO, a cCCP, an EEV, or a compound comprising an EEV by reductive alkylation, the polymer selected should have a single reactive aldehyde so that the degree of polymerization is controlled. See, for example, Kinstler et al., Adv. Drug. Delivery Rev. (2002), 54: 477-485; Roberts et aL, Adv. Drug Delivery Rev. (2002), 54: 459-476; and Zalipsky et al., Adv. Drug Delivery Rev. (1995), 16: 157-182.
[0432] In order to direct covalently link the AC or linker to the CPP, appropriate amino acid residues of the CPP may be reacted with an organic derivatizing agent that is capable of reacting with a selected side chain or theN- or C-termini of an amino acids. Reactive groups on the peptide or conjugate moiety include, e.g., an aldehyde, amino, ester, thiol, a-haloacetyl, maleimido or hydrazino group. Derivatizing agents include, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N -hydroxy succinimide (through lysine residues), glutaraldehyde, succinic anhydride or other agents known in the art.
[0433] Methods of making a therapeutic oligonucleotide and conjugating oligonucleotide to linear CPP are generally described in US Pub. No. 2018/0298383, which is herein incorporated by reference for all purposes. The methods may be applied to the cyclic CPPs disclosed herein.
[0434] Synthetic schemes are provided in FIGS. 3A-3D and FIG. 4.
[0435] Non-limiting examples of compounds that include a CPPs and a reactive group useful for conjugation to an therapeutic moiety, such as an oligonucleotide, a polypeptide, or a small molecule, are shown in Table 3. Example linker groups are also shown. Example reactive groups include tetrafluorophenyl ester (TFP), free carboxylic acid (COOH), an azide (N3) and an alkyne (e.g., a cyclooctyne). In Table 3, n is an integer from 0 to 20; Pipa6 is AcRXRRBRRXRYQFLIRXRBRXRB wherein B is P-Alanine and X is aminohexanoic acid; Dap is 2,3-diaminopropionic acid; NLS is a nuclear localization sequence; |3A is beta alanine; -ss- is a disulfide; PABC is poly(A) binding protein C-terminal domain; Cx where x is a number is an alkyl chain of length x; and BCN is bicyclo [6.1.0]nonyne.
Table 3. Compounds that include a CPPs and a reactive group
Figure imgf000123_0001
Figure imgf000124_0001
[0436] In embodiments, the cCPPs have free carboxylic acid groups that may be utilized for conjugation to a therapeutic moiety, such as an oligonucleotide, a polypeptide, or a small molecule. In embodiments, the EEVs have free carboxylic acid groups that may be utilized for conjugation to the therapeutic moiety.
[0437] FIGS. 3A-3D and FIG. 4 show example conjugation chemistry schemes. The CPPs of the examples may be modified with linker that have reactive groups allowing conjugation. The linkers in each example scheme may not be fully shown and/or are non-limiting. Two example schemes showing conjugation of a CPP or a modified CPP to the modified 5' end of an oligonucleotide via an amide bond is shown in FIG. 3A. An example scheme showing the conjugation of a CPP or modified CPP to the modified 3' end of an oligonucleotide via an amide bond is shown in FIG. 3B. An example scheme showing the conjugation of an azide modified CPP to a 5 '-cyclooctyne modified PMO via strain-promoted azide-alkyne cycloaddition is shown in FIG. 3C. Several azide-alkyne conjugation reactions for conjugating a modified 3' of an oligonucleotide to a modified CPP are shown in FIG. 3D.
[0438] Various examples showing conjugation chemistries using a bifunctional compound that includes a PEG moiety and two reactive handles (e g., activated tetrafluorophenyl ester, alkyne, and N -hydroxysuccinimide ester) are shown in FIG. 4. One of the reactive handles is used to conjugate the AC to the bifunctional molecule and the second reactive handle is used to conjugate the CPP to the bifunctional molecule thereby conjugating the oligonucleotide to the CPP.
Compositions and Formulations
[0439] In embodiments, compositions are provided that include one or more cargo conjugates. In embodiments, the composition is a pharmaceutical composition; that is, a composition designed for administration to a subject.
[0440] In embodiments, pharmaceutically acceptable salts and/or prodrugs of the disclosed cargo conjugates are disclosed. Pharmaceutically acceptable salts include salts of the disclosed cargo conjugates that are prepared with acids or bases, depending on the particular substituents found on the cargo conjugates. Under conditions where the cargo conjugates are sufficiently basic or acidic to form stable nontoxic acid or base salts, administration of the cargo conjugates as salts can be appropriate. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, or magnesium salt. Examples of physiologically acceptable acid addition salts include hydrochloric, hydrobromic, nitric, phosphoric, carbonic, sulfuric, and organic acids like acetic, propionic, benzoic, succinic, fumaric, mandelic, oxalic, citric, tartaric, malonic, ascorbic, alpha-ketoglutaric, alpha-glycophosphoric, maleic, tosyl acid, methanesulfonic, and the like. Thus, disclosed herein are the hydrochloride, nitrate, phosphate, carbonate, bicarbonate, sulfate, acetate, propionate, benzoate, succinate, fumarate, mandelate, oxalate, citrate, tartarate, malonate, ascorbate, alpha-ketoglutarate, alpha-glycophosphate, maleate, tosylate, and mesylate salts. Pharmaceutically acceptable salts of a cargo conjugate can be obtained using standard procedures well known in the art, for example, by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion. Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example calcium) salts of carboxylic acids can also be made.
[0441J In vivo application of the disclosed cargo conjugates, and compositions containing them, can be accomplished by any suitable method and technique presently or prospectively known to those skilled in the art. For example, the disclosed cargo conjugates can be formulated in a physiologically- or pharmaceutically-acceptable form and administered by any suitable route known in the art including, for example, compositions for dropping on the eye or for injecting into the eye (intraocular administration). Administration of the disclosed cargo conjugates or compositions can be a single administration, or at continuous or distinct intervals as can be readily determined by a person skilled in the art.
[0442] The cargo conjugates, and compositions comprising them, can also be administered utilizing liposome technology. These delivery methods can, advantageously, provide a uniform dosage over an extended period of time. The cargo conjugates can also be administered in their salt derivative forms or crystalline forms.
[0443] The cargo conjugates can be formulated according to known methods for preparing pharmaceutically acceptable compositions (pharmaceutical compositions). Formulations are described in detail in a number of sources which are well known and readily available to those skilled in the art. For example, Remington's Pharmaceutical Science by E.W. Martin (1995) describes formulations that can be used in connection with the disclosed methods. In general, the cargo conjugates can be formulated such that an effective amount of the cargo conjugates is combined with a suitable carrier in order to facilitate effective administration of the cargo conjugates. The compositions used can also be in a variety of forms. These include, for example, liquid dosage forms, such as liquid solutions or suspension, and injectable and infusible solutions. The form depends on the intended mode of administration and therapeutic application. The compositions can also include conventional pharmaceutically-acceptable carriers and diluents which are known to those skilled in the art. Examples of carriers or diluents for use with the compounds include water, ethanol, dimethyl sulfoxide, glycerol, alumina, starch, saline, and equivalent carriers and diluents. To provide for the administration of such dosages for the desired therapeutic treatment, compositions can advantageously comprise between about 0.1% and 100% by weight of the total of one or more of the subject cargo conjugates based on the weight of the total composition including carrier or diluent.
[0444] Formulations suitable for administration include, for example, aqueous sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the interstitial fluid of the eye of the intended recipient; and aqueous and nonaqueous sterile suspensions, which can include suspending agents and thickening agents. The formulations can be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and can be stored in a freeze dried (lyophilized) condition requiring only the condition of the sterile liquid carrier, for example, water for injections, prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powder, granules, tablets, etc. It should be understood that in addition to the ingredients mentioned above, the compositions disclosed herein can include other agents conventional in the art having regard to the type of formulation in question.
[0445] Cargo conjugates, and compositions comprising them, can be delivered to a cell either through direct contact with the cell or via a carrier means. Carrier means for delivering compounds and compositions to cells are known in the art and include, for example, encapsulating the composition in a liposome moiety. Another means for delivery of compounds and compositions to a cell can comprise attaching the cargo conjugates to a protein or nucleic acid that is targeted for delivery to the target cell. U.S. Patent No. 6,960,648 and U.S. Application Publication Nos. 20030032594 and 20020120100 disclose amino acid sequences that can be coupled to another composition and that allows the composition to be translocated across biological membranes. U.S. Application Publication No. 20020035243 also describes compositions for transporting biological moieties across cell membranes for intracellular delivery. Cargo conjugates can also be incorporated into polymers, examples of which include poly (D-L lactide-co-glycolide) polymer for intracranial tumors; poly[bis(p-carboxyphenoxy) propane: sebacic acid] in a 20:80 molar ratio (as used in GLIADEL); chondroitin; chitin; and chitosan.
[0446] For the treatment of an ocular disease, the cargo conjugate can be administered to a patient in need of treatment in combination with other active agents designed to treat the disease. These other substances or treatments can be given at the same as or at different times from the cargo conjugates. For example, a pharmaceutical composition comprising a cargo conjugate can further include a second active agent designed to treat the disease.
[0447] Cargo conjugates and compositions containing the same, including pharmaceutically acceptable salts or prodrugs thereof, can be administered by drop, infusion, or injection. In embodiments, the compounds and compositions can be administered parenterally. In embodiments, the compounds and compositions can be administered intravenously. In embodiments, the compounds and compositions can be administered subcutaneously. In embodiments, the compounds and compositions disclosed herein can be administered intraocularly. In embodiments, the compounds and compositions can be administered intravitreally. Solutions of the active agent or its salts can be prepared in water, optionally mixed with a nontoxic surfactant Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations can contain a preservative to prevent the growth of microorganisms.
[0448] The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient, which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. The ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. Optionally, the prevention of the action of microorganisms can be brought about by various other antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. Isotonic agents can be included, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the inclusion of agents that delay absorption, for example, aluminum monostearate and gelatin
[0449] Sterile injectable solutions are prepared by incorporating a cargo conjugate and/or other agent in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation include vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.
[0450] For topical administration, cargo conjugates and compositions containing the same can be applied in as a liquid or solid. However, it will generally be desirable to administer them topically to the eye as compositions, in combination with a ophthalmologically acceptable carrier, which can be a solid or a liquid. In embodiments, compounds are formulated into a solution or suspension for topical application to a surface of the eye, such as to a surface of the cornea.
[0451] Useful liquid carriers include wzater, alcohols or glycols or water-alcohol/glycol blends, in which the compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to modify the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers, for example.
[0452] Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the surface of the eye.
[0453] Also disclosed are pharmaceutical compositions that comprise a compound disclosed herein in combination with a pharmaceutically acceptable carrier, including, but not limited to, pharmaceutical compositions adapted for oral, topical, parenteral or intraocular administration, comprising an amount of a compound The dose administered to a patient, particularly a human, should be sufficient to achieve a therapeutic response in the patient over a reasonable time frame, without lethal toxicity, and causing no more than an acceptable level of side effects or morbidity. One skilled in the art will recognize that dosage will depend upon a variety of factors including the condition (health) of the subject, the body weight of the subject, kind of concurrent treatment, if any, frequency of treatment, therapeutic ratio, as well as the severity and stage of the pathological condition.
[0454] Also disclosed are kits that comprise a cargo conjugate or composition containing the same in one or more containers. The disclosed kits can optionally include pharmaceutically acceptable carriers and/or diluents. A kit can include one or more other components, adjuncts, or adjuvants. A kit includes one or more additional active agents for treating an eye disease. A kit can include instructions or packaging materials that describe how to administer a cargo conjugate or composition of the kit. Containers of the kit can be of any suitable material, e.g., glass, plastic, metal, etc., and of any suitable size, shape, or configuration. A cargo conjugate can be provided in the kit as a solid, such as a tablet, pill, or powder form. A cargo conjugate can be provided in the kit as a liquid or solution. A kit can comprise an ampoule or syringe containing a cargo conjugate or composition containing the same in liquid or solution form.
Methods of Use
[0455] Also provided herein are methods of use of the cargo conjugates or a pharmaceutical compositions comprising one or more cargo conjugates. The method comprises identifying a subject having a disease or disorder of the eye and administering a cargo conjugate or a composition comprising one or more cargo conjugates to the subject. The cargo conjugate includes a therapeutic moiety designed to treat an ocular disease. As such, the method may be a method of treatment of an ocular disease.
[0456] Identifying a subject includes identifying a subject that has an ocular disease. The subject may be a mammal. The subject may be human. A subject that has an ocular disease may display one or more symptoms or clinical signs associated with an ocular disease; may have a genetic signature associated with an ocular disease, for example, may have a mutation in a gene associated with an ocular disease; or both. [0457] Identification of a subject having an ocular disease may include diagnosing the subject with an ocular disease. Diagnosis of an ocular disease may be by way of a physician or other health care provider conducting tests and exams to identify a cause of symptoms displayed by a subject.
[0458] Genetic testing may be used to diagnose a subject with an ocular disease. Genetic testing may include sequencing a portion of the subjects genome to identify genetic anomalies in one or more genes associated with an ocular disease. To identify genetic anomalies, the sequenced portion of a subject’s genome can be compared to a standard genome that does not contain genetic anomalies in the genes associated with the eye. Sequencing technologies and analysis methods are well known.
[0459] Additionally, genetic testing following diagnosis of an ocular disease may be used to identify a particular genetic anomaly causing the disease. Identification of the particular genetic anomaly, such as a mutation that results in the displayed phenotype, may allow one to choose a particular therapeutic moiety designed to have a biological effect given the genetic anomaly. For example, some therapeutic moieties are designed to be effective when a subject displays a particular genetic anomaly of a disease and may not be effective or as effective if the subject displays a different anomaly of the same disease. For example, a subject diagnosed with an ocular disease characterized by one or both of two independent mutations may undergo genetic testing to determine if they have the first mutation, the second mutation, or both. If the subject has the first mutation, a first cargo conjugate having a first therapeutic moiety designed to be effective in a disease state of the first mutation may be administered to the subject. If the subject has the second mutation, a second cargo conjugate having a second therapeutic moiety designed to be effective in a disease state of the second mutation may be administered to the subject. The first cargo conjugate may not be effective or as effective if the subject displays the second mutation and the second cargo conjugate may not be effective or as effective if the subject displays the first mutation.
[0460] The method of administration further includes administering a cargo conjugate or a composition comprising one or more cargo conjugates to the subject. In embodiments, the cargo conjugate or a pharmaceutical composition directly to the eye of the subject; that is, the cargo conjugate or pharmaceutical composition is ocularly administered to the subject. In embodiments, the composition is administered directly to a tissue, such as, for example, to the cornea, sclera, lens, iris, ciliary body, optic nerve, choroid, or retina. [0461] In embodiments, the cargo conjugate or pharmaceutical composition is topically administered. In embodiments, the cargo conjugate or pharmaceutical composition is delivered to the cornea. For example, the pharmaceutical composition may be an eye drop formulation that is contacted with the cornea of the subject’s eye. Other routes of topical administration include drug delivery contacts soaked or coated with a cargo conjugate or pharmaceutical composition. The contact may be designed for and allow a delayed and/or sustained release of the cargo conjugate.
[0462] In embodiments, the cargo conjugate or pharmaceutical composition is periocularly administered. Periocular administration includes drug deposition on the scleral external surface. The drug can diffuse through the scelara. Examples of periocular administration include, but are not limited to, subconjunctival administration, subTenon injection, retrobulbar administration, peribulbar juxtascleral administration, posterior juxtascleral administration, and iontophoresis techniques such as the EYEGATE U system from Kiora Pharmaceuticals, Inc. (Encinitas, CA).
[0463] In embodiments, the cargo conjugate or pharmaceutical composition is intravitreally delivered. Intravitreal injection delivers the injected species into the vitreous humor. Techniques that may be used for intravitreal administration include intravitreal injection (also called intraocular injection) and intravitreal infusion.
[0464] In embodiments, the cargo conjugate or pharmaceutical composition is subretinally administered. Subretinal administration includes delivery of drug or composition to the retina. Examples of subretinal administration include retinal injection (subretinal injection) and implantation of biodegradable drug delivery devices or non-biodegradable medical devices. Subretinal administration may involve subjecting the subject to a pars plana vitrectomy and a retinotomy. Subretinal administration may deliver the drug or composition to the space between or including the retinal pigment epithelium layer and the photoreceptor layer.
[0465] In embodiments, the cargo conjugate or pharmaceutical composition is suprachoroidally administered. Suprachoroidal administration involves depositing a drug or composition in the suprachoroidal space between the scelara and the choroid. Techniques that may be used for suprachoroidal administration include injection and infusion using, for example, a micro needle, a cannulas, or a catheter.
[0466] In embodiments, the cargo conjugate or pharmaceutical composition is intracamerally administered. Intracameral administration involves depositing a drug or composition into the anterior chamber of the eye. Intracameral administration may be accomplished by intracameral injection.
[0467] In embodiments, the cargo conjugate or pharmaceutical composition is delivered to the eye through the use of a medical device. The medical device may be biodegradable or non- biodegradable. The medical device may be implanted at any location within the eye. For example, a cargo conjugate or pharmaceutical composition may be a part of an intravitreal implant, a Cul- de-sac implant, or an episcleral implant.
[0468] Therapeutically effective dosages of the cargo conjugates and pharmaceutical compositions can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art.
[0469] Therapeutically effective dosage ranges for the administration of the cargo conjugates and compositions containing the same are those large enough to produce the desired effect in which the symptoms or disorder are affected. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days.
Certain Definitions
[0470] The type and origin of the molecule are not limited by the case and/or typeface (e.g., bold, italics, etc.). A specific name of gene, transcript, or protein may be denoted as unitalicized and upper case, italicized and upper case, unitalicized and upper case, or unitalicized and lower case. The specific type and origin of the molecule are to be understood in the context of the use of the stated molecule.
[0471] As used in the description and the appended claims, the singular forms “a,” “an ” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a composition” includes mixtures of two or more such compositions, reference to “an agent” includes mixtures of two or more such agents, reference to “the component” includes mixtures of two or more such components, and the like. [0472] The term “about” when immediately preceding a numerical value means a range (e g., plus or minus 10% of that value). For example, “about 50” can mean 45 to 55, “about 25,000” can mean 22,500 to 27,500, etc., unless the context of the disclosure indicates otherwise, or is inconsistent with such an interpretation. For example, in a list of numerical values such as “about 49, about 50, about 55, .. “about 50” means a range extending to less than half the interval(s) between the preceding and subsequent values, e.g., more than 49.5 to less than 52.5. Furthermore, the phrases “less than about” a value or “greater than about” a value should be understood in view of the definition of the term “about” provided herein. Similarly, the term “about” when preceding a series of numerical values or a range of values (e.g., “about 10, 20, 30” or “about 10-30”) refers, respectively to all values in the series, or the endpoints of the range.
[0473] As used herein, the term “cyclic cell penetrating peptide” or “cCPP” refers to a peptide that facilitates the delivery of a cargo, e.g., a therapeutic moiety, into a cell.
[0474] As used herein, the term “endosomal escape vehicle” (EEV) refers to an ocular delivery construct comprising a cCPP, an exocyclic peptide (EP), and a linker.
[0475] As used herein, the term “EEV-conjugate” refers to an endosomal escape vehicle defined herein conjugated by a chemical linkage (i.e., a covalent bond or non-covalent interaction) to a cargo. The cargo can be a therapeutic moiety (e.g., an oligonucleotide, peptide or small molecule) that can be delivered into a cell by the EEV.
[0476] As used herein, the term “exocyclic peptide” (EP) refers to two or more amino acid residues linked by a peptide bond that can be conjugated to a cyclic cell penetrating peptide (cCPP), a linker, a therapeutic moiety, or any combination thereof. The EP, when a part of an ocular delivery construct, may alter the tissue distribution and/or retention of the compound. Typically, the EP comprises at least one positively charged amino acid residue, e.g , at least one lysine residue and/or at least one arginine residue. Non-limiting examples of EP are described herein. The EP can be a peptide that has been identified in the art as a “nuclear localization sequence” (NLS).
[0477] As used herein, “linker” or “L” refers to a moiety that covalently couples two or more components of a delivery construct and/or a cargo construct; includes one or more functional groups that can be used to conjugate one or more components to the linker; or both. For example, a linker can covalently couple one or more moieties (e.g., an exocyclic peptide (EP) and a cargo; a cCPP and an EP; a cCPP and a cargo; a cCPP, an EP, and a cargo) to the cyclic cell penetrating peptide (cCPP). A linker can covalently link a cargo and a delivery construct and/or two or more components of a delivery construct. The linker can comprise a natural or non-natural amino acid or polypeptide. The linker can be a synthetic compound containing two or more appropriate functional groups suitable to conjugate one or more components of a delivery construct and/or a delivery construct to a cargo. The linker can comprise a polyethylene glycol (PEG) moiety. The linker can comprise one or more amino acids. The cCPP may be covalently bound to a cargo via a linker.
[0478] As used herein, the term “oligonucleotide,” “nucleic acid,” and “polynucleotide” refer to an oligomeric compound comprising a plurality of linked nucleotides or nucleosides. One or more nucleotides of an oligonucleotide can be modified. An oligonucleotide can comprise ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). Oligonucleotides can be composed of natural and/or modified nucleobases, sugars and covalent intemucleoside linkages, and can further include non- nucleic acid conjugates.
[0479] The terms “peptide,” “protein,” and “polypeptide” are used interchangeably to refer to a natural or synthetic molecule comprising two or more amino acids linked by the carboxyl group of one amino acid to the alpha amino group of another. Two or more amino acid residues can be linked by the carboxyl group of one amino acid to the alpha amino group. Two or more amino acids of the polypeptide can be joined by a peptide bond. The polypeptide can include a peptide backbone modification in which two or more amino acids are covalently attached by a bond other than a peptide bond. The polypeptide can include one or more non-natural amino acids, amino acid analogs, or other synthetic molecules that are capable of integrating into a polypeptide. The term polypeptide includes naturally occurring and artificially occurring amino acids. The term polypeptide includes peptides, for example, that include from about 2 to about 100 amino acid residues as well as proteins, that include more than about 100 amino acid residues, or more than about 1000 amino acid residues, including, but not limited to therapeutic proteins such as antibodies, enzymes, receptors, soluble proteins and the like.
[0480] The term “therapeutic agent” or “therapeutic moiety” can be used to refer to a cargo that has therapeutic, prophylactic or other biological activity. The therapeutic agent can be a peptide, oligonucleotide or a small molecule. The therapeutic agent can be an enzyme. The therapeutic agent can be an antibody or antigen binding fragment. The therapeutic agent can be an oligonucleotide. The therapeutic agent can be an antisense oligonucleotide. The therapeutic agent can be one or more components of a gene editing machinery (GEM). The therapeutic agent can be an oligonucleotide encoding one or more components of a gene editing machinery (GEM). The therapeutic agent can include gRNA. The therapeutic agent can include a nuclease or an oligonucleotide encoding a nuclease. In embodiments, the nuclease is a Cas nuclease. In embodiments, the nuclease is a Cas9 nuclease. The therapeutic agent can include a ribonucleoprotein (RNP) or aann oligonucleotide encoding a RNP.
[0481] The term “small molecule” refers to an organic compound with pharmacological activity and a molecular weight of less than about 2000 Daltons, or less than about 1000 Daltons, or less than about 500 Daltons. Small molecule therapeutics are typically manufactured by chemical synthesis.
[0482] As used herein, the term “contiguous” refers to two amino acids, which are connected by a covalent bond. For example, in the context of a representative cyclic cell penetrating peptide
(cCPP) such as
Figure imgf000135_0002
, AA1/AA2, AA2/AA3. AA3/AA4,and AA5/AA1 exemplify pairs of contiguous amino acids.
[0483] A residue of a chemical species, as used herein, refers to a derivative of the chemical species that is present in a particular product. To form the product, at least one atom of the species is replaced by a bond to another moiety, such that the product contains a derivative, or residue, of the chemical species. For example, the cyclic cell penetrating peptides (cCPP) can have amino acids (e g., arginine) incorporated therein through formation of one or more peptide bonds. The amino acids incorporated into the cCPP may be referred to residues, or simply as an amino acid.
H
Thus, arginine or an arginine residue refers to
Figure imgf000135_0001
[0484] The term “protonated form thereof' refers to a protonated form of an amino acid. For example, the guanidine group on the side chain of arginine may be protonated to form a guanidinium group. The structure of a protonated form of arginine is
Figure imgf000136_0001
[0485] As used herein, the term “chirality” refers to a molecule that has more than one stereoisomer that differs in the three-dimensional spatial arrangement of atoms, in which one stereoisomer is a non-superimposable mirror image of the other. Amino acids, except for glycine, have a chiral carbon atom adjacent to the carboxyl group. The term “enantiomer” refers to stereoisomers that are chiral. The chiral molecule can be an amino acid residue having a “D” and “L” enantiomer. Molecules without a chiral center, such as glycine, can be referred to as “achiral.” [0486] As used herein, the term “hydrophobic” refers to a moiety that is not soluble in water or has limited solubility in water. Generally, neutral moieties and/or non-polar moieties, or moieties that are predominately neutral and/or non-polar are hydrophobic. Hydrophobicity can be measured by one of the methods disclosed herein.
[0487] As used herein “aromatic” refers to an unsaturated cyclic molecule having 4n + 2 n electrons, wherein n is any integer. The term “non-aromatic” refers to any unsaturated cyclic molecule which does not fall within the definition of aromatic. For example, any linear, branched or cyclic molecule which does not fall within the definition of aromatic is non-aromatic. Examples of non-aromatic amino acids include, but are not limited to, glycine and citrulline.
[0488] “Alkyl”, “alkyl chain” or “alkyl group” refer to a fully saturated, straight or branched hydrocarbon chain radical having from one to forty carbon atoms, and which is attached to the rest of the molecule by a single bond. Alkyls comprising any number of carbon atoms from 1 to 40 are included. An alkyl comprising up to 40 carbon atoms is a C1-C40 alkyl, an alkyl comprising up to 10 carbon atoms is a C1-C10 alkyl, an alkyl comprising up to 6 carbon atoms is a C1-C6 alkyl and an alkyl comprising up to 5 carbon atoms is a C1-C5 alkyl. A C1-C5 alkyl includes Cs alkyls, C* alkyls, C3 alkyls, C2 alkyls and Ci alkyl (z.e., methyl). A Ci-Ce alkyl includes all moieties described above for C1-C5 alkyls but also includes Ce alkyls. A C1-C10 alkyl includes all moieties described above for C1-C5 alkyls and Ci-Ce alkyls, but also includes Ci, Cs, C9 and C10 alkyls. Similarly, a Ci-Cu alkyl includes all the foregoing moieties, but also includes Cn and C12 alkyls. Non-limiting examples of C1-C12 alkyl include methyl, ethyl, zz-propyl, z-propyl, sec-propyl, zz-butyl, z-butyl, sec-butyl, Z-butyl, zz-pentyl, /-amyl, w-hexyl, zz-heptyl, w-octyl, rz-nonyl, zz-decyl, zz-undecyl, andzz- dodecyl. Unless stated otherwise specifically in the specification, an alkyl group can be optionally substituted.
[0489] “Alkylene”, “alkylene chain” or “alkylene group” refers to a fully saturated, straight or branched divalent hydrocarbon chain radical, having from one to forty carbon atoms. Non-limiting examples of C2-C40 alkylene include ethylene, propylene, zz-butylene, ethenylene, propenylene, zz-butenylene, propynylene, zz-butynylene, and the like. Unless stated otherwise specifically in the specification, an alkylene chain can be optionally substituted.
[0490] “Alkenyl”, “alkenyl chain” or “alkenyl group” refers to a straight or branched hydrocarbon chain radical having from two to forty carbon atoms and having one or more carbon-carbon double bonds. Each alkenyl group is attached to the rest of the molecule by a single bond. Alkenyl groups comprising any number of carbon atoms from 2 to 40 are included. An alkenyl group comprising up to 40 carbon atoms is a C2-C40 alkenyl, an alkenyl comprising up to 10 carbon atoms is a C2- Cio alkenyl, an alkenyl group comprising up to 6 carbon atoms is a C2-C6 alkenyl and an alkenyl comprising up to 5 carbon atoms is a C2-C5 alkenyl. A C2-C5 alkenyl includes Cs alkenyls, C4 alkenyls, C3 alkenyls, and C2 alkenyls. A C2-C6 alkenyl includes all moieties described above for C2-C5 alkenyls but also includes C6 alkenyls. A C2-C10 alkenyl includes all moieties described above for C2-C5 alkenyls and C2-C6 alkenyls, but also includes C?, Cs, C9 and C10 alkenyls. Similarly, a C2-C12 alkenyl includes all the foregoing moieties, but also includes Cu and C12 alkenyls. Non-limiting examples of C2-C12 alkenyl include ethenyl (vinyl), 1 -propenyl, 2-propenyl (allyl), iso-propenyl, 2-methyl-l -propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1 -pentenyl, 2- pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1- heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-heptenyl. 6-heptenyl, 1-octenyl, 2-octenyl, 3- octenyl, 4-octenyl, 5-octenyl, 6-octenyl, 7-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 4-nonenyl, 5-nonenyl, 6-nonenyl, 7-nonenyl, 8-nonenyl, 1-decenyl, 2-decenyl, 3-decenyl, 4-decenyl, 5- decenyl, 6-decenyl, 7-decenyl, 8-decenyl, 9-decenyl, 1 -undecenyl, 2-undecenyl, 3-undecenyl, 4- undecenyl, 5-undecenyl, 6-undecenyl, 7-undecenyl, 8-undecenyl, 9-undecenyl, 10-undecenyl, 1- dodecenyl, 2-dodecenyl, 3-dodecenyl, 4-dodecenyl, 5-dodecenyl, 6-dodecenyl, 7-dodecenyl, 8- dodecenyl, 9-dodecenyl, 10-dodecenyl, and 11 -dodecenyl. Unless stated otherwise specifically in the specification, an alkyl group can be optionally substituted.
[0491] “Alkenylene”, “alkenylene chain” or “alkenylene group” refers to a straight or branched divalent hydrocarbon chain radical, having from two to forty carbon atoms, and having one or more carbon-carbon double bonds. Non-limiting examples of C2-C40 alkenylene include ethene, propene, butene, and the like. Unless stated otherwise specifically in the specification, an alkenylene chain can be optionally.
[0492] “Alkoxy” or “alkoxy group” refers to the group -OR, where R is alkyl, alkenyl, alkynyl, cycloalkyl, or heterocyclyl as defined herein. Unless stated otherwise specifically in the specification, an alkoxy group can be optionally substituted.
[0493] “Acyl” or “acyl group” refers to groups -C(O)R, where R is hydrogen, alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl, as defined herein Unless stated otherwise specifically in the specification, acyl can be optionally substituted.
[0494] “Alkylcarbamoyl” or “alkylcarbamoyl group” refers to the group -O-C(O)-NRaRb, where Ra and Rb are the same or different and are independently an alkyl, alkenyl, alkynyl, aryl, heteroaryl, as defined herein, or RaRb can be taken together to form a cycloalkyl group or heterocyclyl group, as defined herein. Unless stated otherwise specifically in the specification, an alkylcarbamoyl group can be optionally substituted.
[0495] “Alkylcarboxamidyl” or “alkylcarboxamidyl group” refers to the group -C(O)-NRaRb, where Ra and Rb are the same or different and are independently an alkyl, alkenyl, alkynyl, aryl, heteroaiyl, cycloalkyl, cycloalkenyl, cycloalkynyl, or heterocyclyl group, as defined herein, or RaRb can be taken together to form a cycloalkyl group, as defined herein. Unless stated otherwise specifically in the specification, an alkylcarboxamidyl group can be optionally substituted.
[0496] “Aryl” refers to a hydrocarbon ring system radical comprising hydrogen, 6 to 18 carbon atoms and at least one aromatic ring. For purposes of this invention, the aryl radical can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which can include fused or bridged ring systems. Aryl radicals include, but are not limited to, aryl radicals derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene, as-indacene, s-indacene, indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene, and triphenylene. Unless stated otherwise specifically in the specification, the term “aryl” is meant to include aryl radicals that are optionally substituted. [0497] “Heteroaryl” refers to a 5- to 20-membered ring system radical comprising hydrogen atoms, one to thirteen carbon atoms, one to six heteroatoms selected from nitrogen, oxygen and sulfur, and at least one aromatic ring For purposes of this invention, the heteroaryl radical can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which can include fused or bridged ring systems; and the nitrogen, carbon or sulfur atoms in the heteroaryl radical can be optionally oxidized; the nitrogen atom can be optionally quatemized. Examples include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, benzooxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[6][l,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl (benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[l,2-a]pyridinyl, carbazolyl, cirmolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 1-oxidopyridinyl, 1-oxidopyrimidinyl, 1-oxidopyrazinyl, 1-oxidopyridazinyl, 1 -phenyl- 1/7-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl, quinoxalinyl, quinolinyl, quinuclidinyl, isoquinolinyl, tetrahydroquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, and thiophenyl (i.e. thienyl). Unless stated otherwise specifically in the specification, a heteroaryl group can be optionally substituted.
[0498] The term “substituted” used herein means any of the above groups (i.e., alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, acyl, alkylcarbamoyl, alkylcarboxamidyl, alkoxycarbonyl, alkylthio, or arylthio) wherein at least one atom is replaced by a non-hydrogen atoms such as, but not limited to: a halogen atom such as F, Cl, Br, and I; an oxygen atom in groups such as hydroxyl groups, alkoxy groups, and ester groups; a sulfur atom in groups such as thiol groups, thioalkyl groups, sulfone groups, sulfonyl groups, and sulfoxide groups; a nitrogen atom in groups such as amines, amides, alkylamines, dialkylamines, arylamines, alkylarylamines, diarylamines, N-oxides, imides, and enamines; a silicon atom in groups such as trialkylsilyl groups, dialkylaryl silyl groups, alkyldiarylsilyl groups, and triarylsilyl groups; and other heteroatoms in various other groups. “Substituted” also means any of the above groups in which one or more atoms are replaced by a higher-order bond (e g., a double- or triple-bond) to a heteroatom such as oxygen in oxo, carbonyl, carboxyl, and ester groups; and nitrogen in groups such as imines, oximes, hydrazones, and nitriles. For example, “substituted” includes any of the above groups in which one or more atoms are replaced with -NRgRh, -NRgC(=O)Rh, -NRgC(=O)NRgRh, -NRgC(=O)ORh, -NRgSChRh, -OC(=O)NRgRn, - ORg, -SRg, -SORg, -SOzRg, -OSOzRg, -SChORg, =NSOiRg, and -SChNRgRh. “Substituted also means any of the above groups in which one or more hydrogen atoms are replaced with -C(=O)Rg, -C(=O)ORg, -C(=O)NRgRh, -CHzSChRg, -CHzSOzNRgRh. In the foregoing, Rg and Rh are the same or different and independently hydrogen, alkyd, alkenyl, alkynyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, haloalkyl, haloalkenyl, haloalkynyl, heterocyclyl, A-heterocyclyl, heterocyclylalkyl, heteroaryl, yV-heteroaryl and/or heteroarylalkyl. “Substituted" further means any of the above groups in which one or more atoms are replaced by an amino, cyano, hydroxyl, imino, nitro, oxo, thioxo, halo, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, haloalkyl, haloalkenyl, haloalkynyl, heterocyclyl, JV-heterocyclyl, heterocyclylalkyl, heteroaryl, A-heteroaryl and/or heteroarylalkyl group. “Substituted” can also mean an amino acid in which one or more atoms on the side chain are replaced by alkyl, alkenyl, alkynyl, acyl, alkylcarboxamidyl, alkoxy carbonyl, carbocyclyl, heterocyclyl, aryl, or heteroaiyl. In addition, each of the foregoing substituents can also be optionally substituted with one or more of the above substituents.
[0499] As used herein, by a “subject" is meant an individual. The subject can be a mammal. Thus, the “subject” can include domesticated animals (e.g., cats, dogs, etc ), livestock (e.g. , cattle, horses, pigs, sheep, goats, etc ), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc ), and birds. “Subject” can also include a mammal, such as a primate or a human. Thus, the subject can be a human or veterinary' patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician.
[0500] The term “inhibit” refers to a decrease in an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This can also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels. [0501] By “reduce” or other forms of the word, such as “reducing” or “reduction,” is meant lowering of an event or characteristic (e.g., tumor growth). It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to. For example, “reduces tumor growth” means reducing the rate of growth of a tumor relative to a standard or a control (e.g., an untreated tumor).
[0502] The term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to reducing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
[0503] The term “therapeutically effective” refers to the amount of the compound or composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.
[0504] The term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio.
[0505] The term “carrier” means a compound, composition, substance, or structure that, when in combination with a compound or composition, aids or facilitates preparation, storage, administration, delivery, effectiveness, selectivity, or any other feature of the compound or composition for its intended use or purpose. For example, a carrier can be selected to reduce any degradation of the active ingredient and any adverse side effects in the subject. [0506] As used herein, the term "pharmaceutically acceptable carrier" refers to sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid and the like. It can also be desirable to include isotonic agents such as sugars, sodium chloride and the like. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable media just prior to use. Suitable inert carriers can include sugars such as lactose.
[0507] “Amino acid” refers to an organic compound that includes an amino group and a carboxylic acid group and has the general formula
Figure imgf000142_0001
where R can be any organic group. An amino acid may be a naturally occurring amino acid or non-naturally occurring amino acid. An amino acid may be a proteogenic amino acid or a non-proteogenic amino acid. An amino acid can be an L-amino acid or a D- amino acid. The term "amino acid side chain" or "side chain" refers to the characterizing substituent (“R”) bound to the a-carbon of a natural or non-natural a-amino acid. An amino acid may be incorporated into a polypeptide via a peptide bond.
[0508] As used herein, an “uncharged” amino acid is an amino acid having a side chain that has a net neutral charge at pH 7.35 to 7.45. Examples of uncharged amino acids include, but are not limited to, glycine and citrulline.
[0509] As used herein, a “charged” amino acid is an amino acid having a side chain having a net charge at a pH of 7.35 to 7.45. An example of a charged amino acid is arginine. [0510] As used herein, “polyethylene glycol” and “PEG” are used interchangeably. “PEGm,” and “PEGm,” are, or are derived from, a molecule of the formula HO(CO)-(CIh)n-(OCH2CH2)m- NH2 where n is any integer from 1 to 5 and m is any integer from 1 to 23. In embodiments, n is 1 or 2. In embodiments, n is 1. In embodiments, n is 2. In embodiments, n is 1 and m is 2. In embodiments, n is 2 and m is 2 In embodiments, n is 1 and m is 4. In embodiments, n is 2 and m is 4. In embodiments, n is 1 and m is 12. In embodiments, n is 2 and m is 12.
[0511] As used herein, “miniPEGm” or “miniPEGm” are, or are derived from, a molecule of the formula HO(CO)-(CH2)n-(OCH2CH2)m-NH2 where n is i and m is any integer from 1 to 23. For example, “miniPEG2” or “miniPEG?” is, or is derived from, (2-[2-[2-aminoethoxy]ethoxy]acetic acid), and “miniPEG4” or “miniPEGi” is, or is derived from, HO(CO)-(CH2)n-(OCH2CH2)m- NH2 where n is 1 and m is 4. “2-[2-[2-aminoethoxy]ethoxy]acetic acid” is also referred to as AEEA, miniPEG or PEG2.
[0512] As used herein, the term “target” refers to a macromolecule associated with a disease. In embodiments, the target is a macromolecule implicated in a disease or pathology of the eye. In embodiments, the target is a polypeptide or protein. In embodiments, the target is an oligonucleotide. In embodiments, the oligonucleotide target comprises DNA. In embodiments, the oligonucleotide target comprises RNA. In embodiments, the oligonucleotide target comprises mRNA. In embodiments, the target is associated with an ocular disease.
[0513] As used herein, the terms “targeting” or “targeted to” refer to selective association of a therapeutic moiety with a target molecule. In embodiments, an antisense oligonucleotide may selectively bind with a target nucleic acid molecule or a region of a target nucleic acid molecule. In embodiments, a peptide may selectively bind with a target protein or region of a target protein. In embodiments, selective binding of a therapeutic moiety with a target molecule is useful for the treatment of a disease, pathologies or other abnormal states or conditions of the eye. In embodiments, the therapeutic moiety includes an antisense oligonucleotide that is capable of hybridizing to a target nucleic acid under physiological conditions. In embodiments, the antisense compound targets a specific portion or site within the target nucleic acid, for example, a portion of the target nucleic acid having at least one identifiable structure, function, or characteristic such as a particular exon or intron, or selected nucleobases or motifs within an exon or intron.
[0514] . As used herein” selectively binds” or “specifi cally binds” means that a therapeutic moiety exhibits enhanced binding to a target molecule as compared to another molecule. With reference to the interaction between an oligonucleotide therapeutic moiety and an oligonucleotide target, a therapeutic oligonucleotide is “specific” for a target oligonucleotide if the therapeutic oligonucleotide has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with a portion of the nucleic acid sequence of the target oligonucleotide when the nucleic acid sequences of the therapeutic oligonucleotide and the target oligonucleotide are aligned. A therapeutic oligonucleotide that is “selectively binds” or “specifically binds” to a target oligonucleotide is one that is capable of hybridizing to the target oligonucleotide of interest and not substantially hybridizing to other oligonucleotide sequences which are not of interest under stringent hybridization/washing conditions. An oligonucleotide which is “gene-specific” is specific for a target oligonucleotide sequence in a gene of interest and does not substantially hybridize to other genes.
[0515] As used herein, “hybridize” or “specifically hybridize” refers to a process where two complementary nucleic acid strands anneal to each in accordance with Watson-Crick base pairing rules. Hybridizations are typically and preferably conducted with probe-length nucleic acid molecules, preferably 20-100 nucleotides in length. Nucleic acid hybridization techniques are well known in the art. See, e.g., Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview, N.Y. Those skilled in the art understand how to determine the appropriate stringency of hybridization/washing conditions such that sequences having at least a desired level of complementarity will stably hybridize, while those having lower complementarity will not. For examples of hybridization conditions and parameters, see, e.g., Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview, N. Y.; Ausubel, F. M. et al. 1994, Current Protocols in Molecular Biology. John Wiley & Sons, Secaucus, N.J.
[0516] With reference to the interaction of an antibody or antigen-binding fragment thereof, or a binding protein, “selectively binds” or “specifically binds” or “specific binding” means that the interaction is dependent upon the presence of a particular structure (e g., an antigenic determinant or epitope) on the molecule. In embodiments, a binding protein or antibody or antigen-binding fragment thereof specifically binds to a target with a KD greater than lO^M, for example, with a KD between 10"*M and 10"12M.
[0517] As used herein, the terms "target nucleic acid sequence," “target polynucleotide sequence,” and “target nucleotide sequence” refer to the nucleic acid sequence or the nucleotide sequence to which a therapeutic moiety, such as an antisense oligonucleotide, binds or hybridizes. Target nucleic acids include, but are not limited, to a portion of a target transcript, target RNA (including, but not limited to pre-mRNA and mRNA or portions thereof), as well as a portion of target nontranslated RNA, such as miRNA. In embodiments, a target nucleic acid can be a portion of a target cellular gene (or mRNA transcribed from such gene) whose expression is associated with a particular disorder or disease state. The term “portion” refers to a defined number of contiguous (i.e., linked) nucleotides of a nucleic acid.
[0518] As used herein, the term “transcript” or “gene transcript” refers an RNA molecule transcribed from DNA and includes, but is not limited to mRNA, pre -mRNA, and partially processed RNA.
[0519] The terms “target transcript” and “target RNA” refer to the pre-mRNA or mRNA transcript that is bound by the therapeutic moiety. The target transcript may include a target nucleotide sequence. In embodiments, the target transcript includes a splice site. In embodiments, the target RNA includes a polyadenylation site or a portion thereof.
[0520] The term “target gene” and “gene of interest5 ’ refer to the gene of which modulation of the expression and/or activity is desired or intended. The target gene may be transcribed into a target transcript that includes a target nucleotide sequence. The target transcript may be translated into a protein of interest.
[0521] The term "target protein" refers to the polypeptide or protein encoded by the target transcript (e.g., target mRNA).
[0522] As used herein, the term “expression," "gene expression," “expression of a gene,” or the like refers to all the functions and steps by which information encoded in a gene is converted into a functional gene product, such as a polypeptide or a non-coding RNA, in a cell. Examples of noncoding RNA include transfer RNA (tRNA) and ribosomal RNA. Gene expression of a polypeptide includes transcription of the gene to form a pre-mRNA, processing of the pre-mRNA to form a mature mRNA, translocating the mature mRNA from the nucleus to the cytoplasm, translation of the mature mRNA into the polypeptide, and assembly of the encoded polypeptide. Expression includes partial expression For example, expression of a gene may be referred to herein as generation of a gene transcript. Translation of a mature mRNA may be referred to herein as expression of the mature mRNA. [0523] As used herein, “modulate” or “modulation” refers to an increase (e g., upregulation) or decrease (e.g., downregulation) of an activity of interest. As used herein, “modulation of gene expression” refers to modulation of one or more of the processes associated with gene expression. For example, modification of gene expression may include modification of one or more of gene transcription, RNA processing, RNA translocation from the nucleus to the cytoplasm, and translation of mRNA into a protein. As used herein, “modulation of protein activity” refers to modulation of protein activity for example, by increasing or decreasing protein expression, increasing or decreasing expression of a fragment of a protein having a biological activity, or by blocking one or more protein-protein or protein-substrate interactions.
[0524] All publications, patents and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications, patents and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
EXAMPLES
[0525] The present disclosure is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the disclosure as set forth herein.
[0526] The following examples illustrate, among other things, that the EEVs described herein may effectively deliver cargo (e.g., a therapeutic moiety) intracellularly when delivered directly to the eye. The examples also illustrate that an EEV-PMO conjugate may achieve broad biodistribution throughout retina after intravitreal injection, including delivery to photoreceptors.
[0527] Abbreviations for reagents used in the Examples are provided as follows HATU (1- [Bis(dimethylamino)methylene]-lH-l,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate); DMSO (dimethylsulfoxide); DIPEA (N,N-Diisopropylethylamine); and DAPI (4',6-diamidino-2-phenylindole).
Example 1: Delivery of Cyanine-5 (Cy5) to the Retina
[0528] In this example, C57B1/6 mice were injected intravitreally with vehicle, EEV conjugated to cyanine-5 (Cy5; a red fluorescent dye), Cy5 alone or no injection. [0529] Table 4 provides a summary of the experimental protocol used to evaluate EEV (EEV1) delivery of Cy5 to the eye.
Table 4: Summary of Experimental Protocol
Figure imgf000147_0001
[0530] As shown in Table 4, compositions comprising vehicle, Cy5, or EEVI -Cy5 were injected into the retina of the left and/or right eyes of six C57BL/6 male mice. After 24 hours, the eyes were harvested, embedded, sectioned, and imaged for Cy5 (red) fluorescence and counterstained with DAPI (blue). No detectable signal above background was detected in untreated, vehicle, and Cy5 groups (data not shown). Distribution throughout the inner and outer nuclear layer, and in one animal retinal pigment epithelium (RPE) was observed for the EEV1-Cy5 group, demonstrating that the EEV effectively intracellularly delivered the Cy5.
[0531] The structure of the EEVI was Ac-PKKKRKV-K(cyclo[Ffd>GrGrQ])-PEGi 2-K(N3)-NH2). Cy5-DBCO (Cy5-azadibenzocyclooctyne) was conjugated to EEVI using strain-promoted azidealkyne cycloaddition, purified via reverse-phase HPLC and lyophilized.
Example 2. Delivery of oligonucleotide to the retina
[0532] Experiments were also performed using (i) a different EEV (EEV2) conjugated to an oligonucleotide (PM01) and to Cy5 and (ii) the PMO conjugated to Cy5. The structure of EEV2 was as follows: Ac-PKKKRKV-miniPEG2-K(cyclo[FGFGRGRQ]-PEGi2-OH. PM01 had a nucleotide sequence of 5'-ATATTGCTATTACCTTAACCCAGAA-3'. The 3' end of the PM01 was attached to the linker (PEG12-OH) of EEV2 via amide conjugation. Briefly, EEV2 and PM01 were dissolved in DMSO, then predissolved HATU and DIPEA were added, and the solution was allowed to react at room temperature (RT) until complete After the reaction was complete, the reaction was quenched with water/acetonitrile before purification and subsequent lyophilization. The Cy5-DBCO was conjugated to the 5' end of the PMO using strain-promoted alkyne-azide cycloaddition using standard protocols, followed by purification and lyophilization.
[0533] For the PMO without the EEV2, Cy5-DBCO was attached to the 5' end of the PMO using strain-promoted alkyne-azide cycloaddition using standard protocols, followed by purification and lyophilization.
[0534] The EEV2-PMO-Cy5 and PMO-Cy5 formulation was injected into the retinas of the mice. No detectable signal above background was detectable in vehicle and Cy5 groups (data not shown). Weak distribution was observed into ganglion cell layer (GCL)/inner nuclear layer (INL) for PMO- Cy5. Broad distribution was observed throughout the retina for EEV2-PMO-Cy5, demonstrating that EEV2 effectively intracellularly delivered the PMO when injected into the eye.
Example 3: Biodistribution of an oligonucleotide in the retina
[0535] Similar to Example 2, C57B1/6 mice were injected via bilateral intravitreally injection with vehicle, or 25 μg an EEV conjugated to a PMO designed to induce exon skipping in mRNA produced in retinal cells. The EEV used was EEV2 (Ac-PKKKRKV-miniPEGi- K(cyclo[FGFGRGRQ]-PEGi2-OH). Similar to Example 2, the 3' end of the PMO was attached to the linker (PEG12-OH) of EEV2 via amide conjugation. Seven days after injection, the mouse eyes were for histology analyzation.
[0536] Harvested eyes were stained with DAPI (blue) and a rabbit anti -PMO antibody (green). The anti-PMO antibody was raised by immunizing a rabbit with a PMO and conducting downstream development using known methods. The stains clearly show the retinal pigment epithelium (RPE), the outer nuclear layer of the retina (ONL), the inner nuclear layer of the retina (INL), and the ganglion cell layer (GCL) of the retina (FIG. 5; see FIG. IB for a schematic of the layers of the retina). The PMO-EEV2 conjugate was localized to the retina (FIG. 5, top). Focusing in on the layers of the retina, the PMO-EEV2 conjugate was distributed in the outer nuclear lever of the retina, the inner nuclear layer of the retina, and the ganglion cell layer of the retina as well as the layers between those explicitly labeled (not all retinal layers could be discreetly determined with the staining method used; FIG. 6). Since EEV2 was able to deliver a PMO to the retina, EEV2 may be useful for delivering PMOs for treating various ocular diseases associated with the retina.
[0537] Another experiment was conducted to analyze the ability of the PMO-EEV2 to induce exon skipping in a gene expressed in the retina. Compared to a PMO only control, exon skipping was observed for cells treated with the PM0-EEV2 conjugate. This indicates that EEV2 is able to penetrate cells of the retina and deliver a PMO to the cytosol.
Example 5: Evaluation of in vivo tolerability of an PMO-EEV5 conjugate
[0538] Similar to Example 2, mice were injected via intravitreally injection with vehicle, or an EEV conjugated to a PMO designed to induce exon skipping in mRNA produced in retinal cells The EEV used was EEV5 (Ac-PKKKRKV-miniPEG2-K(cyclo[FfFGRGRQ])-miniPEG2-K(N3)- NHz). The 3' end of the PMO was attached to the linker (K(N3)) of EEV5 via click chemistry. Seven days after injection, the mouse eyes were harvested and sectioned for histology analyzation.
[0539] FIG. 7 shows the results. Eye tissue treated with DAPI and a GFAP dye. DAPI stains nuclei. The GFAP dye stains glial fibrillary acidic protein, a protein in astrocytes. Intravitreal dosing of the PMO-EEV5 conjugate was well tolerated in mice with no evidence in changes in retinal structure. Additionally, lack of astrocyte infiltration into the retina suggests no inflammation after treatment.
Example 5: Evaluation of various delivery constructs conjugated to a PMO
[0540] Six delivery constructs were conjugated to a PMO designed to induce exon skipping in a gene associated with a retinal disease. The PMO was conjugated to various delivery constructs using amide chemistry when the delivery construct included a terminal carboxylic acid group (- PEGx-OH) or click chemistry when the delivery construct included a terminal azide (Nr). The structure of the delivery constructs (EEVs) is provided in Table 5 below.
[0541] C57BL/6 mice were injected via bilateral intravitreally injection with vehicle, 25 pg of a control, PMO, or PMO conjugated with different EEVs (EEV2-8 listed in Table 5). Each treatment group included 4 mice (8 eyes). The control was an ASO (not a PMO) known to induce exon skipping the gene targeted by the PMO. Seven days post injection, mouse eyes were harvested, exon skipping in six eyes was evaluated by RT-PCR.
Table 5: EEV Constructs
Figure imgf000149_0001
Figure imgf000150_0002
[0542] FIG. 8 shows the results. Conjugates having the delivery constructs EEV5, EEV6, and EEV7 induced the greatest amount of exon skipping. These results indicate a number of delivery constructs described herein are effective in delivering a PMO to appropriate cells in the eye to induce exon skipping in a gene expressed in the retina, suggesting that the delivery constructs may also be suitable for use in delivering other cargo to cells of the eye for treating ocular diseases.
Example 6: Evaluation of various delivery constructs conjugated to a PMO
[0543] Delivery constructs can be conjugated to cargo that includes therapeutic moiety such as a PMO designed to modulate expression of a gene associated with an ocular disease. The cargo can be conjugated to various delivery' constructs using amide chemistry when the delivery construct includes a terminal carboxylic acid group (-PEGx-OH) or click chemistry when the delivery construct includes a terminal azide (Ns). The structure of the delivery constructs (EEVs) that may be tested are provided in Table 6 below.
[0544] Similar to Example 5, C57BL/6 mice may be injected via bilateral intravitreally injection with vehicle, 25 pg of a control, PMO, or PMO conjugated with different EEVs Table 6). A control may be included, for example, an ASO (not a PMO) known to induce exon skipping the gene targeted by the therapeutic moiety. At a suitable time post injection, mouse eyes may be harvested and the impact of the therapeutic moiety evaluated.
Table 6: EEV Sequences
Figure imgf000150_0001

Claims

What is claimed is:
1. A method of delivering a therapeutic agent to an eye of a subject, the method comprising administering a therapeutically effective amount of a cargo conjugate to the eye of the subject, the cargo conjugate comprising:
(a) a cargo comprising the therapeutic agent that selectively binds to a target molecule associated with a disease of the eye; and
(b) an ocular delivery construct comprising an exocyclic peptide (EP), cyclic cell penetrating peptide (cCPP) and one or more linkers, wherein the ocular delivery construct has the structure:
Figure imgf000151_0001
or a protonated form thereof, wherein:
Ri, R2, and Ra are each independently H or an aiyl or heteroaryl side chain of an amino acid; at least two of Ri, R2, and Ra are an aryl or heteroaryl side chain of an amino acid;
R4 and Re are independently H or an amino acid side chain; peptide is an exocyclic peptide (EP) exocyclic peptide (EP) comprising from 2 to 10 amino acids ;
M is a bonding group; each m is independently an integer from 0-3; n is an integer from 0-2; x* is an integer from 0-20; y is an integer from 1-5; q is 1-4; z' is an integer from 1-23; and
Cargo is a therapeutic moiety.
2. The method of claim 1, wherein the amino acid residue comprising an aryl or heteroaryl group is phenylalanine or 3-(2-naphthyl)-alanine.
3. The method of claim 1 or 2, wherein R4 and Re are, independently, H or a side chain of an amino acid selected from arginine, citrulline, serine or histidine.
4. The method of claim 1 or 2, wherein R4 and Re are H.
5. The method of claim 1 or 2, wherein R« and Re are an amino acid side chain of arginine.
6. The method of claim 1 or 2, wherein R» and Re are an amino acid side chain of serine.
7. The method of claim 1 or 2, wherein Rr and Re are an amino acid side chain of histidine.
8. The method of claim 1, wherein the cCPP of the ocular delivery construct has a sequence selected from: FfcDRrRrQ, FGFGRGRQ; GfFGrGrQ, Ffd>GRGRQ; FfFGRGRQ; FfOGrGrQ; FGFGRRRQ; and FGFRRRRQ.
9. The method of claim 1, wherein the wherein the ocular delivery construct prior to conjugated to the cargo is selected from:
(a) cyclo[Ff-Nal-RrRrQ]-PEGz-OH; or cyclo[Ff-Nal-RrRrQ]-PEGz-K(N3)-NH2
(b) Ac-PKKKRKV-PEGx-K(cyclo[GfFGrGrQ])-PEGz-OH; or
Ac-PKKKRKV-PEGx-K(cyclo[GfFGrGrQ])-PEG7-K(N3)-NH2
(c) Ac-PKKKRKV-PEGx-K(cyclo[FfFGRGRQ])-PEGz-OH; or
Ac-PKKKRKV-PEGx’-K(cyclo[FfFGRGRQ])-PEGz-K(N3)-NH2
(d) Ac-PKKKRKV-PEGx--K(cyclo[Ff-Nal-GrGrQ])-PEGz-OH; or
Ac-PKKKRKV- PEGx-K(cyclo[Ff-Nal-GrGrQ])-PEGz-K(N3)-NH2
(e) Ac-PKKKRKV-PEGx-K(cyclo[FGFGRGRQ])-PEGz-OH; or Ac-PKKKRKV-PEGx-K(cyclo[FGFGRGRQ])-PEGz-K(N3)-NH2
(f) Ac-KKKRK-PEGx-K(cyclo(FGFGRGRQ))-PEG7. -OH; or Ac-KKKRK-PEGx-K(cyclo(FGFGRGRQ))-PEGz-K(N3)-NH2
(g) Ac-PKKKRKV-PEGx'-K(cyclo[FGFRRRRQ])-PEGz'-OH; or Ac-PKKKRKV-PEGx-K(cyclo[FGFRRRRQ])-PEGz- K(N3)-NFl2
(h) Ac-PKKKRKV-PEGx'-K(cyclo[FFd>GRGRQ])-PEGz’-OH; or Ac-PKKKRKV-PEGx-K(cyclo[FF<KiRGRQ])-PEGz-K(N3)-NH2
(i) Ac-PKKKRKV-PEGx’-K(cyclo[phF-Fd>SRSRQ])-PEGz-OH; or Ac-PKKKRKV-PEGx--K(cyclo[βhF-FOSRSRQ])-PEGz- K(N3)-NH2; and
G) Ac-PKKKRKV-PEGx-K(cyclo[βhF-FΦDGRGRQ])-PEGz-OH; or Ac-PKKKRKV-PEGx-K(cyclo[βhF-FOGRGRQ])-PEGz- K(N3)-NH2, wherein x' and z' are, independently, an integer from 0 to 12; and -OH is a terminal carboxylic acid.
10. The method of claim 1, wherein the ocular delivery construct prior to conjugation to the cargo is selected from:
(a) cyclo[Ff-Nal-RrRrQ]-PEGi2-OH;
Ac-PKKKRKV-K(cyclo[Ff-Nal-GrGrQ])-PEGi2-K(N3)-NH2;
Ac-PKKKRKV-K(cyclo[Ff-Nal-GrGrQ])-miniPEG2-K(N3)-NH2;
Ac-PKKKRKV- PEG2-K(cyclo[Ff-Nal-GrGrQ])-PEGi2-OH; or
Ac-PKKKRKV-miniPEG2-K(cyclo[FFOGRGRQ])-PEG2-K(N3)-NFl2;
(b) Ac-PKKKRKV- PEG2-K(cyclo[G£FGrGrQ])-PEG2-K(N3)-NH2;
Ac-PKKKRKV- PEG2-K(cyclo[FfFGRGRQ])-PEG2-K(N3)-NFT2;
Ac-PKKKRKV- PEG2-K(cyclo[FGFGRGRQ])-PEGi2-OH;
Ac-PKKKRKV- PEG2-K(cyclo[FGFRRRRQ])-PEGi2-OH;
Ac-PKKKRKV-miniPEG2-K(cyclo[FGFGRGRQ])-PEGi2-K(N3>NH2;
Ac-PKKKRKV-miniPEG2-K(cyclo[FGFGRGRQ])-miniPEG2-K(N3)-NH2; or
Ac-PKKKRKV-miniPEG2-K(cyclo[FfFGRGRQ])-miniPEG2-K(N3)-NH2;
(c) Ac-KKKRK-miniPEG2-K(cyclo(FGFGRGRQ))-miniPEG2-K(N3)-NH2; and
(d) Ac-PKKKRKV-miniPEG2-K(cyclo[βhF-FO>SRSRQ])-PEGi2-OH or
Ac-PKKKRKV-miniPEG2-K(cyclo[phF-F4>GRGRQ])-PEGi2-OH wherein -OH is a terminal carboxylic acid.
11. The method of claim 9 or 10, wherein M comprises
Figure imgf000154_0001
when the ocular delivery construct prior to conjugation to the cargo comprises a terminal carboxylic acid, wherein t' is 0 to 1.
12. The method of claim 9 or 10, wherein M comprises
Figure imgf000154_0002
when the ocular delivery construct prior to conjugation to the cargo comprises a K(N.i).
13. A method of delivering a therapeutic agent to an eye of a subject, the method comprising administering a therapeutically effective amount of a cargo conjugate to the eye of the subject, the cargo conjugate comprising:
(a) a cargo comprising the therapeutic agent that selectively binds to a target molecule associated with a disease of the eye; and
(b) an ocular delivery construct comprising an exocyclic peptide (EP), a first cyclic cell penetrating peptide (cCPP) and one or more linkers, wherein the ocular delivery construct has the structure:
Figure imgf000154_0003
or a protonated form thereof, wherein:
Ri, Ri, and Rs can each independently be H or an amino acid residue having a side chain comprising an aryl or heteroaryl group; at least two of Ri, Rz, and Rs is an aryl or heteroaryl side chain of an amino acid;
R< and Re are independently H or an amino acid side chain;
AAscis an amino acid side chain; nx is 1; q is 1 , 2, 3 or 4; peptide is an exocyclic peptide (EP) exocyclic peptide (EP) comprising from 2 to 10 amino acids;
M is a bonding group; each m is independently an integer from 0-3; n is an integer from 0-2; x' is an integer from 0-20; y is an integer from 1-5; z' is an integer from 1-23; and
Cargo is a therapeutic moiety.
14. The method of claim 13, wherein the amino acid residue comprising an aryl or heteroaryl group is phenylalanine, beta homophenylalanine, or 3-(2-naphthyl)-alanine.
15. The method of claim 13 or 14, wherein R» and Re are, independently, H or a side chain of an amino acid selected from arginine, citrulline, serine or histidine.
16. The method of claim 13 or 14, wherein R.4 and Re are H.
17. The method of claim 13 or 14, wherein R4 and Rs are an amino acid side chain of arginine.
18. The method of claim 13 or 14, wherein R» and Re are an amino acid side chain of serine.
19. The method of claim 13 or 14, wherein R» and Re are an amino acid side chain of histidine.
20. The method of claim 13, wherein the cCPP of the ocular delivery construct has a sequence selected from: βhF-FC>SRSRQ and phF-FΦI>GRGRQ.
21. The method of claim 13, wherein the ocular delivery construct prior to conjugation to the cargo is selected from:
(a) Ac-PKKKRKV-PEGx -K(cyclo[phF-FΦDSRSRQ])-PEGz -OH; or Ac-PKKKRKV-PEGx-K(cyclo[βhF-FΦDSRSRQ])-PEGz- K(N3)-NH2; and
(b) Ac-PKKKRKV-PEGx'-K(cyclo[βhF-F4>GRGRQ])-PEGz-OH; or Ac-PKKKRKV-PEGx-K(cyclo[βhF-FΦGRGRQ])-PEGz'- K(N3)-NH2, wherein x' and z' are, independently, an integer from 0 to 12, and -OH is a terminal carboxylic acid.
22. The method of claim 13, wherein the ocular delivery construct prior to conjugation to the cargo is selected from:
Ac-PKKKRKV-miniPEG2-K(cyclo[βhF-Fd>SRSRQ])-PEGi2-OH and
Ac-PKKKRKV-miniPEG2-K(cyclo[phF-FΦI>GRGRQ])-PEGi2-OH; wherein x' and z' are, independently, an integer from 0 to 12; and -OH is a terminal carboxylic acid.
23. The method of claim 21 or 22, wherein M comprises
Figure imgf000156_0001
when the ocular delivery construct prior to conjugation to the cargo comprises a terminal carboxylic acid, wherein t' is 0 to 1.
24. The method of claim 21 or 22, wherein M comprises
Figure imgf000156_0002
when the ocular delivery construct prior to conjugation to the cargo comprises a K(N3).
25. The method of any one of claims 1 to 24, wherein M comprises ,or
Figure imgf000157_0004
wherein t' is 0 to 10.
Figure imgf000157_0001
26. The method of any one of claims 1 to 25, wherein M comprises
Figure imgf000157_0003
27. The method of any of claims 1 to 25, wherein M comprises
Figure imgf000157_0002
, wherein t' is 0 to
10.
28. The method of any of claims 1 to 27, wherein the EP comprises 1 or 2 amino acids comprising a side chain comprising a guanidine group, or a protonated form thereof.
29. The method of any of claims 1 to 28, wherein the EP comprises 1, 2, 3, or 4 lysine residues.
30. The method of any of claims 1 to 27, wherein the EP comprises one of the following sequences: PKKKRKV; KR; RR; KKK; KGK; KBK; KBR; KRK; KRR; RKK; RRR; KKKK; KKRK; KRKK; KRRK; RKKR; RRRR; KGKK; KKGK; KKKKK, KKKRK; KBKBK; KKKRKV; PGKKRKV; PKGKRKV, PKKGRKV; PKKKGKV; PKKKRGV; or PKKKRKG.
31. The method of any of claims 1 to 30, wherein the cargo comprises a therapeutic oligonucleotide.
32. The method of claim 31, wherein the therapeutic oligonucleotide is an antisense oligonucleotide.
33. The method of claim 32, wherein the antisense oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO).
34. The method of any of claims 1 to 33, wherein the cargo comprises a therapeutic peptide.
35. The method of any one of claims 1 to 34, wherein the disease of the eye is selected from one or more of diabetic retinopathy; glaucoma; retinitis pigmentosa; Usher syndrome; retinal tears or holes; retinal detachment; retinal ischemia; damage associated with laser therapy including photodynamic therapy; surgical light induced iatrogenic retinopathy; drug-induced retinopathies; autosomal dominant optic atrophy; toxic and/or nutritional amblyopias; Leber’s hereditary optic neuropathy; atypical retinitis pigmentosa; Bardet-Biedl syndrome; blue-cone monochromacy; cataracts, central areolar choroidal dystrophy; choroideremia; cone dystrophy; rod dystrophy; rod-cone dystrophy; congenital stationary night blindness; cytomegalovirus retinitis; diabetic macular edema; dominant drusen; giant cell arteritis; Goldmann Favre dystrophy; graves’ ophthalmopathy; gyrate atrophy; iritis; juvenile retinoschisis; Kearns-Sayre syndrome; Lawrence-Moon syndrome; Leber Congenital Amaurosa; wet macular degeneration; dry macular degeneration; macular dystrophy; ocularhistoplasmosis syndrome; Oguchi disease; oxidative damage; proliferative vitreoretinopathy; refsum disease; retinitis punctata albescens; retinopathy of prematurity; rod monochromatism; Usher syndrome such as Usher Syndrome type 2A; scleritis; Sjogren-Larsson syndrome; Sorsby fundus dystrophy; Stargardt disease; choroideremia, optic neuropathy; Bietti crystalline dystrophy; Alport syndrome; X-linked retinoschisis; Macula dystrophy; Achromatopsia; congenital stationary night blindness; Best disease; Pattern dystrophy; and Doyne’s honeycomb dystrophy.
36. The method of any one of claims 1 to 35, wherein administering a cargo conjugate to directly to an eye of the subject comprises topical administration, intravitreal administration, periocular administration, subretinal administration, suprachoroidal administration, or intracameral administration.
37. The method of any of claims 1 to 36, further comprising identifying a subject having an ocular disease.
38. The method of any of claims 1 to 37, wherein the subject is a mammal.
39. The method of claim 38, wherein the mammal is a human.
40. The method of any of claims 1 to 39, wherein the subject is suffering from an ocular disease or disorder, or is at risk of developing, an ocular disease or disorder.
41. The method of claim 40, wherein the subject has a genetic disease or disorder of the eye.
42. The method of any of claims 1 to 41, wherein administration of the compound modulates expression or activity of a target molecule.
43. The method of claim 42, wherein administration of the compound downregulates expression or activity of the target molecule.
44. The method of claim 43, wherein administration of the compound upregulates expression or activity of the target molecule.
45. The method of claim 43, wherein the target molecule is a protein or an oligonucleotide.
46. The method of any one of claims 1 to 41, wherein administration of the compound modulates alternative splicing of a target transcript.
47. The method of any of the preceding claims, wherein the cargo conjugate is administered in a pharmaceutical composition.
PCT/US2023/034113 2022-09-30 2023-09-29 Ocular delivery of therapeutic agents Ceased WO2024073042A1 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US202263411840P 2022-09-30 2022-09-30
US63/411,840 2022-09-30
US202263425807P 2022-11-16 2022-11-16
US63/425,807 2022-11-16

Publications (1)

Publication Number Publication Date
WO2024073042A1 true WO2024073042A1 (en) 2024-04-04

Family

ID=88585308

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2023/034113 Ceased WO2024073042A1 (en) 2022-09-30 2023-09-29 Ocular delivery of therapeutic agents

Country Status (1)

Country Link
WO (1) WO2024073042A1 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN119345385A (en) * 2024-12-24 2025-01-24 浙江大学医学院附属第二医院 Cell-penetrating peptide-modified nano-cerium oxide eye drops, preparation method and application thereof
WO2025038901A1 (en) * 2023-08-17 2025-02-20 Entrada Therapeutics, Inc. Cyclic peptides for delivering therapeutics

Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998039352A1 (en) 1997-03-07 1998-09-11 Takeshi Imanishi Novel bicyclonucleoside and oligonucleotide analogues
WO1999014226A2 (en) 1997-09-12 1999-03-25 Exiqon A/S Bi- and tri-cyclic nucleoside, nucleotide and oligonucleotide analogues
US20020035243A1 (en) 1998-11-26 2002-03-21 Dominik Imfeld Transport system conjugates
US20020120100A1 (en) 2000-10-13 2002-08-29 Christophe Bonny Intracellular delivery of biological effectors
US20030032594A1 (en) 2000-10-13 2003-02-13 Christophe Bonny Intracellular delivery of biological effectors
US20180298383A1 (en) 2011-05-05 2018-10-18 Sarepta Therapeutics, Inc. Peptide Oligonucleotide Conjugates
US20200066459A1 (en) 2017-02-27 2020-02-27 Taiyo Yuden Co., Ltd. Electrochemical device
WO2021127650A1 (en) 2019-12-19 2021-06-24 Entrada Therapeutics, Inc. Compositions for delivery of antisense compounds
WO2021119756A1 (en) * 2019-12-20 2021-06-24 PYC Therapeutics Limited Novel cellular delivery methods
US11225506B2 (en) 2014-05-21 2022-01-18 Entrada Therapeutics, Inc. Cell penetrating peptides and methods of making and using thereof
WO2023081893A1 (en) * 2021-11-08 2023-05-11 Entrada Therapeutics, Inc. Intracellular targeting of oligonucleotides

Patent Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998039352A1 (en) 1997-03-07 1998-09-11 Takeshi Imanishi Novel bicyclonucleoside and oligonucleotide analogues
WO1999014226A2 (en) 1997-09-12 1999-03-25 Exiqon A/S Bi- and tri-cyclic nucleoside, nucleotide and oligonucleotide analogues
US20020035243A1 (en) 1998-11-26 2002-03-21 Dominik Imfeld Transport system conjugates
US20020120100A1 (en) 2000-10-13 2002-08-29 Christophe Bonny Intracellular delivery of biological effectors
US20030032594A1 (en) 2000-10-13 2003-02-13 Christophe Bonny Intracellular delivery of biological effectors
US6960648B2 (en) 2000-10-13 2005-11-01 Universite De Lausanne Intracellular delivery of biological effectors
US20180298383A1 (en) 2011-05-05 2018-10-18 Sarepta Therapeutics, Inc. Peptide Oligonucleotide Conjugates
US11225506B2 (en) 2014-05-21 2022-01-18 Entrada Therapeutics, Inc. Cell penetrating peptides and methods of making and using thereof
US20200066459A1 (en) 2017-02-27 2020-02-27 Taiyo Yuden Co., Ltd. Electrochemical device
WO2021127650A1 (en) 2019-12-19 2021-06-24 Entrada Therapeutics, Inc. Compositions for delivery of antisense compounds
WO2021119756A1 (en) * 2019-12-20 2021-06-24 PYC Therapeutics Limited Novel cellular delivery methods
WO2023081893A1 (en) * 2021-11-08 2023-05-11 Entrada Therapeutics, Inc. Intracellular targeting of oligonucleotides

Non-Patent Citations (35)

* Cited by examiner, † Cited by third party
Title
"Fieser and Fieser's Reagents for Organic Synthesis", vol. 1-40, 1991, JOHN WILEY AND SONS
AUSUBEL, F. M. ET AL.: "Current Protocols in Molecular Biology", 1994, JOHN WILEY & SONS
CHERY, J.: "RNA therapeutics: RNAi and antisense mechanisms and clinical applications", POSTDOC J, vol. 4, no. 7, July 2016 (2016-07-01), pages 35 - 50
CHOSOBRIN, CURR DIAB REP, vol. 14, no. 8, August 2014 (2014-08-01), pages 515
COLLIN ET AL., MOLECULAR THERAPY-NUCLEIC ACIDS, vol. 1, 2012, pages e14
D. EISENBERG ET AL.: "Hydrophobic Moments and Protein Structure", FARADAY SYMP. CHEM. SOC., vol. 17, 1982, pages 109 - 120, XP008070633
DULLA ET AL., INVESTIGATIVE OPTHALMOLOGY & VISUAL SCIENCE, vol. 59, July 2018 (2018-07-01), pages 5315
DULLA ET AL., MOLECULAR THERAPY, vol. 29, no. 8, 4 August 2021 (2021-08-04), pages 2441 - 2455
E.W. MARTIN: "Remington's Pharmaceutical Science", 1995
FERARI ET AL., CURR GENOMICS, vol. 12, no. 4, June 2011 (2011-06-01), pages 238 - 249
FREIER ET AL., NUCLEIC ACIDS RESEARCH, vol. 25, no. 22, 1997, pages 4429 - 4443
GARANTO, GENES (BASEL, vol. 10, no. 6, 14 June 2019 (2019-06-14), pages 452
GOTE ET AL., J PHARM EXP THER, vol. 370, no. 3, September 2019 (2019-09-01), pages 602 - 624
HOLLIGER, PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 6444 - 48
JUNG ET AL., RNA, vol. 20, no. 6, 2014, pages 805 - 814
KHVOROVA ET AL., NATURE BIOTECHNOLOGY, vol. 35, 2017, pages 238 - 248
KHVOROVA, A. ET AL., NAT. BIOTECHNOL., vol. 35, no. 3, March 2017 (2017-03-01), pages 238 - 248
KINSTLER ET AL., ADV. DRUG DELIVERY REV., vol. 54, 2002, pages 459 - 476
KOSHKIN ET AL., TETRAHEDRON, vol. 54, 1998, pages 3607 - 3630
KUSHNAREVA ET AL., CELL DEATH DIS, vol. 7, no. 7, July 2016 (2016-07-01), pages e2309
MOSBY: "Koeppen & Stanton: Berne and Levy Physiology", 2008, ELSEVIER, INC
NANZHANG, FRONT. MICROBIOL., 20 April 2019 (2019-04-20)
PENDSE ET AL.: "Exon 13-skipped USH2A protein retains functional integrity in mice, suggesting an exo-skipping therapeutic approach to treat USH2A-associated disease", BIORXIV, 4 February 2020 (2020-02-04)
POLJAK ET AL., STRUCTURE, vol. 2, 1994, pages 1121 - 23
PRIYA ET AL., INDIAN J OPTHAMOL, vol. 4, no. 9, 6 September 2016 (2016-09-06), pages 620 - 627
PROG RETIN EYE RES, vol. 27, no. 4, July 2008 (2008-07-01), pages 391 - 419
QUAZIMOLDAY, PNAS, vol. 111, no. 13, pages 5024 - 5029
ROBERTS ET AL., NATURE REVIEWS DRUG DISCOVERY, vol. 19, 2020, pages 673 - 694
ROWE-RENDLEMAN ET AL., INVEST OPTHAMOL VIS SCI, vol. 5, no. 4, 5 April 2014 (2014-04-05), pages 2714 - 2730
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", vol. 1-5, 1989, ELSEVIER SCIENCE PUBLISHERS
WIGGSPASQUALE, HUMAN MOL GENET, vol. 26, no. R1, 1 August 2017 (2017-08-01), pages R21 - R27
WILLIAMS ET AL., NUCLEIC ACID CHEM, vol. 42, 2010, pages 1 - 20
WUTSGREENE: "Protective Groups in Organic Synthesis", 2006, WILEY & SONS
ZALIPSKY ET AL., ADV. DRUG DELIVERY REV., vol. 16, 1995, pages 157 - 182
ZHU ET AL.: "RNA-based therapeutics: an overview and prospectus", CELL DEATH & DISEASE, vol. 12, no. 644, 23 July 2022 (2022-07-23)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025038901A1 (en) * 2023-08-17 2025-02-20 Entrada Therapeutics, Inc. Cyclic peptides for delivering therapeutics
CN119345385A (en) * 2024-12-24 2025-01-24 浙江大学医学院附属第二医院 Cell-penetrating peptide-modified nano-cerium oxide eye drops, preparation method and application thereof
CN119345385B (en) * 2024-12-24 2025-05-09 浙江大学医学院附属第二医院 Cell-penetrating peptide modified nano cerium oxide eye drops and preparation method and application thereof

Similar Documents

Publication Publication Date Title
US20250333734A1 (en) Antisense oligonucleotides for the treatment of Stargardt disease
KR20240009393A (en) Cyclic cell penetrating peptide
US20240417429A1 (en) Compositions and methods for skipping exon 45 in duchenne muscular dystrophy
CA3218805A1 (en) Compositions and methods for intracellular therapeutics
WO2024073042A1 (en) Ocular delivery of therapeutic agents
CA3222824A1 (en) Antisense compounds and methods for targeting cug repeats
US20250129365A1 (en) Compounds and methods for skipping exon 44 in duchenne muscular dystrophy
AU2023217717A1 (en) Enhanced dna dendrimers and methods of use thereof
US20250051393A1 (en) Compositions and methods for modulating interferon regulatory factor-5 (irf-5) activity
CN117957022A (en) Antisense compounds and methods for targeting CUG repeat sequences
RU2848055C2 (en) Compounds and methods of skipping exon 44 in duchene muscular dystrophy
US20250011774A1 (en) Antisense compounds and methods for targeting cug repeats
RU2847465C2 (en) Compositions and methods of skipping exon 45 in duchenne muscular dystrophy
WO2025038902A2 (en) Intracellular targeting of oligonucleotides
WO2025038901A1 (en) Cyclic peptides for delivering therapeutics
CN118284434A (en) Compositions and methods for skipping exon 45 in Duchenne muscular dystrophy
CN118215504A (en) Compounds and methods for skipping exon 44 in duchenne muscular dystrophy
WO2025072246A9 (en) Compounds and methods for skipping exon 50 in duchenne muscular dystrophy
JP2025537167A (en) Calcium voltage-gated channel accessory subunit gamma 1 (CACNG1) binding proteins and CACNG1-mediated delivery to skeletal muscle
OA21931A (en) Antisense compounds and methods for targeting CUG repeats.
KR20180095694A (en) Nucleic acid oligomers and uses thereof

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23797966

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 23797966

Country of ref document: EP

Kind code of ref document: A1