WO2023131648A1 - Nanoparticulate compositions for gene therapy - Google Patents

Nanoparticulate compositions for gene therapy Download PDF

Info

Publication number
WO2023131648A1
WO2023131648A1 PCT/EP2023/050181 EP2023050181W WO2023131648A1 WO 2023131648 A1 WO2023131648 A1 WO 2023131648A1 EP 2023050181 W EP2023050181 W EP 2023050181W WO 2023131648 A1 WO2023131648 A1 WO 2023131648A1
Authority
WO
WIPO (PCT)
Prior art keywords
polymer
group
composition according
branched
nanoparticulate composition
Prior art date
Application number
PCT/EP2023/050181
Other languages
French (fr)
Inventor
Wenxin Wang
Zhonglei He
Sigen A
Yinghao Li
Irene LARA-SAEZ
Xianqing WANG
Hongyun Tai
Original Assignee
Branca Bunus Limited
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 Branca Bunus Limited filed Critical Branca Bunus Limited
Publication of WO2023131648A1 publication Critical patent/WO2023131648A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • 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
    • A61K47/58—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 by reactions only involving carbon-to-carbon unsaturated bonds, e.g. poly[meth]acrylate, polyacrylamide, polystyrene, polyvinylpyrrolidone, polyvinylalcohol or polystyrene sulfonic acid resin
    • 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
    • A61K47/595—Polyamides, e.g. nylon
    • 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
    • A61K47/56—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
    • A61K47/61—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 the organic macromolecular compound being a polysaccharide or a derivative thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6933—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained by reactions only involving carbon to carbon, e.g. poly(meth)acrylate, polystyrene, polyvinylpyrrolidone or polyvinylalcohol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6935—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides 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
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6939—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being a polysaccharide, e.g. starch, chitosan, chitin, cellulose or pectin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle

Definitions

  • Nanoparticulate compositions for gene therapy are described.
  • the present invention relates to a nanoparticulate composition for gene therapy. Also contemplated are methods of treating skin genetic disorders such as Recessive dystrophic epidermolysis bullosa (RDEB).
  • RDEB Recessive dystrophic epidermolysis bullosa
  • polymer-based and lipid-based vectors are the two most common vectors, which carry distinct advantages, but both contribute to gene therapy development.
  • PAEs poly( ⁇ -amino ester)s
  • RNA also called large RNA, mainly include messenger RNA (mRNA) and long non-coding RNA (IncRNA), are the typical nucleic acids susceptible to auto- hydrolysis, even without the presence of enzymes or any specific condition.
  • mRNA messenger RNA
  • IncRNA long non-coding RNA
  • RNA and double strands DNA are much more stable due to double strands structures or lack of 2' OH groups, but they still can be susceptible to hydrolysis under acidic conditions with the presence of enzymes or other particular conditions.
  • Polymers for delivery of DNA are described in WANG YAO ET AL ( “Effects of Branching Strategy on the Gene Transfection of Highly Branched Poly(-amino ester)s”, CHINESE JOURNAL OF POLYMER SCIENCE, CHINESE CHEMICAL SOCIETY AND INSTITUTE OF CHEMISTRY, CAS, BEIJING, vol. 38, no.
  • anti-cleavage units which have not been described before, incorporated into polymers, can encapsulate nucleic acids and form a stable-particle that presents a high stability against acidic hydrolysis, enzyme hydrolysis, and especially, auto- hydrolysis, without the need for inclusion of lipids to form a hydrophobic environment.
  • the stable-particle has been found to be capable of delivering long RNA and, similarly, stabilised delivery of other nucleic acids.
  • the polymers specifically carry diamine moieties (anti-cleavage units) that can both condense the nucleic acids via electrostatic interaction and protect each phosphodiester bond from hydrolysis and loss of function.
  • Example of suitable anti-cleavage units are provided in Figure 1.
  • the Applicant has also discovered that polymers having about 10 to about 150 diamine moieties per polymer backbone provides for excellent mRNA protection and transfection efficacy (Figure 9).
  • the invention provides a composition comprising a nucleic acid complexed within a polymer, wherein the polymer comprises a diamine moiety incorporated into the polymer as a terminal group, and in which the diamine moiety comprises two terminal amine groups separated by a linear or branched, optionally substituted, hydrocarbon backbone.
  • the hydrocarbon backbone is a saturated hydrocarbon backbone. In any embodiment, the hydrocarbon backbone has 3, 4 or 5 carbon atoms, with or without hydrocarbon branching from the hydrocarbon backbone.
  • the polymer comprises the plurality of diamine moieties as terminal endcap groups.
  • the polymer comprises the plurality of diamine moieties as polymer side-chain terminal groups.
  • the polymer comprises 5 to 200 diamine moieties per polymer backbone.
  • the polymer comprises about 10 to about 150 diamine moieties per polymer backbone.
  • the polymer is a cationic polymer.
  • the polymer is a linear polymer.
  • the polymer is a branched polymer.
  • the polymer is a hyperbranched polymer.
  • the hyperbranched polymer is a 3-branching or 4-branching hyperbranched polymer.
  • the polymer is a comb polymer.
  • the polymer is a brush polymer.
  • the polymer is a poly(beta amino ester) polymer (hereafter “PAE polymer”).
  • the polymer is a PEG polymer. In any embodiment, the polymer is a poly(glycidyl methacrylate) polymer.
  • the polymer is a hyaluronic acid polymer.
  • the polymer is a poly(amidoamine) polymer (hereafter “PAMAM polymer”).
  • the polymer is an oligomer combination of a PAE and PEG polymers.
  • each diamine terminal group each, independently, has a chemical formula R1-L1-R2: in which:
  • R1 and R2 are each independently selected from a primary, secondary or tertiary amine group
  • L1 is a linker
  • L1 is a straight or branched, optionally substituted, hydrocarbon chain having 3, 4 or 5 carbon atoms.
  • L1 is saturated.
  • L1 is selected from the group consisting of: CH 2 -(CH 2 )y-CH(X1)-CH 2 ; CH 2 -CH(X2)-(CH 2 )y-CH 2 ; CH 2 -CH(X2)-CH(X1)-CH 2 ; CH(X3)-CH(X2)-CH(X1)-CH 2 ; and, CH(X3)-CH(X2)-CH(X1)-CH(X4); wherein, y is at each occurrence 0, 1 or 2;
  • X1 is independently selected from the group consisting of: hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl (straight and branched), hexyl (straight and branched), -OH -F, -Cl, -Br, -I, -SH, -N 3 , -NO 2 and -COOH;
  • X2 is independently selected from the group consisting of: methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl (straight and branched), hexyl (straight and branched), -OH -F, -Cl, -Br, -I, -SH, -N 3 , -NO 2 and -COOH;
  • X3 is independently selected from the group consisting of: methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl (straight and branched), hexyl (straight and branched), -OH -F, -Cl, -Br, -I, -SH, -N 3 , -NO 2 and -COOH;
  • X4 is independently selected from the group consisting of: methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl (straight and branched), hexyl (straight and branched), -OH -F, -Cl, -Br, -I, -SH, -N 3 , -NO 2 and -COOH.
  • L1 is selected from the group consisting of: (CH 2 ) 3 ; (CH 2 ) 4 , (CH 2 ) 5 ,CH 2 - CH 2 -CH(CH 3 )-CH 2 ; CH 2 -CH(CH 3 )-CH(CH 3 )-CH 2 ; CH(CH 3 )-CH(CH 3 )-CH(CH 3 )-CH 2 ; CH(CH 3 )-CH(CH 3 )-CH(CH 3 )-CH(CH 3 ); and, CH(CH 3 )-CH 2 -CH 2 -CH 2 .
  • L1 is selected from the group consisting of: (CH 2 ) 3 ; CH 2 -CH 2 -CH(CH 3 )- CH 2 .
  • R1 has the structure R1 ’-N-R1 ”, in which R1 ’ and R1 ” are each independently selected from the group consisting of: H and CH 2 Z1 ; wherein
  • Z1 is independent selected from the group consisting of: H, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl (straight and branched), hexyl (straight and branched), -OH, -CH 2 OH, -F, -Cl, -Br, -I, -SH, -N 3 , -NO 2 , -COOH and a polymer, wherein at least one of R1 and R2 is linked to the polymer, wherein R1 ’ and R1 ” are the same or different.
  • R1 has a structure R1 ’-N-R1 ”, in which R1 ’ and R1 ” are each independently selected from the group consisting of: H; CH 3 ; CH 2 OH; CH 2 CH 2 OH; and a polymer, wherein R1 ’ and R1 ” are the same or different.
  • R2 has the structure R2’-N-R2”, in which R2’ and R2” are each independently selected form the group consisting of: H and CH 2 Z2; wherein
  • Z2 is independently selected from the group consisting of: H, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl (straight and branched), hexyl (straight and branched), -OH, -CH 2 OH, -F, -Cl, -Br, -I, -SH, -N 3 , -NO 2 , -COOH and a polymer, wherein at least one of R1 and R2 is linked to the polymer, wherein R2’ and R2” are the same or different.
  • R2 has a structure selected from:
  • R2’-N-R2 in which R2’ and R2” are each independently selected from the group consisting of: H; CH 3 ; CH 2 OH; CH 2 CH 2 OH; and a polymer; and a heterocyclic group containing two ring nitrogen atoms; wherein R2’ and R2” are the same or different.
  • R2, or R1 , or R2 and R1 have a structure selected from: a substituted or unsubstituted piperazinyl group; a substituted or unsubstituted piperidinyl group; a substituted or unsubstituted pyrrolidinyl group; a substituted or unsubstituted pyrazolidinyl group; and a substituted or unsubstituted imidazolidinyl group.
  • each diamine terminal group each, independently, has a chemical formula R1-L1-R2: in which:
  • R1 has a structure R1 ’-N-R1 ”, in which R1’ and R1 ” are each independently selected from the group consisting of: H; CH 3 ; CH 2 OH; CH 2 CH 2 OH; and a polymer;
  • L1 is selected from the group consisting of: (CH 2 ) 3 ; and CH 2 -CH 2 -CH(CH 3 )CH 2 ;
  • R2 has a structure selected from:
  • R2’-N-R2 in which R2’ and R2” are each independently selected from the group consisting of: H; CH 3 ; CH 2 OH; CH 2 CH 2 OH; and a polymer; and a heterocyclic group containing two ring nitrogen atoms.
  • the heterocyclic group containing two ring nitrogen atoms has a structure:
  • L1 does not contain an ether group.
  • the diamine terminal group has a molecular weight of less than 2000 Da, 1500 Da, 1000 Da, 500 Da, 400 Da, 300 Da, 250 Da or 200 Da.
  • the composition is a non-viral gene delivery vector. In any embodiment, the composition is substantially free of lipid.
  • the nucleic acid is a single stranded nucleic acid.
  • the nucleic acid is an RNA molecule.
  • the RNA molecule is a long RNA molecule.
  • the long RNA molecule is selected from a messenger RNA molecule (mRNA) and a long non-coding RNA molecule (IncRNA).
  • mRNA messenger RNA molecule
  • IncRNA long non-coding RNA molecule
  • the composition is a pharmaceutical composition and comprises a a suitable pharmaceutical excipient.
  • the composition is microparticulate.
  • the composition is nanoparticulate.
  • the nucleic acid is provided in the form of a gene editing ribonucleoprotein system.
  • the gene editing ribonucleoprotein system is a Cas9-gRNA ribonucleoprotein system, such as a CRISPR-Cas9 gene editing system, which is typically configured to induce deletion of a targeted genomic sequence including excision of a mutation or exon in a gene, replace a mutation in a gene, or produce a knock-down or knock-out of a gene.
  • CRISPR-Cas9 gene editing system such as a CRISPR-Cas9 gene editing system
  • Other gene editing ribonucleoprotein systems include for example alternative CRISPR-Cas derivatives such as Cas12a, Cas14, CRISPR Base editors, zinc finger nuclease systems and TALEN systems.
  • the gene editing ribonucleoprotein system is a CRISPR- Cas9 gene editing system.
  • the gene editing ribonucleoprotein system is configured for exon- excision.
  • the gene editing ribonucleoprotein system is a CRISPR-Cas9 gene editing system.
  • the gene editing ribonucleoprotein system is configured to excise exon 80 of the COL7A1 gene encoding for Collagen VII protein.
  • the nanoparticulate composition has an average dimension of 50-500, 50-400, 50-300, 100-400, 100-300, 150-250, and ideally about 200 nm.
  • Methods of measuring the average size of the nanoparticulate compositions are for example a dynamic light scattering system or transmission electron microscopy.
  • PDI polydispersity index
  • a Malvern Zetasizer Nano ZS equipped with a scattering angle of 173° can be used. Nanoparticulate size measurements are performed in a clear plastic disposable cuvette.
  • the invention also provides a method of making a composition of the invention comprising the steps of: providing a solution of nucleic acid in a buffer; providing a solution of polymer in a suitable non-aqueous solvent, in which the polymer comprises a diamine moiety incorporated into the polymer as a terminal group; and in which the diamine moiety comprises two terminal amine groups separated by a linear or branched, optionally substituted, hydrocarbon backbone; and mixing the solutions such that there is optionally an excess of mass of the polymer over that of the nucleic acid in the mixture; and typically resting the mixture to allow the composition to form.
  • the polymer solution is prepared by dissolving the polymer in an alcohol such as ethanol.
  • the polymer is dissolved in the solvent at a concentration of 10-200 mg/ml, preferably 50-150 mg/ml, and ideally about 100 mg/ml.
  • the polymer solution comprises 0.1 to 100 g of polymer. system typically containing 0.1 to 100 ⁇ g of ribonucleoprotein complex.
  • the nucleic acid buffer is sodium acetate.
  • the first and second solutions are mixed at a volumetric ratio of about 1-100:1-100, such that there is an excess of mass of the polymer over that of the nucleic acid.
  • the amount of polymer in the second solution is 1 to 100 times more than the nucleic acid in terms of mass, and in which the first and second solutions are mixed at a volumetric ratio of about 1-100:1-100.
  • the polymer comprises 10 to 150 diamine moieties per polymer backbone.
  • the invention also provides a composition of the invention, for use in a method of treatment of a genetic disease in a subject, in which a therapeutically effective amount of the composition is administered to the subject.
  • the genetic disease is characterised by a mutation in a gene of the individual.
  • the nucleic acid is configured to edit a gene of the individual. Editing may comprise non-homologous end joining (NHEJ) (i.e. for large or small genomic deletions or exon excision), knock down or knock out a gene, and homology direct repair (HDR).
  • NHEJ non-homologous end joining
  • HDR homology direct repair
  • the editing comprises deleting or replacing the mutation or a section of the gene including the mutation.
  • the composition is administered topically or by sub-dermal injection.
  • the genetic disease is selected from a skin genetic disorder.
  • skin genetic disorder examples include Epidermolysis Bullosa (EB), Recessive dystrophic epidermolysis bullosa (RDEB), Epidermolytic Palmoplantar Keratoderma, Hailey-Hailey’s disease, Darier’s disease, Localized Autosomal Recessive Hypotrichosis.
  • Additional skin diseases may include: alternative EB subtypes such as Simplex EB and Junctional EB, Epidermolytic Palmoplantar Keratoderma, Hailey-Hailey’s disease, Darier’s disease and, Localized Autosomal Recessive Hypotrichosis,
  • the genetic disease is Recessive Dystrophic subtype of Epidermolysis Bullosa (RDEB), and wherein the gene editing ribonucleoprotein system is configured for collagen VII exon 80 excision.
  • the gene editing ribonucleoprotein system is a CRISPR-Cas9 gene editing system.
  • the invention provides a method of treating a genetic disease in a subject comprising a step of administering a composition according to the invention to the individual.
  • the composition of the invention comprises a gene editing ribonucleoprotein system typically comprising a CRISPR nuclease complexed with a cationic polymer.
  • the CRISPR nuclease protein is Cas9 or a Cas9 derivative.
  • the invention provides a method of treating a skin genetic disease in a subject comprising a step of administering a composition according to the invention to the skin of the individual by topical administration or sub-dermal injection, in which the composition of the invention comprises a gene editing ribonucleoprotein system typically comprising a CRISPR nuclease complexed with a cationic polymer.
  • the CRISPR nuclease protein is Cas9 or a Cas9 derivative.
  • the invention provides a method of genetically modifying a cell ex-vivo or in-vitro comprising a step of incubating the cell with a composition according to the invention, whereby the nucleic acid forming part of the composition genetically modifies the cell.
  • the method comprises a step of isolating the cell from a subject, and then implanting the genetically modified cell into the subject.
  • the subject has a genetic disease characterised by a mutation in a gene in the cell, wherein the nucleic acid is configured to correct the mutation or delete the mutation or all or part of an exon containing the mutation.
  • the invention provides a method of genetically modifying a sample of tissue ex-vivo or in-vitro comprising a step of incubating the tissue with a composition according to the invention, whereby the nucleic acid genetically modifies at least some of the cells of the tissue.
  • the method comprises a step of isolating the tissue from a subject, and then implanting the genetically modified tissue into the subject.
  • the subject has a genetic disease characterised by a mutation in a gene in a cell of the tissue, wherein the nucleic acid is configured to correct the mutation or delete the mutation or all or part of an exon containing the mutation.
  • the invention provides a cell or tissue genetically modified in-vitro or ex- vivo according to a method of the invention.
  • FIGURE 1 Scheme defining the concept of Stable-Particles, and examples of the example library of specific anti-cleavage units .
  • R can be the same or different and selected from the above structures, while at least one of the R groups links to polymer moiety.that can be integrated into polymers.
  • FIGURE 2 Route of action of the Stable-Particles.
  • FIGURE 3 Formation of the Stable-Particles for transfection
  • FIGURE 4 Figure 4.
  • the mRNA entrapment of HPAE-stable was higher and more stable than HPAE-Control and Xfect, although all groups had a high mRNA entrapment close to 100% after a short time incubation ( ⁇ 10 min) (Fig. 4a).
  • the nanoparticle sizes of Stable-Particles before and after lyophilization were similar and both slightly smaller than HPAE-Control- and Xfect- formed mRNA-complex (Fig. 4b).
  • the sizes of Stable-Particles before and after lyophilization were stable in aqueous solutions after 4 h incubation (Fig. 4c).
  • FIGURE 5 Cell viability of HEK293 after 48 hours transfection with Stable-Particles.
  • Transfected Human Embryonic Kidney cells (HEK293) with Stable-Particles at different doses (0.1 to 0.5 ⁇ g mRNA, 20:1 w/w) for luciferase expression showed high viability, comparable to untreated ones (set as 100% viability) and superior to Lipo MessengerMAX.
  • FIGURE 6 Luminescence 48-hour post-transfection of HEK293 with Stable-Particles. The luminescence intensities represent the expression of luciferase protein after 48-hour post- transfection of HEK293. As different doses with 0.1 to 0.5 ⁇ g mRNA, the Stable-Particles showed excellent efficacy similar to or higher than Lipo MessengerMAX and one to two orders of magnitude superior to Xfect (Fig. 7).
  • FIGURE 7 Cellular uptake of HPAE-stable formulated Stable-Particle and Xfect formulated mRNA-complex. The uptake and intracellular transportation of the Stable-Particle was visualized using fluorescently labelled red mRNA, showing excellent uptake and intracellular allocation of Stable-Particle (Fig. 7). On the contrary, the commercial linear PAE transfection reagent Xfect also successfully delivered mRNA into cells but failed to produce high efficacy compared to the Stable-Particle, presenting the unique and robust mRNA protection from the Stable-Particle comparable with leading lipid-based vectors (Fig. 6 and Fig. 7).
  • FIGURE 8 Efficacy of liquid and lyophilized Stable-Particle store at different temperatures.
  • Stable-Particles in the solution can be stored at -20 and -80 degrees Celsius for long terms, but not at room temperature or 4 degrees Celsius due to the spontaneous hydrolysis/degradation of abundant ester bonds in HPAE-stable polymer material, besides mRNA hydrolysis. After lyophilization, Stable-Particles remained 100% efficacy for long terms when stored at 4, -20 and -80 degrees Celsius, and remained majority efficacy at room temperature even after 24 weeks, which is considered stable.
  • FIGURE 9 (a) Luminescence 48-hour post-transfection of HEK293 with HPAEs with different amounts of anti-cleavage units, (b) mRNA entrapment of HPAEs with different amounts of anti-cleavage units.
  • the term “comprise,” or variations thereof such as “comprises” or “comprising,” are to be read to indicate the inclusion of any recited integer (e.g. a feature, element, characteristic, property, method/process step or limitation) or group of integers (e.g. features, element, characteristics, properties, method/process steps or limitations) but not the exclusion of any other integer or group of integers.
  • the term “comprising” is inclusive or open-ended and does not exclude additional, unrecited integers or method/process steps.
  • the term “disease” is used to define any abnormal condition that impairs physiological function and is associated with specific symptoms.
  • the term is used broadly to encompass any disorder, illness, abnormality, pathology, sickness, condition or syndrome in which physiological function is impaired irrespective of the nature of the aetiology (or indeed whether the aetiological basis for the disease is established). It therefore encompasses conditions arising from infection, trauma, injury, surgery, radiological ablation, age, poisoning or nutritional deficiencies.
  • treatment refers to an intervention (e.g. the administration of an agent to a subject) which cures, ameliorates or lessens the symptoms of a disease or removes (or lessens the impact of) its cause(s) (for example, the reduction in accumulation of pathological levels of lysosomal enzymes).
  • the term is used synonymously with the term “therapy”.
  • treatment refers to an intervention (e.g. the administration of an agent to a subject) which prevents or delays the onset or progression of a disease or reduces (or eradicates) its incidence within a treated population.
  • the term treatment is used synonymously with the term “prophylaxis”.
  • an effective amount or a therapeutically effective amount of an agent defines an amount that can be administered to a subject without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio, but one that is sufficient to provide the desired effect, e.g. the treatment or prophylaxis manifested by a permanent or temporary improvement in the subject's condition.
  • the amount will vary from subject to subject, depending on the age and general condition of the individual, mode of administration and other factors. Thus, while it is not possible to specify an exact effective amount, those skilled in the art will be able to determine an appropriate "effective" amount in any individual case using routine experimentation and background general knowledge.
  • a therapeutic result in this context includes eradication or lessening of symptoms, reduced pain or discomfort, prolonged survival, improved mobility and other markers of clinical improvement.
  • a therapeutic result need not be a complete cure. Improvement may be observed in biological I molecular markers, clinical or observational improvements.
  • the methods of the invention are applicable to humans, large racing animals (horses, camels, dogs), and domestic companion animals (cats and dogs).
  • the term subject defines any subject, particularly a mammalian subject, for whom treatment is indicated.
  • Mammalian subjects include, but are not limited to, humans, domestic animals, farm animals, zoo animals, sport animals, pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, camels, bison, cattle, cows; primates such as apes, monkeys, orangutans, and chimpanzees; canids such as dogs and wolves; felids such as cats, lions, and tigers; equids such as horses, donkeys, and zebras; food animals such as cows, pigs, and sheep; ungulates such as deer and giraffes; and rodents such as mice, rats, hamsters and guinea pigs.
  • the subject is a human.
  • the term “equine” refers to mammals of the family Equidae, which includes horses, donkeys, asses, kiang and zebra.
  • “Nucleic acid” as used herein refers to ribonucleic acid molecules, deoxyribonucleic acid molecules, and artificial nucleic acid molecules, that comprise phosphodiester bonds and are generally susceptible to auto-hydrolysis, acid hydrolysis or enzyme hydrolysis.
  • the nucleic acid molecule is typically single stranded.
  • Exemplary nucleic acids for use in the present invention include long RNA molecules, including messenger RNA (mRNA) and long non-coding RNA (IncRNA).
  • nucleic acid containing complexes such as nucleoproteins including gene editing ribonucleotproteins.
  • the nucleic acid is capable of editing a gene in a cell (gene editing nucleic acid), for example by deletion, excision or replacement of a mutation, a section of a gene, or an exon, knock down or knock out a gene, and homology directed repair (HDR).
  • Polymer The polymer for use in the present invention may be a linear, branched, hyperbranched, comb or brush polymer.
  • the polymer may be selected from a poly-beta amino ester (PAE) polymer, polyethylene glycol (PEG) polymer, an oligomer combination of PAE and PEG, a poly(amidoamine) polymer, a polyacrylate polymer, a polyaspartamide polymer, a natural polymer such as chitosan, or any derivative thereof.
  • PAE poly-beta amino ester
  • PEG polyethylene glycol
  • PEG poly(amidoamine) polymer
  • polyacrylate polymer a polyaspartamide polymer
  • a natural polymer such as chitosan, or any derivative thereof.
  • Diamine moiety means a chemical group having two terminal amine groups linked by a hydrocarbon backbone that may be linear or branched and is optionally substituted.
  • the amine groups may each, independently, be primary, secondary or tertiary amine groups.
  • Examples of diamine moieties suitable for use in the present invention are provided in Figure 1.
  • the diamine moieties can be incorporated into polymers as terminal groups (i.e. the backbone, endcaps or side chains) to acquire optimal interaction with nucleic acids, or other polymer parts.
  • the anti-cleavage units are optimally based on specific diamines, ideally with three to five carbons between two amine groups, and optionally including substitutions that do not hinder intramolecular affinity between the anti-cleavage units and phosphodiester bonds.
  • the optimal carbon numbers depend on the structures of amine groups. With the optimal amine distance and structure, the anti- cleavage units can bind to negatively charged phosphodiester bonds of nucleic acids, restrict strand movement, and explicitly block the attack from 2'OH to prevent hydrolysis, if any.
  • the structures of the anti-cleavage units should fit into the space around phosphodiester bonds to present the most effective hydrolysis-blocking ability, which can be varied with different nucleic acids.
  • the units shall not carry structure/groups or too many charged atoms/groups that hinder optimal interaction with secondary/condensed secondary structures of the nucleic acids (Fig. 1). Structures of polymers carrying anti-cleavage units should be flexible and can have enough units interacting with as much as possible phosphodiester bonds within the nucleic acids for optimal stabilisation.
  • the anti-cleavage units can be the examples given in Fig. 1.
  • Terminal group as applied to the position in which the diamine moieties are incorporated into the polymer refers to diamine moieties being incorporated as endcap and side-chain terminal groups.
  • X diamine moieties per polymer backbone refers to the average amounts of diamine moieties conjugated on polymer backbones after the endcapping reaction. Due to the nature of the polymerisation reaction, the molecular weights and available terminals for endcap conjugation vary slightly between each polymer molecule. Therefore, the average amounts of diamine moieties are controlled by endcap monomer feeding ratios and backbone branching degrees and can be measured and calculated by the Nuclear Magnetic Resonance (NMR) interpretation of the final polymer solution.
  • NMR Nuclear Magnetic Resonance
  • Comb polymer refers to polymer molecules consists of a main chain with linear side chains, and where there are two or more three-way branch points 1 .
  • “Brush polymer” refers to polymer molecules consists of a main chain with linear side chain and where one or more of the branch points are at least four-way 2 .
  • “Cationic polymer” refers to polymers with positive charges.
  • Poly-beta amino ester or “PAE” polymer refers to the polymer formed from the Michael- type addition of bifunctional amines to diacrylate esters 3 .
  • Poly-beta amino ester hyperbranched polymer refers to a cationic polymer formed by random polymerisation between branched monomers (for example monomers having three, four or more reactive sites that can react with acrylate or amine groups), diacrylate groups and first and second amine components to provide a highly branched poly(B-amino ester) (HPAE) having a 3-D structure and multiple end groups.
  • HPAE highly branched poly(B-amino ester)
  • the term includes 3- branching hyperbranched polymers and 4-branching hyperbranched polymers.
  • Polyethylene glycol or “PEG” polymer refers to polymers obtained by polymerizing with monomers containing PEG units (eg. polyethylene glycol) diacrylate), where PEG refer to a oligomer or polymer of ethylene oxide 4 .
  • “3-branching hyperbranched polymer” refers to polymers formed by reacting a monomer with three reacting sites that can react with acrylate or amine groups (three-branching monomer) with a diacrylate and first and second amine components.
  • the polymer is formed using a oligomer combination approach, in which the diacrylate and first amine components are reacted together to form a first oligomer, the first oligomer and second amine component are reacted together to form a second oligomer, and the second oligomer and four branching monomer are reacted together to form the hyperbranched polymer of the invention.
  • This oligomer combination approach is described in detail in Zeng et al (Nano. Lett.
  • the four-branching monomer, diacrylate component, and first amine are reacted together in a Michael Addition reaction to form a first polymer, and the first polymer and second amine component (endcapping amine) are reacted together in a Michael Addition reaction to form the hyperbranched polymer of the invention.
  • Examples of 3-branching hyperbranched polymers are described in US2017216455 and Zeng et al.
  • “4-branching hyperbranched polymer” refers to polymers formed by reacting a monomer with four reacting sites that can react with acrylate or amine groups (four-branching monomer) with a diacrylate and first and second amine components. They are described in
  • Gene editing ribonucleoprotein system or “gene editing RNP system” refers to a complex formed by a ribosomal protein bound to one or more sequences of nucleic acid that is capable of editing a gene in a mammal, for example by deleting or replacing a mutation in a gene or a segment of a gene (such as an exon), inserting an oligonucleotide into a gene (insertional mutagenesis), or modulating the expression of a gene (knock-down or knock- out mutation).
  • the nucleic acid may be RNA in a format consisting of but no limited to, crRNA, tracRNA, sgRNA.
  • the nucleic acid is a sgRNA comprising crRNA and tracrRNA.
  • the ribosomal protein may be a CRISPR nuclease protein, e.g. Cas9, Cas12a, Cas14 or a Cas variant, for example modified versions of nuclease dead (dCas9).
  • the gene editing ribonucleoprotein system can be further complimented with the addition of nucleic acids in the form of DNA or RNA or a combination of both. Complimentary nucleic acids can be incorporated into the gene editing ribonucleoprotein system to induce gene augmentation, gene silencing, gene addition, gene knockdown, gene knockout, gene editing via homology directed repair.
  • the nucleic acid may be employed in a format consisting of but not limited to RNA oligonucleotides, antisense oligonucleotides.
  • DNA may be employed in a format consisting of but not limited to DNA oligonucleotides, antisense oligonucleotides, single-strand DNA donor oligo, plasmid DNA.
  • the gene editing system may be a CRISPR-associated Cas system (Sander and Joung (2014) CRISPR-Cas systems for editing, regulating and targeting genomes Nature Biotechnology 32(4): 347-355)), a TALEN system (Boch J (February 2011); "TALES of genome targeting". Nature Biotechnology. 29 (2): 135-6.
  • the gene editing system is configured to perform insertational mutagenesis on a cell, for example OBLIGARE systems, and CRISPR-Cpf1 systems (Maresca et al.
  • Obligate Ligation-Gated Recombination (ObLiGaRe): Custom-designed nuclease- mediated targeted integration through nonhomologous end joining Genome Res. 23: 539- 546; see also WO2014/033644), Fagerlund et al. (2015) The Cpf1 CRISPR-Cas protein expands genome-editing tools Genome Biology 16: 251-253; Ledford (2015) Bacteria yield new gene cutter Smaller CRISPR enzyme should simplify genome editing Nature 526: 17).
  • the gene editing ribonucleoprotein system of the invention may be employed in gene addition, gene replacement, gene knockdown and gene editing.
  • Gene replacement is defined as the provision of a functional healthy copy of a gene to replace a dysfunctional mutant containing gene which has given rise to a disease.
  • Gene addition is defined as the supplementation of therapeutic genes that target a specific aspect of a disease mechanism.
  • Gene knockdown is defined as the process of inhibiting a target genes capability to synthesize a toxic/dysfunctional protein which gives rise to a disease.
  • Gene editing is defined as the process whereby a target genes nucleotide sequence is altered resulting in either a loss of function/correction/manipulation of gene expression.
  • Such gene editing systems consists of but are not limited to i) clustered, regularly interspaced, palindromic repeats (CRISPR)-associated (Cas) system; (ii) a transcription activator-like effector nuclease (TALEN) system; or (iii) a zinc finger nuclease (ZFN) system.
  • CRISPR clustered, regularly interspaced, palindromic repeats
  • TALEN transcription activator-like effector nuclease
  • ZFN zinc finger nuclease
  • Linker means any linker group, including an aryl or alkyl group. Preferred linkers include O, NH, CH 2 , alkyl, lower alkyl, alkoxy, lower alkoxy, O-alkyl, CH 2 O, CH 2 NH, and CH 2 NHCOCH 2 , CO, COO. “Nanoparticulate composition” refers to a composition is the nano-size range.
  • the particulate composition has a particle size of less than 2 pm, 1 .5 pm, 1000 nm, for example 20-900 nm, 50-800 nm, 50-700 nm, 50-600 nm, 50-500 nm, 50-400 nm, 50-300 nm, 100-300 nm, 150-250 nm, or about 200 nm.
  • Gene therapy/editing The present invention may be used to edit a portion of the genome of a cell or replace a portion of the genome of a cell with an exogenous DNA insert in an orientation-specific manner.
  • the invention may be used to edit or replace a defective portion of a disease-causing gene (i.e. for gene repair), or to insertionally inactivate (i.e. silence) a gene the expression of which is associated with a disease, or to edit or modify a gene for example to delete disease causing mutations or modify or add in residues required for normal functioning of a gene.
  • a disease-causing gene i.e. for gene repair
  • insertionally inactivate i.e. silence
  • the invention finds application in gene therapy, as herein defined.
  • Gene therapy according to the invention may target all of the cells in an organism, or may be targeted to a subset of cells (e.g. to selected organs, tissues or cells).
  • Gene therapy according to the invention may target somatic cells specifically.
  • Gene therapy according to the invention may exclude the targeting of germ line cells. It may exclude the targeting of totipotent cells. It may exclude the targeting of human embryos.
  • the method may be applied ex vivo to isolated organs, tissues or cells (e.g. to blood, blood cells, immune cells, bone marrow cells, skin cells, nervous tissue, muscle etc.).
  • isolated organs, tissues or cells e.g. to blood, blood cells, immune cells, bone marrow cells, skin cells, nervous tissue, muscle etc.
  • Gene therapy finds application in the treatment of any genetically inherited disorder, particularly those arising from single gene mutations.
  • gene therapy finds particular application in the treatment of lysosomal storage diseases, muscular dystrophies, cystic fibrosis, Marfan syndrome, sickle cell anaemia, dwarfism, phenylketonuria, neurofibromatosis, Huntington’s disease, osteogenesis imperfecta, thalassemia and hemochromatosis.
  • diseases which may be suitable for gene therapy according to the invention include diseases and disorders of: blood, coagulation, heterogenous skin disease, cell proliferation and dysregulation, neoplasia (including cancer), inflammatory processes, immune system (including autoimmune diseases), metabolism, liver, kidney, musculoskeletal, neurological, neuronal and ocular tissues.
  • Exemplary skin diseases include recessive dystrophic epidermolysis bullosa (RDEB), a rare heterogenous skin disease caused by biallelic loss-of-function mutations in the COL7A1 gene.
  • Additional skin diseases may include: alternative EB subtypes such as Simplex EB and Junctional EB, Epidermolytic Palmoplantar Keratoderma, Hailey-Hailey’s disease, Darier’s disease and Localized Autosomal Recessive Hypotrichosis.
  • Exemplary blood and coagulation diseases and disorders include: anaemia, bare lymphocyte syndrome, bleeding disorders, deficiencies of factor H, factor H-like 1 , factor V, factor VIII, factor VII, factor X, factor XI, factor XII, factor XIIIA, factor XIIIB, Fanconi anaemia, haemophagocytic lymphohistiocytosis, haemophilia A, haemophilia B, haemorrhagic disorder, leukocyte deficiency, sickle cell anaemia and thalassemia.
  • immune related diseases and disorders include: AIDS; autoimmune lymphoproliferative syndrome; combined immunodeficiency; HIV -1 ; HIV susceptibility or infection; immunodeficiency and severe combined immunodeficiency (SCIDs).
  • Autoimmune diseases which can be treated according to the invention include Grave’s disease, rheumatoid arthritis, Hashimoto’s thyroiditis, vitiligo, type I (early onset) diabetes, pernicious anaemia, multiple sclerosis, glomerulonephritis, systemic lupus E (SLE, lupus) and Sjogren syndrome.
  • autoimmune diseases include scleroderma, psoriasis, ankylosing spondilitis, myasthenia gravis, pemphigus, polymyositis, dermomyositis, uveitis, Guillain-Barre syndrome, Crohn's disease and ulcerative colitis (frequently referred to collectively as inflammatory bowel disease (IBD)).
  • IBD inflammatory bowel disease
  • exemplary diseases include: amyloid neuropathy; amyloidosis; cystic fibrosis; lysosomal storage diseases; hepatic adenoma; hepatic failure; neurologic disorders; hepatic lipase deficiency; hepatoblastoma, cancer or carcinoma; medullary cystic kidney disease; phenylketonuria; polycystic kidney; or hepatic disease.
  • Exemplary musculoskeletal diseases and disorders include: muscular dystrophy (e.g.
  • Exemplary neurological and neuronal diseases and disorders include: ALS, Alzheimer's disease; autism; fragile X syndrome, Huntington's disease, Parkinson's disease, Schizophrenia, secretase related disorders, trinucleotide repeat disorders, Kennedy's disease, Friedrich's ataxia, Machado-Joseph's disease, spinocerebellar ataxia, myotonic dystrophy and dentatorubral pallidoluysian atrophy (DRPLA).
  • ALS Alzheimer's disease
  • autism fragile X syndrome
  • Huntington's disease Huntington's disease
  • Parkinson's disease Parkinson's disease
  • Schizophrenia secretase related disorders
  • trinucleotide repeat disorders Kennedy's disease, Friedrich's ataxia, Machado-Joseph's disease, spinocerebellar ataxia, myotonic dystrophy and dentatorubral pallidoluysian atrophy (DRPLA).
  • Exemplary ocular diseases include: age related macular degeneration, corneal clouding and dystrophy, cornea plana congenital, glaucoma, Leber’s congenital amaurosis and macular dystrophy.
  • Gene therapy according to the invention finds particular application in the treatment of lysosomal storage disorders. Listed below are exemplary lysosomal storage disorders and the corresponding defective enzymes:
  • Gaucher disease Acid beta-glucosidase or glucocerebrosidase
  • GMI-gangliosidosis Acid beta-galactosidase
  • Tay-Sachs disease beta-Hexosaminidase A
  • Hurler-Scheie disease alpha-L-lduronidase
  • Morquio disease A N-Acetylgalactosamine-6-sulfate sulfatase
  • Gene therapy according to the invention also finds particular application in the treatment of proteostatic diseases including both aggregative and misfolding proteostatic diseases, for example prion diseases, various amyloidoses and neurodegenerative disorders (e.g. Parkinson’s disease, Alzheimer’s disease and Huntington’s disease), certain forms of diabetes, emphysema, cancer and cystic fibrosis.
  • proteostatic diseases including both aggregative and misfolding proteostatic diseases, for example prion diseases, various amyloidoses and neurodegenerative disorders (e.g. Parkinson’s disease, Alzheimer’s disease and Huntington’s disease), certain forms of diabetes, emphysema, cancer and cystic fibrosis.
  • Cystic fibrosis occurs when there is a mutation in the CFTR gene leading to reduced ion channel activity (via increased clearance of the misfolded CFTR proteins).
  • Gene therapy according to the invention finds particular application in the treatment of expanded CAG repeat diseases. These diseases stem from the expansion of CAG repeats in particular genes with the encoded proteins having corresponding polyglutamine tracts which lead to aggregation and accumulation in the nuclei and cytoplasm of neurons.
  • Aggregated amino-terminal fragments of mutant huntingtin are toxic to neuronal cells and are thought to mediate neurodegeneration. Examples include Huntington's disease (HD), which is characterized by selective neuronal cell death primarily in the cortex and striatum. CAG expansions have also been found in at least seven other inherited neurodegenerative disorders, including for example spinal and bulbar muscular atrophy (SBMA), Kennedy’s disease, some forms of amyotrophic lateral sclerosis (ALS), dentatorubral pallidoluysian atrophy (DRPLA) and spinocerebellar ataxia (SCA) types 1 , 2, 3, 6 and 7.
  • SBMA spinal and bulbar muscular atrophy
  • ALS amyotrophic lateral sclerosis
  • DRPLA dentatorubral pallidoluysian atrophy
  • SCA spinocerebellar ataxia
  • Gene therapy according to the invention finds particular application in the treatment of any neoplasia, including proliferative disorders, benign, pre-cancerous and malignant neoplasia, hyperlasia, metaplasia and dysplasia.
  • the invention therefore finds application in the treatment of proliferative disorders which include, but are not limited to cancer, cancer metastasis, smooth muscle cell proliferation, systemic sclerosis, cirrhosis of the liver, adult respiratory distress syndrome, idiopathic cardiomyopathy, lupus erythematosus, retinopathy (e.g.
  • Neoplasia involving smooth muscle cell proliferation include hyperproliferation of cells in the vasculature (e.g. intimal smooth muscle cell hyperplasia, restenosis and vascular occlusion, including in particular stenosis following biologically- or mechanically-mediated vascular injury, such as angioplasty).
  • intimal smooth muscle cell hyperplasia can include hyperplasia in smooth muscle other than the vasculature (e.g.
  • Non-cancerous proliferative disorders also include hyperproliferation of cells in the skin such as psoriasis and its varied clinical forms, Reiter's syndrome, pityriasis rubra pilaris and hyperproliferative variants of disorders of keratinization (including actinic keratosis, senile keratosis and scleroderma). Particularly preferred is the treatment of malignant neoplasia (cancer).
  • cancer malignant neoplasia
  • composition of the invention may be adapted for topical, oral, rectal, parenteral, intramuscular, intraperitoneal, intra-arterial, intrabronchial, subcutaneous, subdermal, intradermal, intravenous, nasal, vaginal, buccal, ocular or sublingual routes of administration.
  • parenteral intramuscular, intraperitoneal, intra-arterial, intrabronchial, subcutaneous, subdermal, intradermal, intravenous, nasal, vaginal, buccal, ocular or sublingual routes of administration.
  • intramuscular intraperitoneal
  • intra-arterial intrabronchial
  • subcutaneous, subdermal, intradermal, intravenous, nasal, vaginal, buccal, ocular or sublingual routes of administration for oral administration, particular use is made of compressed tablets, pills, tablets, drops, and capsules.
  • these compositions contain from 0.01 to 250 mg and more preferably from 0.1-10 mg, of active ingredient per dose.
  • Other forms of administration comprise solutions or emulsions which
  • compositions of the present invention may also be in form of suspensions, emulsions, lotions, ointments, creams, gels, sprays, nebulizers, solutions or dusting powders.
  • the composition of the invention may be formulated for topical delivery.
  • Topical delivery generally means delivery to the skin, but can also mean delivery to a body lumen lined with epithelial cells, for example the lungs or airways, the gastrointestinal tract, the buccal cavity.
  • formulations for topical delivery are described in Topical drug delivery formulations edited by David Osborne and Antonio Aman, Taylor & Francis, the complete contents of which are incorporated herein by reference.
  • compositions or formulations for delivery to the airways are described in O’Riordan et al (Respir Care, 2002, Nov. 47), EP2050437, W02005023290, US2010098660, and US20070053845.
  • Composition and formulations for delivering active agents to the ileum, especially the proximal ileum include microparticles and microencapsulates where the active agent is encapsulated within a protecting matrix formed of polymer or dairy protein that is acid resistant but prone to dissolution in the more alkaline environment of the ileum. Examples of such delivery systems are described in EP1072600.2 and EP13171757.1 .
  • An alternative means of transdermal administration is by use of a skin patch.
  • the active ingredient can be incorporated into a cream consisting of an aqueous emulsion of polyethylene glycols or liquid paraffin or into a hydrogel.
  • the active ingredient can also be incorporated, at a concentration of between 1 and 10% by weight, into an ointment consisting of a white wax or white soft paraffin base together with such stabilisers and preservatives as may be required.
  • Injectable forms may contain between 10-1000 mg, preferably between 10-250 mg, of active ingredient per dose.
  • compositions may be formulated in unit dosage form, i.e., in the form of discrete portions containing a unit dose, or a multiple or sub-unit of a unit dose.
  • the agent may be administered at a dose of from 0.01 to 50 mg/kg body weight, such as from 0.1 to 10 mg/kg, more preferably from 0.1 to 1 mg/kg body weight.
  • pharmaceutically acceptable excipient refers to a diluent, adjuvant, excipient, or vehicle with which the polyplex is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like.
  • the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, or skin penetration enhancers. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.
  • a base polymer (PGMA) can be obtained through controlled radical polymerisation.
  • the base polymer is then further capped with diamine 103 to obtain PGMA-103.
  • the base polymer HA-A is prepared by reacting hyaluronic acid and glycidyl acrylate. The base polymer is then further capped with diamine 103 to obtain HA-A-103.
  • Example 3 Synthesis of poly(amidomine) polymer with terminal diamine stabilising group
  • An embodiment of a PAMAM polymer with terminal diamine stabilising groups was prepared according to Scheme 3 below.
  • the PAMAM is prepared by divergent synthesis, which refers to the sequential “growth” of a dendrimer layer by layer.
  • the example PAMAM polymer uses diamine 103 as initiator core to react with methyl acrylate via Michael addition. More diamine 103 is then coupled with ester-termination to form generation 1 PAMAM.
  • a hyperbranched-based polymer (HB-PEG) can be obtained through controlled radical polymerisation.
  • the base polymer is then further capped with diamine 103 to obtain Hyperbranched-PEG-103.
  • a brush-shaped based polymer (Brush-PEG) can be obtained through controlled radical polymerisation.
  • the base polymer is then further capped with diamine 103 to obtain Brush- PEG-103.
  • the arms are prepared as linear polymer or long monomers (eg. PEGDA). Then the arms are attached to the core (eg. 103) to obtain the base polymer. The base polymer is then further capped with diamine 103 to obtain Star-PEG-103.
  • PEGDA long monomers
  • the base polymer is then further capped with diamine 103 to obtain Star-PEG-103.
  • PEG polymer with terminal diamine stabilising groups was prepared according to Scheme 7 below. Through Michael addition of diamine and diacrylate monomers, a linear based polymer can be obtained. The base polymer is then further capped with diamine 103 to obtain linear- PEG-103.
  • Backbone monomers are mixed in DMSO for polymerization at 90 °C to form hyperbranched backbones via Michael addition, and then 103 diamines are added and serve as endcap monomers to coupling with acrylate terminations to obtain hyperbranched PAE polymer with terminal stabilising groups.
  • HPAEs contain different amounts of anti-cleavage units.
  • Example 9 (comparative) - Synthesis of a hyperbranched PAE polymer without terminal diamine stabilising group (HPAE control)
  • a hyperbranched HPAE was synthesized with a non-stabilising terminal diamine endcap in which the linker connecting the two amines contains carbon atoms and three oxygen atoms, according to Scheme 9 below.
  • Backbone monomers are mixed in DMSO for polymerization at 90 °C to form hyperbranched backbones via Michael addition. Then non-stabilising terminal 122 diamines are added and serve as endcap monomer to coupling with acrylate terminations to obtain the control hyperbranched PAE polymer.
  • the luciferase mRNA (Luc mRNA) was formulated with the HPAE-stable and HPAE-control polymers in different mixture buffer options, then either lyophilized with presenting of sugar lyoprotectant or used directly (Fig. 3).
  • the mixture buffer combinations for the Stable- Particles were compelling, and extended buffer choices can be suitable for different uses.
  • the Stable-Particles formed by mixing polymer ethanol solution and mRNA sodium acetate solution was selected as the example in the following sections.
  • RiboGreenTM Assay is used to quantify mRNA entrapment. Luc mRNA and polymers were formulated to form polyplex solutions. Then, the polyplex solution samples were incubated with RiboGreenTM working solution at room temperature for 5 min and measured at 485 nm for fluorescence. After the first measurement, the RiboGreenTM mixed samples are then incubated at 37 °C for 1 , 2, 3 and 4 hours before fluorescence reading at 485 nm. Negative controls containing the same amount of polymers and RiboGreenTM and the positive control containing the same amount of mRNA and RiboGreenTM were also measured simultaneously. The mRNA entrapment is then calculated by:
  • the sizes of polyplexes formulated by the different polymers and Luc mRNA were measured using Zetasizer Pro (Malvern Panalytical).
  • the Zetasizer Pro contains He-Ne (633 nm) laser with 4 mW max powder. The temperature of the samples was controlled at 25 °C. 100 pl of the polyplexes solution was prepared and then measured freshly or after lyophilisation, stored at room temperature for 24 hours and final reconstitution. The lyophilisation and reconstitution methods are described in sections below. After loading polyplex solution samples in the disposable polystyrene cuvettes (Malvern Panalytical, ZEN0040), the samples were measured immediately and then incubated at 37°C for 1 , 2, 3 and 4 hours. The dynamic light scattering (DLS) with back angle was used to determine the particle size at each time point.
  • DLS dynamic light scattering
  • alamarBlueTM assay which provided a quantitative measurement of cell proliferation and metabolic health.
  • Cell viability has been assessed 48-72 hrs post transfection experiments in cells.
  • Culture media is removed from cells in a well plate and cells are washed with (hanks balanced salt solution) HBSS per well.
  • 100 ⁇ l of alamarBlueTM working solution (10% alamarBlueTM in HBSS) is added to each well and allowed to incubate under normal cell culture conditions for 2 hrs protected from light.
  • the alamarBlueTM solution is transferred to a fresh flat bottomed 96 well plate and absorbance at 570 nm and 600 nm is recorded on a SpectraMax M3 multi-plate reader. Wells containing alamarBlueTM reagent only are subtracted from each sample as a background reading. Untreated cells are used to normalize fluorescence values and plotted as 100% viable.
  • Cells are seeded 24hr-48hrs prior to transfections to allow attachment to well plates and flasks. Cells are seeded at optimized cell densities.
  • polymer- DNA complexes are prepared and after complexation, are mixed with the appropriate cell media such that the final polyplex solution is no more than 20% of the overall media volume.
  • Cell media containing polymer-DNA complexes are added to cells and after 4hrs is removed and replaced with fresh media to remove complexes.
  • trehalose is added to polyplex solution samples to the final trehalose concentration of 10% (w/w). All samples are frozen at -80 °C overnight and then immediately subjected to freeze-dry with a Christ Alpha 1 -2 LDplus Freeze-Dryer at -55 °C for 24 h. Afterwards, the polyplexes are stored at room temperature, 4 °C, -20 °C or -80 °C for 24 hours, or 1 , 2, 4, 6, 12, 24 weeks, respectively. Meanwhile, trehalose is added to the other group of polyplex solution samples to 10% (w/w), and then directly stored at room temperature, 4 °C, -20 °C or -80 °C for indicated periods. The lyophilised samples are reconstituted with the original volume of pure water and used for transfection along with thawed frozen samples to identify the change of size or change of transfection efficacy.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Nanotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biochemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Dermatology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

A nanoparticulate composition comprising a nucleic acid complexed within a polymer, wherein the polymer comprises a diamine moiety incorporated into the polymer as a terminal group, and in which the diamine moiety comprises two terminal amine groups separated by a linear or branched, optionally substituted, hydrocarbon backbone having 3 to 5 carbon atoms with or without hydrocarbon branching from the hydrocarbon backbone. The polymer comprises the plurality of diamine moieties as terminal endcap groups or polymer side-chain terminal groups. The or each diamine terminal group each, independently, has a chemical formula R1-L1-R2 in which R1 and R2 are each independently selected from a primary, secondary or tertiary amine group and L1 is a straight or branched, optionally substituted, hydrocarbon chain having 3, 4 or 5 carbon atoms with or without hydrocarbon branching from the hydrocarbon backbone. The use of the nanoparticulate composition in gene therapy of a subject is also disclosed.

Description

TITLE
Nanoparticulate compositions for gene therapy
Field of the Invention
The present invention relates to a nanoparticulate composition for gene therapy. Also contemplated are methods of treating skin genetic disorders such as Recessive dystrophic epidermolysis bullosa (RDEB).
Background to the Invention
Among non-viral gene delivery vectors, polymer-based and lipid-based vectors are the two most common vectors, which carry distinct advantages, but both contribute to gene therapy development.
Although many polymeric gene delivery vectors usually carry much larger payloads but can lack protective efficacy for cargos, exceptionally long RNA, which is sensitive to hydrolysis or other damaging factors, compared to lipid-based vectors. Even the poly(β -amino ester)s (PAEs), one of the most promising candidates, were not ideal for delivering mRNA susceptible to auto-hydrolysis, unless the PAEs are designed to be lipid-like and co- formulated with lipid-based components to form a more hydrophobic structure to protect mRNA from hydrolysis.
Hydrolysis is a crucial process for the intracellular and extracellular manipulation of genetic materials, but also the main reason for the function loss of nucleic acids during transfection. Long RNA, also called large RNA, mainly include messenger RNA (mRNA) and long non-coding RNA (IncRNA), are the typical nucleic acids susceptible to auto- hydrolysis, even without the presence of enzymes or any specific condition. The auto- hydrolysis happens because the ribose in RNA has a hydroxyl group at the 2' position, which can be deprotonated in aqueous solutions and attack the adjacent phosphorus in the phosphodiester bond of the RNA backbone, leading to backbone cleavage and subsequently loss of functions. Compared to long RNA, short double strands RNA and double strands DNA are much more stable due to double strands structures or lack of 2' OH groups, but they still can be susceptible to hydrolysis under acidic conditions with the presence of enzymes or other particular conditions. Polymers for delivery of DNA are described in WANG YAO ET AL ( “Effects of Branching Strategy on the Gene Transfection of Highly Branched Poly(-amino ester)s”, CHINESE JOURNAL OF POLYMER SCIENCE, CHINESE CHEMICAL SOCIETY AND INSTITUTE OF CHEMISTRY, CAS, BEIJING, vol. 38, no. 8, 17 April 2020 (2020-04-17), pages 830-839), DEZHONG ZHOU ET AL ( SCIENCE ADVANCES, vol. 2, no. 6, 1 June 2016 (2016-06-01) page e1600102), WO2021/058492A1 , WO2010/132879A2 and GREGORY T ZUGATES ET AL (“Rapid Optimization of Gene Delivery by Parallel End-modification of Poly(-amino ester)s”, MOLECULAR THERAPY, vol. 15, no. 7, 1 July 2007 (2007-07-01), pages 1306-1312).
It is an object of the invention to overcome at least one of the above-referenced problems.
Summary of the Invention
The Applicant has unexpectedly discovered that anti-cleavage units, which have not been described before, incorporated into polymers, can encapsulate nucleic acids and form a stable-particle that presents a high stability against acidic hydrolysis, enzyme hydrolysis, and especially, auto- hydrolysis, without the need for inclusion of lipids to form a hydrophobic environment. The stable-particle has been found to be capable of delivering long RNA and, similarly, stabilised delivery of other nucleic acids. The polymers specifically carry diamine moieties (anti-cleavage units) that can both condense the nucleic acids via electrostatic interaction and protect each phosphodiester bond from hydrolysis and loss of function. Example of suitable anti-cleavage units are provided in Figure 1. The Applicant has also discovered that polymers having about 10 to about 150 diamine moieties per polymer backbone provides for excellent mRNA protection and transfection efficacy (Figure 9).
In a first aspect, the invention provides a composition comprising a nucleic acid complexed within a polymer, wherein the polymer comprises a diamine moiety incorporated into the polymer as a terminal group, and in which the diamine moiety comprises two terminal amine groups separated by a linear or branched, optionally substituted, hydrocarbon backbone.
In any embodiment, the hydrocarbon backbone is a saturated hydrocarbon backbone. In any embodiment, the hydrocarbon backbone has 3, 4 or 5 carbon atoms, with or without hydrocarbon branching from the hydrocarbon backbone.
In any embodiment, the polymer comprises the plurality of diamine moieties as terminal endcap groups.
In any embodiment, the polymer comprises the plurality of diamine moieties as polymer side-chain terminal groups.
In any embodiment, the polymer comprises 5 to 200 diamine moieties per polymer backbone.
In any embodiment, the polymer comprises about 10 to about 150 diamine moieties per polymer backbone.
In any embodiment, the polymer is a cationic polymer.
In any embodiment, the polymer is a linear polymer.
In any embodiment, the polymer is a branched polymer.
In any embodiment, the polymer is a hyperbranched polymer.
In one embodiment, the hyperbranched polymer is a 3-branching or 4-branching hyperbranched polymer.
In any embodiment, the polymer is a comb polymer.
In any embodiment, the polymer is a brush polymer.
In any embodiment, the polymer is a poly(beta amino ester) polymer (hereafter “PAE polymer”).
In any embodiment, the polymer is a PEG polymer. In any embodiment, the polymer is a poly(glycidyl methacrylate) polymer.
In any embodiment, the polymer is a hyaluronic acid polymer.
In any embodiment, the polymer is a poly(amidoamine) polymer (hereafter “PAMAM polymer”).
In any embodiment, the polymer is an oligomer combination of a PAE and PEG polymers.
In any embodiment, the or each diamine terminal group each, independently, has a chemical formula R1-L1-R2: in which:
R1 and R2 are each independently selected from a primary, secondary or tertiary amine group; and
L1 is a linker.
In any embodiment, L1 is a straight or branched, optionally substituted, hydrocarbon chain having 3, 4 or 5 carbon atoms.
In any embodiment, L1 is saturated.
In any embodiment, L1 is selected from the group consisting of: CH2-(CH2)y-CH(X1)-CH2; CH2-CH(X2)-(CH2)y-CH2; CH2-CH(X2)-CH(X1)-CH2; CH(X3)-CH(X2)-CH(X1)-CH2; and, CH(X3)-CH(X2)-CH(X1)-CH(X4); wherein, y is at each occurrence 0, 1 or 2;
X1 is independently selected from the group consisting of: hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl (straight and branched), hexyl (straight and branched), -OH -F, -Cl, -Br, -I, -SH, -N3, -NO2 and -COOH;
X2 is independently selected from the group consisting of: methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl (straight and branched), hexyl (straight and branched), -OH -F, -Cl, -Br, -I, -SH, -N3, -NO2 and -COOH;
X3 is independently selected from the group consisting of: methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl (straight and branched), hexyl (straight and branched), -OH -F, -Cl, -Br, -I, -SH, -N3, -NO2 and -COOH; X4 is independently selected from the group consisting of: methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl (straight and branched), hexyl (straight and branched), -OH -F, -Cl, -Br, -I, -SH, -N3, -NO2 and -COOH.
In any embodiment, L1 is selected from the group consisting of: (CH2)3; (CH2)4, (CH2)5,CH2- CH2-CH(CH3)-CH2; CH2-CH(CH3)-CH(CH3)-CH2; CH(CH3)-CH(CH3)-CH(CH3)-CH2; CH(CH3)-CH(CH3)-CH(CH3)-CH(CH3); and, CH(CH3)-CH2-CH2-CH2.
In any embodiment, L1 is selected from the group consisting of: (CH2)3; CH2-CH2-CH(CH3)- CH2.
In any embodiment, R1 has the structure R1 ’-N-R1 ”, in which R1 ’ and R1 ” are each independently selected from the group consisting of: H and CH2Z1 ; wherein
Z1 is independent selected from the group consisting of: H, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl (straight and branched), hexyl (straight and branched), -OH, -CH2OH, -F, -Cl, -Br, -I, -SH, -N3, -NO2, -COOH and a polymer, wherein at least one of R1 and R2 is linked to the polymer, wherein R1 ’ and R1 ” are the same or different.
In any embodiment, R1 has a structure R1 ’-N-R1 ”, in which R1 ’ and R1 ” are each independently selected from the group consisting of: H; CH3; CH2OH; CH2CH2OH; and a polymer, wherein R1 ’ and R1 ” are the same or different.
In any embodiment, R2 has the structure R2’-N-R2”, in which R2’ and R2” are each independently selected form the group consisting of: H and CH2Z2; wherein
Z2 is independently selected from the group consisting of: H, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl (straight and branched), hexyl (straight and branched), -OH, -CH2OH, -F, -Cl, -Br, -I, -SH, -N3, -NO2, -COOH and a polymer, wherein at least one of R1 and R2 is linked to the polymer, wherein R2’ and R2” are the same or different.
In any embodiment, R2 has a structure selected from:
R2’-N-R2”, in which R2’ and R2” are each independently selected from the group consisting of: H; CH3; CH2OH; CH2CH2OH; and a polymer; and a heterocyclic group containing two ring nitrogen atoms; wherein R2’ and R2” are the same or different.
In any embodiment, R2, or R1 , or R2 and R1 , have a structure selected from: a substituted or unsubstituted piperazinyl group; a substituted or unsubstituted piperidinyl group; a substituted or unsubstituted pyrrolidinyl group; a substituted or unsubstituted pyrazolidinyl group; and a substituted or unsubstituted imidazolidinyl group.
In any embodiment, the or each diamine terminal group each, independently, has a chemical formula R1-L1-R2: in which:
R1 has a structure R1 ’-N-R1 ”, in which R1’ and R1 ” are each independently selected from the group consisting of: H; CH3; CH2OH; CH2CH2OH; and a polymer;
L1 is selected from the group consisting of: (CH2)3; and CH2-CH2-CH(CH3)CH2;
R2 has a structure selected from:
R2’-N-R2”, in which R2’ and R2” are each independently selected from the group consisting of: H; CH3; CH2OH; CH2CH2OH; and a polymer; and a heterocyclic group containing two ring nitrogen atoms.
In any embodiment, the heterocyclic group containing two ring nitrogen atoms has a
Figure imgf000008_0001
structure:
In any embodiment, L1 does not contain an ether group.
In any embodiment, the diamine terminal group has a molecular weight of less than 2000 Da, 1500 Da, 1000 Da, 500 Da, 400 Da, 300 Da, 250 Da or 200 Da.
In any embodiment, the composition is a non-viral gene delivery vector. In any embodiment, the composition is substantially free of lipid.
In any embodiment, the nucleic acid is a single stranded nucleic acid.
In any embodiment, the nucleic acid is an RNA molecule.
In any embodiment, the RNA molecule is a long RNA molecule.
In any embodiment, the long RNA molecule is selected from a messenger RNA molecule (mRNA) and a long non-coding RNA molecule (IncRNA).
In any embodiment, the composition is a pharmaceutical composition and comprises a a suitable pharmaceutical excipient.
In any embodiment, the composition is microparticulate.
In any embodiment, the composition is nanoparticulate.
In any embodiment, the nucleic acid is provided in the form of a gene editing ribonucleoprotein system.
In any embodiment, the gene editing ribonucleoprotein system is a Cas9-gRNA ribonucleoprotein system, such as a CRISPR-Cas9 gene editing system, which is typically configured to induce deletion of a targeted genomic sequence including excision of a mutation or exon in a gene, replace a mutation in a gene, or produce a knock-down or knock-out of a gene. Other gene editing ribonucleoprotein systems that may be employed with the present invention include for example alternative CRISPR-Cas derivatives such as Cas12a, Cas14, CRISPR Base editors, zinc finger nuclease systems and TALEN systems.
In any preferred embodiment, the gene editing ribonucleoprotein system is a CRISPR- Cas9 gene editing system.
In any embodiment, the gene editing ribonucleoprotein system is configured for exon- excision. In one embodiment, the gene editing ribonucleoprotein system is a CRISPR-Cas9 gene editing system. In one embodiment, the gene editing ribonucleoprotein system is configured to excise exon 80 of the COL7A1 gene encoding for Collagen VII protein.
In any embodiment, the nanoparticulate composition has an average dimension of 50-500, 50-400, 50-300, 100-400, 100-300, 150-250, and ideally about 200 nm. Methods of measuring the average size of the nanoparticulate compositions are for example a dynamic light scattering system or transmission electron microscopy. To measure nanoparticulate size, and polydispersity index (PDI), which provides a measurement of nanoparticle uniformity in solution, a Malvern Zetasizer Nano ZS (Malvern Instrument) equipped with a scattering angle of 173° can be used. Nanoparticulate size measurements are performed in a clear plastic disposable cuvette.
The invention also provides a method of making a composition of the invention comprising the steps of: providing a solution of nucleic acid in a buffer; providing a solution of polymer in a suitable non-aqueous solvent, in which the polymer comprises a diamine moiety incorporated into the polymer as a terminal group; and in which the diamine moiety comprises two terminal amine groups separated by a linear or branched, optionally substituted, hydrocarbon backbone; and mixing the solutions such that there is optionally an excess of mass of the polymer over that of the nucleic acid in the mixture; and typically resting the mixture to allow the composition to form.
In one embodiment, the polymer solution is prepared by dissolving the polymer in an alcohol such as ethanol.
In one embodiment, the polymer is dissolved in the solvent at a concentration of 10-200 mg/ml, preferably 50-150 mg/ml, and ideally about 100 mg/ml.
In one embodiment, the polymer solution comprises 0.1 to 100 g of polymer. system typically containing 0.1 to 100 μg of ribonucleoprotein complex.
In one embodiment, the nucleic acid buffer is sodium acetate.
In one embodiment, the first and second solutions are mixed at a volumetric ratio of about 1-100:1-100, such that there is an excess of mass of the polymer over that of the nucleic acid.
In one embodiment, the amount of polymer in the second solution is 1 to 100 times more than the nucleic acid in terms of mass, and in which the first and second solutions are mixed at a volumetric ratio of about 1-100:1-100.
In one embodiment, the polymer comprises 10 to 150 diamine moieties per polymer backbone.
The invention also provides a composition of the invention, for use in a method of treatment of a genetic disease in a subject, in which a therapeutically effective amount of the composition is administered to the subject.
In any embodiment, the genetic disease is characterised by a mutation in a gene of the individual.
In any embodiment, the nucleic acid is configured to edit a gene of the individual. Editing may comprise non-homologous end joining (NHEJ) (i.e. for large or small genomic deletions or exon excision), knock down or knock out a gene, and homology direct repair (HDR). In a preferred embodiment, the editing comprises deleting or replacing the mutation or a section of the gene including the mutation.
In one embodiment, the composition is administered topically or by sub-dermal injection.
In one embodiment, the genetic disease is selected from a skin genetic disorder. Examples include Epidermolysis Bullosa (EB), Recessive dystrophic epidermolysis bullosa (RDEB), Epidermolytic Palmoplantar Keratoderma, Hailey-Hailey’s disease, Darier’s disease, Localized Autosomal Recessive Hypotrichosis. Additional skin diseases may include: alternative EB subtypes such as Simplex EB and Junctional EB, Epidermolytic Palmoplantar Keratoderma, Hailey-Hailey’s disease, Darier’s disease and, Localized Autosomal Recessive Hypotrichosis,
In one embodiment, the genetic disease is Recessive Dystrophic subtype of Epidermolysis Bullosa (RDEB), and wherein the gene editing ribonucleoprotein system is configured for collagen VII exon 80 excision. Preferably, the gene editing ribonucleoprotein system is a CRISPR-Cas9 gene editing system.
In another aspect, the invention provides a method of treating a genetic disease in a subject comprising a step of administering a composition according to the invention to the individual. In any embodiment, the composition of the invention comprises a gene editing ribonucleoprotein system typically comprising a CRISPR nuclease complexed with a cationic polymer. Typically, the CRISPR nuclease protein is Cas9 or a Cas9 derivative.
In another aspect, the invention provides a method of treating a skin genetic disease in a subject comprising a step of administering a composition according to the invention to the skin of the individual by topical administration or sub-dermal injection, in which the composition of the invention comprises a gene editing ribonucleoprotein system typically comprising a CRISPR nuclease complexed with a cationic polymer. Typically, the CRISPR nuclease protein is Cas9 or a Cas9 derivative.
In another aspect, the invention provides a method of genetically modifying a cell ex-vivo or in-vitro comprising a step of incubating the cell with a composition according to the invention, whereby the nucleic acid forming part of the composition genetically modifies the cell. In one embodiment, the method comprises a step of isolating the cell from a subject, and then implanting the genetically modified cell into the subject. In one embodiment, the subject has a genetic disease characterised by a mutation in a gene in the cell, wherein the nucleic acid is configured to correct the mutation or delete the mutation or all or part of an exon containing the mutation.
In another aspect, the invention provides a method of genetically modifying a sample of tissue ex-vivo or in-vitro comprising a step of incubating the tissue with a composition according to the invention, whereby the nucleic acid genetically modifies at least some of the cells of the tissue. In one embodiment, the method comprises a step of isolating the tissue from a subject, and then implanting the genetically modified tissue into the subject. In one embodiment, the subject has a genetic disease characterised by a mutation in a gene in a cell of the tissue, wherein the nucleic acid is configured to correct the mutation or delete the mutation or all or part of an exon containing the mutation.
In another aspect, the invention provides a cell or tissue genetically modified in-vitro or ex- vivo according to a method of the invention.
Other aspects and preferred embodiments of the invention are defined and described in the other claims set out below.
Brief Description of the Figures
FIGURE 1 : Scheme defining the concept of Stable-Particles, and examples of the example library of specific anti-cleavage units . R can be the same or different and selected from the above structures, while at least one of the R groups links to polymer moiety.that can be integrated into polymers.
FIGURE 2: Route of action of the Stable-Particles.
FIGURE 3: Formation of the Stable-Particles for transfection
FIGURE 4: Figure 4. (a) mRNA entrapment of HPAE-stable and control groups; (b) Nanoparticle sizes of HPAE-stable and control groups; (c) Nanoparticle stability of HPAE- stable before and after lyophilization. The mRNA entrapment of HPAE-stable was higher and more stable than HPAE-Control and Xfect, although all groups had a high mRNA entrapment close to 100% after a short time incubation (<10 min) (Fig. 4a). The nanoparticle sizes of Stable-Particles before and after lyophilization were similar and both slightly smaller than HPAE-Control- and Xfect- formed mRNA-complex (Fig. 4b). The sizes of Stable-Particles before and after lyophilization were stable in aqueous solutions after 4 h incubation (Fig. 4c).
FIGURE 5: Cell viability of HEK293 after 48 hours transfection with Stable-Particles. Transfected Human Embryonic Kidney cells (HEK293) with Stable-Particles at different doses (0.1 to 0.5 μg mRNA, 20:1 w/w) for luciferase expression showed high viability, comparable to untreated ones (set as 100% viability) and superior to Lipo MessengerMAX. FIGURE 6: Luminescence 48-hour post-transfection of HEK293 with Stable-Particles. The luminescence intensities represent the expression of luciferase protein after 48-hour post- transfection of HEK293. As different doses with 0.1 to 0.5 μg mRNA, the Stable-Particles showed excellent efficacy similar to or higher than Lipo MessengerMAX and one to two orders of magnitude superior to Xfect (Fig. 7).
FIGURE 7: Cellular uptake of HPAE-stable formulated Stable-Particle and Xfect formulated mRNA-complex. The uptake and intracellular transportation of the Stable-Particle was visualized using fluorescently labelled red mRNA, showing excellent uptake and intracellular allocation of Stable-Particle (Fig. 7). On the contrary, the commercial linear PAE transfection reagent Xfect also successfully delivered mRNA into cells but failed to produce high efficacy compared to the Stable-Particle, presenting the unique and robust mRNA protection from the Stable-Particle comparable with leading lipid-based vectors (Fig. 6 and Fig. 7).
FIGURE 8: Efficacy of liquid and lyophilized Stable-Particle store at different temperatures. Stable-Particles in the solution can be stored at -20 and -80 degrees Celsius for long terms, but not at room temperature or 4 degrees Celsius due to the spontaneous hydrolysis/degradation of abundant ester bonds in HPAE-stable polymer material, besides mRNA hydrolysis. After lyophilization, Stable-Particles remained 100% efficacy for long terms when stored at 4, -20 and -80 degrees Celsius, and remained majority efficacy at room temperature even after 24 weeks, which is considered stable.
FIGURE 9: (a) Luminescence 48-hour post-transfection of HEK293 with HPAEs with different amounts of anti-cleavage units, (b) mRNA entrapment of HPAEs with different amounts of anti-cleavage units.
Detailed Description of the Invention
All publications, patents, patent applications and other references mentioned herein are hereby incorporated by reference in their entireties for all purposes as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference and the content thereof recited in full. Definitions and general preferences
Where used herein and unless specifically indicated otherwise, the following terms are intended to have the following meanings in addition to any broader (or narrower) meanings the terms might enjoy in the art:
Unless otherwise required by context, the use herein of the singular is to be read to include the plural and vice versa. The term "a" or "an" used in relation to an entity is to be read to refer to one or more of that entity. As such, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.
As used herein, the term "comprise," or variations thereof such as "comprises" or "comprising," are to be read to indicate the inclusion of any recited integer (e.g. a feature, element, characteristic, property, method/process step or limitation) or group of integers (e.g. features, element, characteristics, properties, method/process steps or limitations) but not the exclusion of any other integer or group of integers. Thus, as used herein the term "comprising" is inclusive or open-ended and does not exclude additional, unrecited integers or method/process steps.
As used herein, the term “disease” is used to define any abnormal condition that impairs physiological function and is associated with specific symptoms. The term is used broadly to encompass any disorder, illness, abnormality, pathology, sickness, condition or syndrome in which physiological function is impaired irrespective of the nature of the aetiology (or indeed whether the aetiological basis for the disease is established). It therefore encompasses conditions arising from infection, trauma, injury, surgery, radiological ablation, age, poisoning or nutritional deficiencies.
As used herein, the term "treatment" or "treating" refers to an intervention (e.g. the administration of an agent to a subject) which cures, ameliorates or lessens the symptoms of a disease or removes (or lessens the impact of) its cause(s) (for example, the reduction in accumulation of pathological levels of lysosomal enzymes). In this case, the term is used synonymously with the term “therapy”. Additionally, the terms "treatment" or "treating" refers to an intervention (e.g. the administration of an agent to a subject) which prevents or delays the onset or progression of a disease or reduces (or eradicates) its incidence within a treated population. In this case, the term treatment is used synonymously with the term “prophylaxis”.
As used herein, an effective amount or a therapeutically effective amount of an agent defines an amount that can be administered to a subject without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio, but one that is sufficient to provide the desired effect, e.g. the treatment or prophylaxis manifested by a permanent or temporary improvement in the subject's condition. The amount will vary from subject to subject, depending on the age and general condition of the individual, mode of administration and other factors. Thus, while it is not possible to specify an exact effective amount, those skilled in the art will be able to determine an appropriate "effective" amount in any individual case using routine experimentation and background general knowledge. A therapeutic result in this context includes eradication or lessening of symptoms, reduced pain or discomfort, prolonged survival, improved mobility and other markers of clinical improvement. A therapeutic result need not be a complete cure. Improvement may be observed in biological I molecular markers, clinical or observational improvements. In a preferred embodiment, the methods of the invention are applicable to humans, large racing animals (horses, camels, dogs), and domestic companion animals (cats and dogs).
In the context of treatment and effective amounts as defined above, the term subject (which is to be read to include "individual", "animal", "patient" or "mammal" where context permits) defines any subject, particularly a mammalian subject, for whom treatment is indicated. Mammalian subjects include, but are not limited to, humans, domestic animals, farm animals, zoo animals, sport animals, pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, camels, bison, cattle, cows; primates such as apes, monkeys, orangutans, and chimpanzees; canids such as dogs and wolves; felids such as cats, lions, and tigers; equids such as horses, donkeys, and zebras; food animals such as cows, pigs, and sheep; ungulates such as deer and giraffes; and rodents such as mice, rats, hamsters and guinea pigs. In preferred embodiments, the subject is a human. As used herein, the term “equine” refers to mammals of the family Equidae, which includes horses, donkeys, asses, kiang and zebra. “Nucleic acid” as used herein refers to ribonucleic acid molecules, deoxyribonucleic acid molecules, and artificial nucleic acid molecules, that comprise phosphodiester bonds and are generally susceptible to auto-hydrolysis, acid hydrolysis or enzyme hydrolysis. The nucleic acid molecule is typically single stranded. Exemplary nucleic acids for use in the present invention include long RNA molecules, including messenger RNA (mRNA) and long non-coding RNA (IncRNA). The term also encompasses nucleic acid containing complexes such as nucleoproteins including gene editing ribonucleotproteins. Typically, the nucleic acid is capable of editing a gene in a cell (gene editing nucleic acid), for example by deletion, excision or replacement of a mutation, a section of a gene, or an exon, knock down or knock out a gene, and homology directed repair (HDR).
“Polymer”: The polymer for use in the present invention may be a linear, branched, hyperbranched, comb or brush polymer. The polymer may be selected from a poly-beta amino ester (PAE) polymer, polyethylene glycol (PEG) polymer, an oligomer combination of PAE and PEG, a poly(amidoamine) polymer, a polyacrylate polymer, a polyaspartamide polymer, a natural polymer such as chitosan, or any derivative thereof.
“Diamine moiety” means a chemical group having two terminal amine groups linked by a hydrocarbon backbone that may be linear or branched and is optionally substituted. The amine groups may each, independently, be primary, secondary or tertiary amine groups. Examples of diamine moieties suitable for use in the present invention are provided in Figure 1. The diamine moieties can be incorporated into polymers as terminal groups (i.e. the backbone, endcaps or side chains) to acquire optimal interaction with nucleic acids, or other polymer parts. The inventors discovered that the anti-cleavage units are optimally based on specific diamines, ideally with three to five carbons between two amine groups, and optionally including substitutions that do not hinder intramolecular affinity between the anti-cleavage units and phosphodiester bonds. The optimal carbon numbers depend on the structures of amine groups. With the optimal amine distance and structure, the anti- cleavage units can bind to negatively charged phosphodiester bonds of nucleic acids, restrict strand movement, and explicitly block the attack from 2'OH to prevent hydrolysis, if any. Furthermore, the structures of the anti-cleavage units should fit into the space around phosphodiester bonds to present the most effective hydrolysis-blocking ability, which can be varied with different nucleic acids. In addition, the units shall not carry structure/groups or too many charged atoms/groups that hinder optimal interaction with secondary/condensed secondary structures of the nucleic acids (Fig. 1). Structures of polymers carrying anti-cleavage units should be flexible and can have enough units interacting with as much as possible phosphodiester bonds within the nucleic acids for optimal stabilisation. In some embodiments, the anti-cleavage units can be the examples given in Fig. 1.
“Terminal group” as applied to the position in which the diamine moieties are incorporated into the polymer refers to diamine moieties being incorporated as endcap and side-chain terminal groups.
“X diamine moieties per polymer backbone” refers to the average amounts of diamine moieties conjugated on polymer backbones after the endcapping reaction. Due to the nature of the polymerisation reaction, the molecular weights and available terminals for endcap conjugation vary slightly between each polymer molecule. Therefore, the average amounts of diamine moieties are controlled by endcap monomer feeding ratios and backbone branching degrees and can be measured and calculated by the Nuclear Magnetic Resonance (NMR) interpretation of the final polymer solution.
“Comb polymer” refers to polymer molecules consists of a main chain with linear side chains, and where there are two or more three-way branch points1.
“Brush polymer” refers to polymer molecules consists of a main chain with linear side chain and where one or more of the branch points are at least four-way2.
“Cationic polymer” refers to polymers with positive charges. E.g. LPAE, HPAE, LBPAE, poly-beta amino ester hyperbranched polymers, hyperbranched polymers, hyperbranched poly-beta amino ester polymers, and hyperbranched PEG polymers.
“Poly-beta amino ester” or “PAE” polymer refers to the polymer formed from the Michael- type addition of bifunctional amines to diacrylate esters3.
“Poly-beta amino ester hyperbranched polymer” refers to a cationic polymer formed by random polymerisation between branched monomers (for example monomers having three, four or more reactive sites that can react with acrylate or amine groups), diacrylate groups and first and second amine components to provide a highly branched poly(B-amino ester) (HPAE) having a 3-D structure and multiple end groups. The term includes 3- branching hyperbranched polymers and 4-branching hyperbranched polymers. “Polyethylene glycol” or “PEG” polymer refers to polymers obtained by polymerizing with monomers containing PEG units (eg. polyethylene glycol) diacrylate), where PEG refer to a oligomer or polymer of ethylene oxide4.
“3-branching hyperbranched polymer” refers to polymers formed by reacting a monomer with three reacting sites that can react with acrylate or amine groups (three-branching monomer) with a diacrylate and first and second amine components. In one embodiment, the polymer is formed using a oligomer combination approach, in which the diacrylate and first amine components are reacted together to form a first oligomer, the first oligomer and second amine component are reacted together to form a second oligomer, and the second oligomer and four branching monomer are reacted together to form the hyperbranched polymer of the invention. This oligomer combination approach is described in detail in Zeng et al (Nano. Lett. 2019 19, 381-391). In another embodiment, the four-branching monomer, diacrylate component, and first amine are reacted together in a Michael Addition reaction to form a first polymer, and the first polymer and second amine component (endcapping amine) are reacted together in a Michael Addition reaction to form the hyperbranched polymer of the invention. Examples of 3-branching hyperbranched polymers are described in US2017216455 and Zeng et al.
“4-branching hyperbranched polymer” refers to polymers formed by reacting a monomer with four reacting sites that can react with acrylate or amine groups (four-branching monomer) with a diacrylate and first and second amine components. They are described in
“Gene editing ribonucleoprotein system” or “gene editing RNP system” refers to a complex formed by a ribosomal protein bound to one or more sequences of nucleic acid that is capable of editing a gene in a mammal, for example by deleting or replacing a mutation in a gene or a segment of a gene (such as an exon), inserting an oligonucleotide into a gene (insertional mutagenesis), or modulating the expression of a gene (knock-down or knock- out mutation). The nucleic acid may be RNA in a format consisting of but no limited to, crRNA, tracRNA, sgRNA. Generally the nucleic acid is a sgRNA comprising crRNA and tracrRNA. The ribosomal protein may be a CRISPR nuclease protein, e.g. Cas9, Cas12a, Cas14 or a Cas variant, for example modified versions of nuclease dead (dCas9). The gene editing ribonucleoprotein system can be further complimented with the addition of nucleic acids in the form of DNA or RNA or a combination of both. Complimentary nucleic acids can be incorporated into the gene editing ribonucleoprotein system to induce gene augmentation, gene silencing, gene addition, gene knockdown, gene knockout, gene editing via homology directed repair. In some embodiments the nucleic acid may be employed in a format consisting of but not limited to RNA oligonucleotides, antisense oligonucleotides. DNA may be employed in a format consisting of but not limited to DNA oligonucleotides, antisense oligonucleotides, single-strand DNA donor oligo, plasmid DNA. The gene editing system may be a CRISPR-associated Cas system (Sander and Joung (2014) CRISPR-Cas systems for editing, regulating and targeting genomes Nature Biotechnology 32(4): 347-355)), a TALEN system (Boch J (February 2011); "TALES of genome targeting". Nature Biotechnology. 29 (2): 135-6. doi:10.1038/nbt.1767. PMID 21301438), a meganuclease system, or a zinc finger nuclease (ZFN) system (Carroll, D (2011) "Genome engineering with zinc-finger nucleases". Genetics Society of America. 188 (4): 773 78doi:10.1534/genetics.111.131433. PMC 3176093. PMID 21828278). In one embodiment, the gene editing system is configured to perform insertational mutagenesis on a cell, for example OBLIGARE systems, and CRISPR-Cpf1 systems (Maresca et al. (2013) Obligate Ligation-Gated Recombination (ObLiGaRe): Custom-designed nuclease- mediated targeted integration through nonhomologous end joining Genome Res. 23: 539- 546; see also WO2014/033644), Fagerlund et al. (2015) The Cpf1 CRISPR-Cas protein expands genome-editing tools Genome Biology 16: 251-253; Ledford (2015) Bacteria yield new gene cutter Smaller CRISPR enzyme should simplify genome editing Nature 526: 17). The gene editing ribonucleoprotein system of the invention may be employed in gene addition, gene replacement, gene knockdown and gene editing. Gene replacement is defined as the provision of a functional healthy copy of a gene to replace a dysfunctional mutant containing gene which has given rise to a disease. Gene addition is defined as the supplementation of therapeutic genes that target a specific aspect of a disease mechanism. Gene knockdown is defined as the process of inhibiting a target genes capability to synthesize a toxic/dysfunctional protein which gives rise to a disease. Gene editing is defined as the process whereby a target genes nucleotide sequence is altered resulting in either a loss of function/correction/manipulation of gene expression. Such gene editing systems consists of but are not limited to i) clustered, regularly interspaced, palindromic repeats (CRISPR)-associated (Cas) system; (ii) a transcription activator-like effector nuclease (TALEN) system; or (iii) a zinc finger nuclease (ZFN) system.
“Linker” means any linker group, including an aryl or alkyl group. Preferred linkers include O, NH, CH2, alkyl, lower alkyl, alkoxy, lower alkoxy, O-alkyl, CH2O, CH2NH, and CH2NHCOCH2, CO, COO. “Nanoparticulate composition” refers to a composition is the nano-size range. In one embodiment, the particulate composition has a particle size of less than 2 pm, 1 .5 pm, 1000 nm, for example 20-900 nm, 50-800 nm, 50-700 nm, 50-600 nm, 50-500 nm, 50-400 nm, 50-300 nm, 100-300 nm, 150-250 nm, or about 200 nm.
“Gene therapy/editing”: The present invention may be used to edit a portion of the genome of a cell or replace a portion of the genome of a cell with an exogenous DNA insert in an orientation-specific manner.
Thus, the invention may be used to edit or replace a defective portion of a disease-causing gene (i.e. for gene repair), or to insertionally inactivate (i.e. silence) a gene the expression of which is associated with a disease, or to edit or modify a gene for example to delete disease causing mutations or modify or add in residues required for normal functioning of a gene.
Thus, the invention finds application in gene therapy, as herein defined.
Gene therapy according to the invention may target all of the cells in an organism, or may be targeted to a subset of cells (e.g. to selected organs, tissues or cells).
Gene therapy according to the invention may target somatic cells specifically.
Gene therapy according to the invention may exclude the targeting of germ line cells. It may exclude the targeting of totipotent cells. It may exclude the targeting of human embryos.
In cases where gene therapy according to the invention is applied to selected organs, tissues or cells, the method may be applied ex vivo to isolated organs, tissues or cells (e.g. to blood, blood cells, immune cells, bone marrow cells, skin cells, nervous tissue, muscle etc.).
Gene therapy finds application in the treatment of any genetically inherited disorder, particularly those arising from single gene mutations. Thus, gene therapy finds particular application in the treatment of lysosomal storage diseases, muscular dystrophies, cystic fibrosis, Marfan syndrome, sickle cell anaemia, dwarfism, phenylketonuria, neurofibromatosis, Huntington’s disease, osteogenesis imperfecta, thalassemia and hemochromatosis.
Other diseases which may be suitable for gene therapy according to the invention include diseases and disorders of: blood, coagulation, heterogenous skin disease, cell proliferation and dysregulation, neoplasia (including cancer), inflammatory processes, immune system (including autoimmune diseases), metabolism, liver, kidney, musculoskeletal, neurological, neuronal and ocular tissues.
Exemplary skin diseases include recessive dystrophic epidermolysis bullosa (RDEB), a rare heterogenous skin disease caused by biallelic loss-of-function mutations in the COL7A1 gene. Additional skin diseases may include: alternative EB subtypes such as Simplex EB and Junctional EB, Epidermolytic Palmoplantar Keratoderma, Hailey-Hailey’s disease, Darier’s disease and Localized Autosomal Recessive Hypotrichosis.
Exemplary blood and coagulation diseases and disorders include: anaemia, bare lymphocyte syndrome, bleeding disorders, deficiencies of factor H, factor H-like 1 , factor V, factor VIII, factor VII, factor X, factor XI, factor XII, factor XIIIA, factor XIIIB, Fanconi anaemia, haemophagocytic lymphohistiocytosis, haemophilia A, haemophilia B, haemorrhagic disorder, leukocyte deficiency, sickle cell anaemia and thalassemia.
Examples of immune related diseases and disorders include: AIDS; autoimmune lymphoproliferative syndrome; combined immunodeficiency; HIV -1 ; HIV susceptibility or infection; immunodeficiency and severe combined immunodeficiency (SCIDs). Autoimmune diseases which can be treated according to the invention include Grave’s disease, rheumatoid arthritis, Hashimoto’s thyroiditis, vitiligo, type I (early onset) diabetes, pernicious anaemia, multiple sclerosis, glomerulonephritis, systemic lupus E (SLE, lupus) and Sjogren syndrome. Other autoimmune diseases include scleroderma, psoriasis, ankylosing spondilitis, myasthenia gravis, pemphigus, polymyositis, dermomyositis, uveitis, Guillain-Barre syndrome, Crohn's disease and ulcerative colitis (frequently referred to collectively as inflammatory bowel disease (IBD)). Other exemplary diseases include: amyloid neuropathy; amyloidosis; cystic fibrosis; lysosomal storage diseases; hepatic adenoma; hepatic failure; neurologic disorders; hepatic lipase deficiency; hepatoblastoma, cancer or carcinoma; medullary cystic kidney disease; phenylketonuria; polycystic kidney; or hepatic disease.
Exemplary musculoskeletal diseases and disorders include: muscular dystrophy (e.g.
Duchenne and Becker muscular dystrophies), osteoporosis and muscular atrophy.
Exemplary neurological and neuronal diseases and disorders include: ALS, Alzheimer's disease; autism; fragile X syndrome, Huntington's disease, Parkinson's disease, Schizophrenia, secretase related disorders, trinucleotide repeat disorders, Kennedy's disease, Friedrich's ataxia, Machado-Joseph's disease, spinocerebellar ataxia, myotonic dystrophy and dentatorubral pallidoluysian atrophy (DRPLA).
Exemplary ocular diseases include: age related macular degeneration, corneal clouding and dystrophy, cornea plana congenital, glaucoma, Leber’s congenital amaurosis and macular dystrophy.
Gene therapy according to the invention finds particular application in the treatment of lysosomal storage disorders. Listed below are exemplary lysosomal storage disorders and the corresponding defective enzymes:
Pompe disease: Acid alpha-glucosidase
Gaucher disease: Acid beta-glucosidase or glucocerebrosidase
Fabry disease: alpha-Galactosidase A
GMI-gangliosidosis: Acid beta-galactosidase
Tay-Sachs disease: beta-Hexosaminidase A
Sandhoff disease: beta-Hexosaminidase B
Niemann-Pick disease: Acid sphingomyelinase
Krabbe disease: Galactocerebrosidase
Farber disease: Acid ceramidase
Metachromatic leukodystrophy: Arylsulfatase A
Hurler-Scheie disease: alpha-L-lduronidase
Hunter disease: lduronate-2-sulfatase
Sanfilippo disease A: Heparan N-sulfatase Sanfilippo disease B: alpha-N-Acetylglucosaminidase
Sanfilippo disease C: Acetyl-CoA: alpha-glucosaminide N-acetyltransferase
Sanfilippo disease D: N-Acetylglucosamine-6-sulfate sulfatase
Morquio disease A: N-Acetylgalactosamine-6-sulfate sulfatase
Morquio disease B: Acid beta-galactosidase
Maroteaux-Lamy disease: Arylsulfatase B
Sly disease: beta-Glucuronidase alpha-Mannosidosis: Acid alpha-mannosidase beta-Mannosidosis: Acid beta-mannosidase
Fucosidosis: Acid alpha-L-fucosidase
Sialidosis: Sialidase
Schindler-Kanzaki disease: alpha-N-acetylgalactosaminidase
Gene therapy according to the invention also finds particular application in the treatment of proteostatic diseases including both aggregative and misfolding proteostatic diseases, for example prion diseases, various amyloidoses and neurodegenerative disorders (e.g. Parkinson’s disease, Alzheimer’s disease and Huntington’s disease), certain forms of diabetes, emphysema, cancer and cystic fibrosis.
Gene therapy according to the invention finds particular application in the treatment of cystic fibrosis. Cystic fibrosis occurs when there is a mutation in the CFTR gene leading to reduced ion channel activity (via increased clearance of the misfolded CFTR proteins).
Gene therapy according to the invention finds particular application in the treatment of expanded CAG repeat diseases. These diseases stem from the expansion of CAG repeats in particular genes with the encoded proteins having corresponding polyglutamine tracts which lead to aggregation and accumulation in the nuclei and cytoplasm of neurons.
Aggregated amino-terminal fragments of mutant huntingtin are toxic to neuronal cells and are thought to mediate neurodegeneration. Examples include Huntington's disease (HD), which is characterized by selective neuronal cell death primarily in the cortex and striatum. CAG expansions have also been found in at least seven other inherited neurodegenerative disorders, including for example spinal and bulbar muscular atrophy (SBMA), Kennedy’s disease, some forms of amyotrophic lateral sclerosis (ALS), dentatorubral pallidoluysian atrophy (DRPLA) and spinocerebellar ataxia (SCA) types 1 , 2, 3, 6 and 7. Gene therapy according to the invention finds particular application in the treatment of any neoplasia, including proliferative disorders, benign, pre-cancerous and malignant neoplasia, hyperlasia, metaplasia and dysplasia. The invention therefore finds application in the treatment of proliferative disorders which include, but are not limited to cancer, cancer metastasis, smooth muscle cell proliferation, systemic sclerosis, cirrhosis of the liver, adult respiratory distress syndrome, idiopathic cardiomyopathy, lupus erythematosus, retinopathy (e.g. diabetic retinopathy), cardiac hyperplasia, benign prostatic hyperplasia, ovarian cysts, pulmonary fibrosis, endometriosis, fibromatosis, hematomas, lymphangiomatosis, sarcoidosis and desmoid tumours. Neoplasia involving smooth muscle cell proliferation include hyperproliferation of cells in the vasculature (e.g. intimal smooth muscle cell hyperplasia, restenosis and vascular occlusion, including in particular stenosis following biologically- or mechanically-mediated vascular injury, such as angioplasty). Moreover, intimal smooth muscle cell hyperplasia can include hyperplasia in smooth muscle other than the vasculature (e.g. blockage of the bile duct, bronchial airways and in the kidneys of patients with renal interstitial fibrosis). Non-cancerous proliferative disorders also include hyperproliferation of cells in the skin such as psoriasis and its varied clinical forms, Reiter's syndrome, pityriasis rubra pilaris and hyperproliferative variants of disorders of keratinization (including actinic keratosis, senile keratosis and scleroderma). Particularly preferred is the treatment of malignant neoplasia (cancer).
Administration
The composition of the invention may be adapted for topical, oral, rectal, parenteral, intramuscular, intraperitoneal, intra-arterial, intrabronchial, subcutaneous, subdermal, intradermal, intravenous, nasal, vaginal, buccal, ocular or sublingual routes of administration. For oral administration, particular use is made of compressed tablets, pills, tablets, drops, and capsules. Preferably, these compositions contain from 0.01 to 250 mg and more preferably from 0.1-10 mg, of active ingredient per dose. Other forms of administration comprise solutions or emulsions which may be injected intravenously, intra- arterial, subcutaneously, intradermally, intraperitoneally or intramuscularly, and which are prepared from sterile or sterilisable solutions. The pharmaceutical compositions of the present invention may also be in form of suspensions, emulsions, lotions, ointments, creams, gels, sprays, nebulizers, solutions or dusting powders. The composition of the invention may be formulated for topical delivery. Topical delivery generally means delivery to the skin, but can also mean delivery to a body lumen lined with epithelial cells, for example the lungs or airways, the gastrointestinal tract, the buccal cavity. In particular, formulations for topical delivery are described in Topical drug delivery formulations edited by David Osborne and Antonio Aman, Taylor & Francis, the complete contents of which are incorporated herein by reference. Compositions or formulations for delivery to the airways are described in O’Riordan et al (Respir Care, 2002, Nov. 47), EP2050437, W02005023290, US2010098660, and US20070053845. Composition and formulations for delivering active agents to the ileum, especially the proximal ileum, include microparticles and microencapsulates where the active agent is encapsulated within a protecting matrix formed of polymer or dairy protein that is acid resistant but prone to dissolution in the more alkaline environment of the ileum. Examples of such delivery systems are described in EP1072600.2 and EP13171757.1 . An alternative means of transdermal administration is by use of a skin patch. For example, the active ingredient can be incorporated into a cream consisting of an aqueous emulsion of polyethylene glycols or liquid paraffin or into a hydrogel. The active ingredient can also be incorporated, at a concentration of between 1 and 10% by weight, into an ointment consisting of a white wax or white soft paraffin base together with such stabilisers and preservatives as may be required.
Injectable forms may contain between 10-1000 mg, preferably between 10-250 mg, of active ingredient per dose.
Compositions may be formulated in unit dosage form, i.e., in the form of discrete portions containing a unit dose, or a multiple or sub-unit of a unit dose.
A person of ordinary skill in the art can easily determine an appropriate dose of one of the instant compositions to administer to a subject without undue experimentation. Typically, a physician will determine the actual dosage which will be most suitable for an individual patient and it will depend on a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy. The dosages disclosed herein are exemplary of the average case. There can of course be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention. Depending upon the need, the agent may be administered at a dose of from 0.01 to 50 mg/kg body weight, such as from 0.1 to 10 mg/kg, more preferably from 0.1 to 1 mg/kg body weight.
The term "pharmaceutically acceptable excipient" refers to a diluent, adjuvant, excipient, or vehicle with which the polyplex is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, or skin penetration enhancers. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.
Exemplification
The invention will now be described with reference to specific examples. These are merely exemplary and for illustrative purposes only: they are not intended to be limiting in any way to the scope of the monopoly claimed or to the invention described. These examples constitute the best mode currently contemplated for practicing the invention.
Example 1 - Synthesis of polvtalycidyl methacrylate) polymer with terminal diamine stabilising group
An embodiment of a poly(glycidyl methacrylate) polymer with terminal diamine stabilising groups was prepared according to Scheme 1 below.
A base polymer (PGMA) can be obtained through controlled radical polymerisation. The base polymer is then further capped with diamine 103 to obtain PGMA-103.
Example 2 - Synthesis of hyaluronic acid polymer with terminal diamine stabilising group
An embodiment of a hyaluronic acid polymer with terminal diamine stabilising groups was prepared according to Scheme 2 below.
The base polymer HA-A is prepared by reacting hyaluronic acid and glycidyl acrylate. The base polymer is then further capped with diamine 103 to obtain HA-A-103.
Example 3 - Synthesis of poly(amidomine) polymer with terminal diamine stabilising group An embodiment of a PAMAM polymer with terminal diamine stabilising groups was prepared according to Scheme 3 below.
The PAMAM is prepared by divergent synthesis, which refers to the sequential “growth” of a dendrimer layer by layer. The example PAMAM polymer uses diamine 103 as initiator core to react with methyl acrylate via Michael addition. More diamine 103 is then coupled with ester-termination to form generation 1 PAMAM.
Example 4 - Synthesis of a hyperbranched PEG polymer with terminal diamine stabilising group
An embodiment of a PEG polymer with terminal diamine stabilising groups was prepared according to Scheme 4 below.
A hyperbranched-based polymer (HB-PEG) can be obtained through controlled radical polymerisation. The base polymer is then further capped with diamine 103 to obtain Hyperbranched-PEG-103.
Example 5 - Synthesis of a brush PEG polymer with terminal diamine stabilising group
An embodiment of a PEG polymer with terminal diamine stabilising groups was prepared according to Scheme 5 below.
A brush-shaped based polymer (Brush-PEG) can be obtained through controlled radical polymerisation. The base polymer is then further capped with diamine 103 to obtain Brush- PEG-103.
Example 6 - Synthesis of a star PEG polymer with terminal diamine stabilising group
An embodiment of a PEG polymer with terminal diamine stabilising groups was prepared according to Scheme 6 below.
The arms are prepared as linear polymer or long monomers (eg. PEGDA). Then the arms are attached to the core (eg. 103) to obtain the base polymer. The base polymer is then further capped with diamine 103 to obtain Star-PEG-103.
Example 7 - Synthesis of a linear PEG polymer with terminal diamine stabilising group
An embodiment of a PEG polymer with terminal diamine stabilising groups was prepared according to Scheme 7 below. Through Michael addition of diamine and diacrylate monomers, a linear based polymer can be obtained. The base polymer is then further capped with diamine 103 to obtain linear- PEG-103.
Example 8 - Synthesis of a hyperbranched PAE polymer with terminal diamine stabilising group (HPAE stable)
An embodiment of a hyperbranched PAE polymer with terminal diamine stabilising groups was prepared according to Scheme 8 below.
Backbone monomers are mixed in DMSO for polymerization at 90 °C to form hyperbranched backbones via Michael addition, and then 103 diamines are added and serve as endcap monomers to coupling with acrylate terminations to obtain hyperbranched PAE polymer with terminal stabilising groups.
Using the example monomers, a series of HPAE polymers containing different amounts of 103 endcap moieties have been synthesised (as seen in the table below).
Table 1. HPAEs contain different amounts of anti-cleavage units.
Figure imgf000029_0001
The polymers were tested in HEK293 cells for the transfection of luciferase mRNA and their mRNA entrapment efficacy. Figure 9 shows that a specific range, about 10 to about 150 moieties, of anti-cleavage unit amounts provides for optimal mRNA protection and transfection efficacy.
Example 9 (comparative) - Synthesis of a hyperbranched PAE polymer without terminal diamine stabilising group (HPAE control)
As a control, a hyperbranched HPAE was synthesized with a non-stabilising terminal diamine endcap in which the linker connecting the two amines contains carbon atoms and three oxygen atoms, according to Scheme 9 below.
Backbone monomers are mixed in DMSO for polymerization at 90 °C to form hyperbranched backbones via Michael addition. Then non-stabilising terminal 122 diamines are added and serve as endcap monomer to coupling with acrylate terminations to obtain the control hyperbranched PAE polymer.
Figure imgf000031_0001
Figure imgf000032_0001
Figure imgf000033_0001
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
Figure imgf000037_0001
Synthesis of composition of the invention
The luciferase mRNA (Luc mRNA) was formulated with the HPAE-stable and HPAE-control polymers in different mixture buffer options, then either lyophilized with presenting of sugar lyoprotectant or used directly (Fig. 3). The mixture buffer combinations for the Stable- Particles were compelling, and extended buffer choices can be suitable for different uses. The Stable-Particles formed by mixing polymer ethanol solution and mRNA sodium acetate solution was selected as the example in the following sections.
As an example:
1. Dilute mRNA in sodium acetate solution to desired concentrations;
2. Dilute polymers in ethanol in a separate tube to desired concentrations;
3. Add diluted polymer solution into diluted mRNA solution with a volume ratio of 1 :1 ;
4. Incubate for the required time (i.e. 1-10 min) to form Stable-Particles;
5. Use for transfection freshly or lyophilization for storage as drug products. mRNA entrapment, size and stability
RiboGreen™ Assay is used to quantify mRNA entrapment. Luc mRNA and polymers were formulated to form polyplex solutions. Then, the polyplex solution samples were incubated with RiboGreen™ working solution at room temperature for 5 min and measured at 485 nm for fluorescence. After the first measurement, the RiboGreen™ mixed samples are then incubated at 37 °C for 1 , 2, 3 and 4 hours before fluorescence reading at 485 nm. Negative controls containing the same amount of polymers and RiboGreen™ and the positive control containing the same amount of mRNA and RiboGreen™ were also measured simultaneously. The mRNA entrapment is then calculated by:
Figure imgf000038_0001
The sizes of polyplexes formulated by the different polymers and Luc mRNA were measured using Zetasizer Pro (Malvern Panalytical). The Zetasizer Pro contains He-Ne (633 nm) laser with 4 mW max powder. The temperature of the samples was controlled at 25 °C. 100 pl of the polyplexes solution was prepared and then measured freshly or after lyophilisation, stored at room temperature for 24 hours and final reconstitution. The lyophilisation and reconstitution methods are described in sections below. After loading polyplex solution samples in the disposable polystyrene cuvettes (Malvern Panalytical, ZEN0040), the samples were measured immediately and then incubated at 37°C for 1 , 2, 3 and 4 hours. The dynamic light scattering (DLS) with back angle was used to determine the particle size at each time point.
Transfected Cell Viability
Evaluation of cell cytotoxicity induced by different polyplex conditions has been assessed using the alamarBlue™ assay, which provided a quantitative measurement of cell proliferation and metabolic health. Cell viability has been assessed 48-72 hrs post transfection experiments in cells. Culture media is removed from cells in a well plate and cells are washed with (hanks balanced salt solution) HBSS per well. Following this, 100 μl of alamarBlue™ working solution (10% alamarBlue™ in HBSS) is added to each well and allowed to incubate under normal cell culture conditions for 2 hrs protected from light. After incubation, the alamarBlue™ solution is transferred to a fresh flat bottomed 96 well plate and absorbance at 570 nm and 600 nm is recorded on a SpectraMax M3 multi-plate reader. Wells containing alamarBlue™ reagent only are subtracted from each sample as a background reading. Untreated cells are used to normalize fluorescence values and plotted as 100% viable.
Cell Transfection Efficiency
Cells are seeded 24hr-48hrs prior to transfections to allow attachment to well plates and flasks. Cells are seeded at optimized cell densities. On the day of transfection, polymer- DNA complexes are prepared and after complexation, are mixed with the appropriate cell media such that the final polyplex solution is no more than 20% of the overall media volume. Cell media containing polymer-DNA complexes are added to cells and after 4hrs is removed and replaced with fresh media to remove complexes.
Thermal stability of fresh and lyophilized Stable-Particles
For lyophilisation, trehalose is added to polyplex solution samples to the final trehalose concentration of 10% (w/w). All samples are frozen at -80 °C overnight and then immediately subjected to freeze-dry with a Christ Alpha 1 -2 LDplus Freeze-Dryer at -55 °C for 24 h. Afterwards, the polyplexes are stored at room temperature, 4 °C, -20 °C or -80 °C for 24 hours, or 1 , 2, 4, 6, 12, 24 weeks, respectively. Meanwhile, trehalose is added to the other group of polyplex solution samples to 10% (w/w), and then directly stored at room temperature, 4 °C, -20 °C or -80 °C for indicated periods. The lyophilised samples are reconstituted with the original volume of pure water and used for transfection along with thawed frozen samples to identify the change of size or change of transfection efficacy.
Equivalents
The foregoing description details presently preferred embodiments of the present invention. Numerous modifications and variations in practice thereof are expected to occur to those skilled in the art upon consideration of these descriptions. Those modifications and variations are intended to be encompassed within the claims appended hereto.
References
1 . Abbasi, M., Faust, L. & Wilhelm, M. Comb and Bottlebrush Polymers with Superior Rheological and Mechanical Properties. Adv. Mater. 31 , 1806484 (2019).
2. Milner, S. T. Polymer brushes. Science (80-. ). 251 , 905-914 (1991).
3. Cordeiro, R. A., Serra, A., Coelho, J. F. J. & Faneca, H. Poly(β -amino ester)-based gene delivery systems: From discovery to therapeutic applications. Journal of Controlled Release vol. 310 155-187 (2019).
4. D’souza, A., delivery, R. S.-E. opinion on drug & 2016, undefined. Polyethylene glycol (PEG): a versatile polymer for pharmaceutical applications. Taylor Fr. 13, 1257- 1275 (2016).

Claims

CLAIMS:
1. A nanoparticulate composition comprising a nucleic acid complexed within a polymer, wherein the polymer comprises a diamine moiety incorporated into the polymer as a terminal group, and in which the or each diamine terminal group each, independently, has a chemical formula R1-L1-R2: in which:
R1 and R2 are each independently selected from a primary, secondary or tertiary amine group; and
L1 is a straight or branched, optionally substituted, hydrocarbon chain having 3, 4 or 5 carbon atoms with or without hydrocarbon branching from the hydrocarbon chain.
2. A nanoparticulate composition according to Claim 1 , in which the nucleic acid is a long RNA molecule.
3. A nanoparticulate composition according to Claim 1 , in which the nucleic acid is a long RNA molecule selected from a messenger RNA molecule (mRNA) and a long non-coding RNA molecule (IncRNA).
4. A nanoparticulate composition according to any preceding Claim, in which the hydrocarbon chain is a saturated hydrocarbon chain.
5. A nanoparticulate composition according to any preceding Claim, in which the polymer comprises a plurality of diamine moieties as terminal endcap groups or polymer side-chain terminal groups.
6. A nanoparticulate composition according to any preceding Claim, in which the polymer is Selected from a hyperbranched polymer, a comb polymer and a brush polymer.
7. A nanoparticulate composition according to any preceding Claim, in which the polymer is selected from the group consisting of: a poly(beta amino ester) polymer; a PEG polymer; a poly(glycidyl methacrylate) polymer; a hyaluronic acid polymer; a poly(amidoamine) polymer; and an oligomer combination of a PAE and PEG polymers.
8. A nanoparticulate composition according to any preceding Claim, in which L1 is selected from the group consisting of: CH2-(CH2)y-CH(X1)-CH2; CH2-CH(X2)-(CH2)y-CH2; CH2- CH(X2)-CH(X1)-CH2; CH(X3)-CH(X2)-CH(X1)-CH2; and, CH(X3)-CH(X2)-CH(X1)-CH(X4); wherein, y is at each occurrence 0, 1 or 2;
X1 is independently selected from the group consisting of: hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl (straight and branched), hexyl (straight and branched), -OH -F, -Cl, -Br, -I, -SH, -N3, -NO2 and -COOH;
X2 is independently selected from the group consisting of: methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl (straight and branched), hexyl (straight and branched), -OH -F, -Cl, -Br, -I, -SH, -N3, -NO2 and -COOH;
X3 is independently selected from the group consisting of: methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl (straight and branched), hexyl (straight and branched), -OH -F, -Cl, -Br, -I, -SH, -N3, -NO2 and -COOH;
X4 is independently selected from the group consisting of: methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl (straight and branched), hexyl (straight and branched), -OH -F, -Cl, -Br, -I, -SH, -N3, -NO2 and -COOH.
9. A nanoparticulate composition according to any preceding Claim, in which L1 is selected from the group consisting of: (CH2)3; (CH2)4, (CH2)5,CH2-CH2-CH(CH3)-CH2; CH2-CH(CH3)- CH(CH3)-CH2; CH(CH3)-CH(CH3)-CH(CH3)-CH2; CH(CH3)-CH(CH3)-CH(CH3)-CH(CH3); and, CH(CH3)-CH2-CH2-CH2.
10. A nanoparticulate composition according to any preceding Claims, in which R1 has the structure R1 ’-N-R1 ”, in which R1 ’ and R1 ” are each independently selected from the group consisting of: H and CH2Z1 ; wherein
Z1 is independent selected from the group consisting of: H, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl (straight and branched), hexyl (straight and branched), -OH, -CH2OH, -F, -Cl, -Br, -I, -SH, -N3, -NO2, -COOH and a polymer, wherein at least one of R1 and R2 is linked to the polymer, wherein R1 ’ and R1 ” are the same or different.
1 1 . A nanoparticulate composition according to any preceding Claim, in which R1 has a structure R1 ’-N-R1 ”, in which R1 ’ and R1 ” are each independently selected from the group consisting of: H; CH3; CH2OH; CH2CH2OH; and a polymer, wherein R1’ and R1 ” are the same or different.
12. A nanoparticulate composition according to any preceding Claim, in which R2 has the structure R2’-N-R2”, in which R2’ and R2” are each independently selected form the group consisting of: H and CH2Z2; wherein
Z2 is independently selected from the group consisting of: H, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl (straight and branched), hexyl (straight and branched), -OH, -CH2OH, -F, -Cl, -Br, -I, -SH, -N3, -NO2, -COOH and a polymer, wherein at least one of R1 and R2 is linked to the polymer, wherein R2’ and R2” are the same or different.
13. A nanoparticulate composition according to any preceding Claim, in which R2 has a structure selected from:
R2’-N-R2”, in which R2’ and R2” are each independently selected from the group consisting of: H; CH3; CH2OH; CH2CH2OH; and a polymer; and a heterocyclic group containing two ring nitrogen atoms; wherein R2’ and R2” are the same or different.
14. A nanoparticulate composition according to any preceding Claim, in which R2, or R1 , or R2 and R1 , have a structure selected from: a substituted or unsubstituted piperazinyl group; a substituted or unsubstituted piperidinyl group; a substituted or unsubstituted pyrrolidinyl group; a substituted or unsubstituted pyrazolidinyl group; and a substituted or unsubstituted imidazolidinyl group.
15. A nanoparticulate composition according to Claim 1 , in which: the nucleic acid is a long RNA molecule; and the polymer is selected from the group consisting of: a poly(beta amino ester) polymer; a PEG polymer; a poly(glycidyl methacrylate) polymer; a hyaluronic acid polymer; a poly(amidoamine) polymer; and an oligomer combination of a PAE and PEG polymers.
16. A nanoparticulate composition according to any preceding Claim, in which: the nucleic acid is a mRNA molecule; and the polymer is a poly(beta amino ester) polymer.
17. A nanoparticulate composition comprising a nucleic acid complexed within a polymer, wherein the polymer comprises a diamine moiety incorporated into the polymer as a terminal group, and in which the diamine moiety comprises two terminal amine groups separated by a linear or branched, optionally substituted, hydrocarbon backbone having 3 to 5 carbon atoms with or without hydrocarbon branching from the hydrocarbon backbone, wherein the nucleic acid is a long RNA molecule.
18. A nanoparticulate composition according to Claim 17, in which the polymer comprises about 10 to about 150 diamine moieties per polymer backbone.
19. A nanoparticulate composition according to any preceding Claim, in which the long RNA molecule is a mRNA molecule.
20. A pharmaceutical composition comprising a nanoparticulate composition according to any of Claims 1 to 19 in combination with a suitable pharmaceutical excipient.
21. A pharmaceutical composition according to Claim 20, for use in a method of treating a genetic disorder in a subject by gene therapy.
PCT/EP2023/050181 2022-01-05 2023-01-05 Nanoparticulate compositions for gene therapy WO2023131648A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP22150378 2022-01-05
EP22150378.2 2022-01-05

Publications (1)

Publication Number Publication Date
WO2023131648A1 true WO2023131648A1 (en) 2023-07-13

Family

ID=80218443

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2023/050181 WO2023131648A1 (en) 2022-01-05 2023-01-05 Nanoparticulate compositions for gene therapy

Country Status (1)

Country Link
WO (1) WO2023131648A1 (en)

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005023290A2 (en) 2003-05-23 2005-03-17 Pestka Biomedical Laboratories, Inc. Uses of interferons for the treatment of severe acute respiratory syndrome and other viral infections
US20070053845A1 (en) 2004-03-02 2007-03-08 Shiladitya Sengupta Nanocell drug delivery system
EP2050437A1 (en) 2007-10-15 2009-04-22 Laboratoires SMB Improved pharmaceutical dry powder compositions for inhalation.
US20100098660A1 (en) 2007-05-18 2010-04-22 Research Foundation Of State University Of New York And New York University Method of treating tuberculosis with interferons
WO2010132879A2 (en) 2009-05-15 2010-11-18 The Johns Hopkins University Multicomponent degradable cationic polymers
WO2014033644A2 (en) 2012-08-28 2014-03-06 Novartis Ag Methods of nuclease-based genetic engineering
US20170216455A1 (en) 2014-08-06 2017-08-03 National University Of Ireland, Galway HYPERBRANCHED POLY (ß-AMINO ESTER) FOR GENE THERAPY
WO2021058492A1 (en) 2019-09-25 2021-04-01 University College Dublin Nanoparticle compositions for gene therapy

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005023290A2 (en) 2003-05-23 2005-03-17 Pestka Biomedical Laboratories, Inc. Uses of interferons for the treatment of severe acute respiratory syndrome and other viral infections
US20070053845A1 (en) 2004-03-02 2007-03-08 Shiladitya Sengupta Nanocell drug delivery system
US20100098660A1 (en) 2007-05-18 2010-04-22 Research Foundation Of State University Of New York And New York University Method of treating tuberculosis with interferons
EP2050437A1 (en) 2007-10-15 2009-04-22 Laboratoires SMB Improved pharmaceutical dry powder compositions for inhalation.
WO2010132879A2 (en) 2009-05-15 2010-11-18 The Johns Hopkins University Multicomponent degradable cationic polymers
WO2014033644A2 (en) 2012-08-28 2014-03-06 Novartis Ag Methods of nuclease-based genetic engineering
US20170216455A1 (en) 2014-08-06 2017-08-03 National University Of Ireland, Galway HYPERBRANCHED POLY (ß-AMINO ESTER) FOR GENE THERAPY
WO2021058492A1 (en) 2019-09-25 2021-04-01 University College Dublin Nanoparticle compositions for gene therapy

Non-Patent Citations (20)

* Cited by examiner, † Cited by third party
Title
ABBASI, M.FAUST, L.WILHELM, M.: "Comb and Bottlebrush Polymers with Superior Rheological and Mechanical Properties", ADV. MATER., vol. 31, 2019, pages 1806484
BOCH J: "TALEs of genome targeting", NATURE BIOTECHNOLOGY, vol. 29, no. 2, February 2011 (2011-02-01), pages 135 - 6, XP055077654, DOI: 10.1038/nbt.1767
CARROLL, D: "Genome engineering with zinc-finger nucleases", GENETICS SOCIETY OF AMERICA, vol. 188, no. 4, 2011, pages 773 - 78, XP055171682, DOI: 10.1534/genetics.111.131433
CORDEIRO, R. A.SERRA, A.COELHO, J. F. J.FANECA, H.: "Poly([3-amino ester)-based gene delivery systems: From discovery to therapeutic applications", JOURNAL OF CONTROLLED RELEASE, vol. 310, 2019, pages 155 - 187
DEZHONG ZHOU ET AL, SCIENCE ADVANCES, vol. 2, no. 6, 1 June 2016 (2016-06-01), pages e1600102, XP055539263, DOI: 10.1126/sciadv.1600102 *
DEZHONG ZHOU ET AL., SCIENCE ADVANCES, vol. 2, no. 6, 1 June 2016 (2016-06-01), pages e1600102
D'SOUZA, A.: "Polyethylene glycol (PEG): a versatile polymer for pharmaceutical applications", vol. 13, 2016, TAYLOR FR, article "delivery, R. S.-E. opinion on drug & 2016, undefined", pages: 1257 - 1275
FAGERLUND ET AL.: "The Cpf1 CRISPR-Cas protein expands genome-editing tools", GENOME BIOLOGY, vol. 16, 2015, pages 251 - 253
GREGORY T ZUGATES ET AL.: "Rapid Optimization of Gene Delivery by Parallel End-modification of Poly(-amino ester)s", MOLECULAR THERAPY, vol. 15, no. 7, 1 July 2007 (2007-07-01), pages 1306 - 1312, XP055261837, DOI: 10.1038/sj.mt.6300132
GREGORY T ZUGATES ET AL: "Rapid Optimization of Gene Delivery by Parallel End-modification of Poly(β-amino ester)s", MOLECULAR THERAPY, vol. 15, no. 7, 1 July 2007 (2007-07-01), US, pages 1306 - 1312, XP055261837, ISSN: 1525-0016, DOI: 10.1038/sj.mt.6300132 *
KACZMAREK JAMES C ET AL: "Systemic delivery of mRNA and DNA to the lung using polymer-lipid nanoparticles", BIOMATERIALS, ELSEVIER, AMSTERDAM, NL, vol. 275, 10 June 2021 (2021-06-10), XP086706259, ISSN: 0142-9612, [retrieved on 20210610], DOI: 10.1016/J.BIOMATERIALS.2021.120966 *
LEDFORD: "Bacteria yield new gene cutter Smaller CRISPR enzyme should simplify genome editing", NATURE, vol. 526, 2015, pages 17
MARESCA: "Obligate Ligation-Gated Recombination (ObLiGaRe): Custom-designed nuclease-mediated targeted integration through nonhomologous end joining", GENOME RES, vol. 23, 2013, pages 539 - 546, XP055077484, DOI: 10.1101/gr.145441.112
MILNER, S. T.: "Polymer brushes", SCIENCE, vol. 251, no. 80, 1991, pages 905 - 914, XP000941345, DOI: 10.1126/science.251.4996.905
O'RIORDAN ET AL., RESPIR CARE, November 2002 (2002-11-01)
SANDER, JOUNG: "CRISPR-Cas systems for editing, regulating and targeting genomes", NATURE BIOTECHNOLOGY, vol. 32, no. 4, 2014, pages 347 - 355, XP055481941, DOI: 10.1038/nbt.2842
WANG YAO ET AL.: "CHINESE JOURNAL OF POLYMER SCIENCE", vol. 38, 17 April 2020, CHINESE CHEMICAL SOCIETY AND INSTITUTE OF CHEMISTRY, article "Effects of Branching Strategy on the Gene Transfection of Highly Branched Poly(-amino ester)s", pages: 830 - 839
WANG YAO ET AL: "Effects of Branching Strategy on the Gene Transfection of Highly Branched Poly(-amino ester)s", CHINESE JOURNAL OF POLYMER SCIENCE, CHINESE CHEMICAL SOCIETY AND INSTITUTE OF CHEMISTRY, CAS, BEIJING, vol. 38, no. 8, 17 April 2020 (2020-04-17), pages 830 - 839, XP037165668, ISSN: 0256-7679, [retrieved on 20200417], DOI: 10.1007/S10118-020-2393-Y *
ZENG ET AL., NANO. LETT., vol. 19, 2019, pages 381 - 391
ZENG, 4-BRANCHING HYPERBRANCHED POLYMER

Similar Documents

Publication Publication Date Title
Luo et al. Arginine functionalized peptide dendrimers as potential gene delivery vehicles
Qiu et al. Developing biodegradable lipid nanoparticles for intracellular mRNA delivery and genome editing
Tang et al. Efficient in vitro siRNA delivery and intramuscular gene silencing using PEG-modified PAMAM dendrimers
EP4034587B1 (en) Hyperbranched cationic polymers useful as nucleic acid delivery vectors for transfecting cells
Lin et al. An acid-labile block copolymer of PDMAEMA and PEG as potential carrier for intelligent gene delivery systems
Gao et al. Gene-carried chitosan-linked-PEI induced high gene transfection efficiency with low toxicity and significant tumor-suppressive activity
Guo et al. Image-guided and tumor-targeted drug delivery with radiolabeled unimolecular micelles
US10172956B2 (en) Polymeric nanoparticles
US20130149783A1 (en) Cleavable modifications to reducible poly (amido ethylenimines)s to enhance nucleotide delivery
Tanaka et al. Development of lipid-like materials for RNA delivery based on intracellular environment-responsive membrane destabilization and spontaneous collapse
AU2014300980A1 (en) Compositions for introducing RNA into cells
Zhao et al. Tumor acidity-induced sheddable polyethylenimine-poly (trimethylene carbonate)/DNA/polyethylene glycol-2, 3-dimethylmaleicanhydride ternary complex for efficient and safe gene delivery
WO2008058457A1 (en) A biodegradable crosslinked polyethyleneimine and its uses
Ravina et al. Core− shell dendriplexes with sterically induced stoichiometry for gene delivery
Lee et al. General structure–activity relationship for poly (glycoamidoamine) s: the effect of amine density on cytotoxicity and DNA delivery efficiency
US20220259597A1 (en) Oligonucleotide antagonists for rna guided genome editing
Wang et al. Tris [2-(acryloyloxy) ethyl] isocyanurate cross-linked low-molecular-weight polyethylenimine as gene delivery carriers in cell culture and dystrophic mdx mice
Cao et al. Divalent folate modification on PEG: an effective strategy for improving the cellular uptake and targetability of PEGylated polyamidoamine–polyethylenimine copolymer
Wang et al. Circumvent PEGylation dilemma by implementing matrix metalloproteinase-responsive chemistry for promoted tumor gene therapy
Boyle et al. Molecular additives significantly enhance glycopolymer-mediated transfection of large plasmids and functional CRISPR-Cas9 transcription activation ex vivo in primary human fibroblasts and induced pluripotent stem cells
US20220340933A1 (en) Nanoparticle compositions for gene therapy
Fang et al. Inhalable Bottlebrush Polymer Bioconjugates as Vectors for Efficient Pulmonary Delivery of Oligonucleotides
WO2023131648A1 (en) Nanoparticulate compositions for gene therapy
EP2907876B1 (en) Reduction stimuli-responsive gene vector system and preparation and use thereof
EP4433031A1 (en) A composition comprising a therapeutically active agent packaged within a drug delivery vehicle

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: 23700446

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 2023700446

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2023700446

Country of ref document: EP

Effective date: 20240805