WO2024156871A1 - Mixtures of lipid nanoparticles and cell penetrating peptides - Google Patents

Mixtures of lipid nanoparticles and cell penetrating peptides Download PDF

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WO2024156871A1
WO2024156871A1 PCT/EP2024/051912 EP2024051912W WO2024156871A1 WO 2024156871 A1 WO2024156871 A1 WO 2024156871A1 EP 2024051912 W EP2024051912 W EP 2024051912W WO 2024156871 A1 WO2024156871 A1 WO 2024156871A1
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membrane
cpp
lnp
cargo
permeable
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French (fr)
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Ülo Langel
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Glasspearl Oü
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
    • A61K47/6455—Polycationic oligopeptides, polypeptides or polyamino acids, e.g. for complexing nucleic acids
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
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    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3513—Protein; Peptide
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    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific
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    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/40—Vectors comprising a peptide as targeting moiety, e.g. a synthetic peptide, from undefined source

Definitions

  • the present invention relates to a biobarrier-permeable constructs for transport of cargo such as oligonucleotides across a lipid membrane and subsequent delivery of cargo into cells as well through the in vivo epithelial barriers such as blood-brain-barrier, using the nanoparticle mixtures of lipid nanoparticles and cell penetrating peptides embedding the cargo.
  • the present invention also relates to therapeutic uses of such a construct, for example in the vaccination and in the treatment of cancer.
  • Nucleic acid delivery issues are many since most therapies require the transmembrane delivery of relatively large amounts of oligonucleotides since the RNA or mRNA are supposed to cause the cellular immune response.
  • packaging/conjugation of nanostructures between the nucleic acid payloads and the carrier system protection of nucleic acids from enzymatic degradation, promoting cellular entry or trans-barrier delivery, and targeting the delivery to specific tissue types and sites within the cell in order to be used for gene therapy and gene editing purposes.
  • the severe COVID-19 is characterized by dysregulated cytokine release profile, dysfunctional immune responses, and hypercoagulation with a high risk of progression to multi-organ failure and death (Premeaux et al., 2022).
  • Non-viral delivery systems offer improved biosafety and flexibility, and have been tremendously advanced during the recent year’s development.
  • there is still a need for optimized efficient therapeutic delivery systems that are both non-toxic and highly efficient.
  • the membrane-permeable constructs of the present invention the mixtures of LNP and CPPs of the PepFect and NickFect types, with varying ratios in range of 0-100 % or 100-0 %, respectively, have improved and are more efficient at delivery of cargo, several types of oligonucleotides (ON) : antisense ON, siRNA, miRNA, plasmid and mRNA into the intracellular compartment as well as through in vivo biobarriers.
  • ON oligonucleotides
  • the constructs synergistically increase the cargo transduction in vitro and in vivo, exposing significantly less toxic effects.
  • the membrane-permeable constructs may be used for vaccination and targeted gene therapy in e.g. tumours.
  • the constructs of the present invention are more effective for cell cargo delivery, preferably for oligonucleotide delivery.
  • the constructs are more effective at all ON delivery than existing CPPs or LNPs on their own.
  • the membrane permeable constructs of the present invention have low toxicity and less side effects due to the decreased amounts of LNP and CPP and are expected to be cheaper to synthesise compared to existing CPPs or LNPs.
  • a membrane-permeable construct for transport of ON cargo across a lipid membrane and subsequent delivery of cargo into cells, wherein the construct comprises a known cell penetrating amino acid sequence of the PepFect or NickFect types (See above).
  • a membrane-permeable construct for transport of ON cargo across a lipid membrane and subsequent delivery of cargo into cells, wherein the construct comprises a known cell penetrating amino acid sequence of the PepFect or NickFect types (see above) and the known LNP described in Yue-Xuan Li et al. 2022 .
  • the present invention relates to a membrane-permeable construct for transport of cargo across a lipid membrane and subsequent delivery of cargo into cells, the membrane permeable construct comprising a lipid nanoparticle (LNP), and a cell penetrating peptide (CPP).
  • LNP lipid nanoparticle
  • CPP cell penetrating peptide
  • the present invention relates to a method for producing a membrane-permeable construct, the method comprising the steps of providing a lipid nanoparticle (LNP), providing a cell penetrating peptide (CPP), and mixing the lipid nanoparticle (LNP) with the cell penetrating peptide (CPP), wherein the mixing of the lipid nanoparticle (LNP) and the cell penetrating peptide produces the membrane-permeable construct.
  • LNP lipid nanoparticle
  • CPP cell penetrating peptide
  • a complex comprising the construct of the invention and a ON cargo non-covalently interacting therewith (e.g. interacting via ionic interactions).
  • a method of vaccination and of treating cancer in an individual comprising administering an effective amount of the pharmaceutical composition of the invention to an individual.
  • LNP lipid nanoparticles
  • CPP cell penetrating peptides
  • LNPs are composed of ionizable cationic lipids, phospholipids, cholesterol and polyethylene glycol (PEG)-lipids. LNPs generate ⁇ 50 nm particles with narrow size distributions, this size is crucial to allowing these particles to pass through the fenestrated liver vasculature. Critical chemical issues for clinical translation have been ionizable cationic lipid optimization, diffusible PEG-lipid design and a robust, scalable manufacturing process. LNP technology is now facilitating mRNA applications for protein replacement therapy and vaccine development (Kulkarni et al., 2021 ) against influenza virus vaccine, Zika virus in mice and non-human primates, P. falciparum, personalized vaccines for cancer immunotherapy, and SARS-CoV-2.
  • mRNA/LNP therapeutic design used with the COVID vaccines have several side effects, including, inflammatory responses, immunogenicity and allergic reactions and developers are trying to optimize these delivery systems to reduce mRNA ending up in the liver and reduce the inflammatory side effects.
  • the CPP based delivery system disclosed herein is advantageous over known LNP based delivery systems in that it has increased efficiency with respect to cargo delivery, is non-inflammatory and can therefore be used for inflammatory diseases, such as, but not limited to immune diseases and neurodegenerative disease treatments without exacerbating disease symptoms.
  • the CPP based delivery system also does not cause unexpected immunogenecity and are robust to produce and stable for everyday use. Thus, there is a need for ways to deliver therapeutics to patients without triggering indesirable side effects and symptoms of disease. It is well known that while all known LNPs activate the Toll receptors, which causes both an inflammatory response and cellular immune responses, the CPP disclosed herein are known not to be ligands of the Toll receptors.
  • CPP based delivery systems are not just an alternative to existing delivery systems, but represent an improvement in delivery of therapeutic molecules to patients in general and with a particular advantage when the delivery of therapeutics is related to diseases or disorders with inflammatory components or symptoms.
  • CPPs in combination with LNPs can also alleviate the side effects often associated with LNP mediated delivery of therapeutics alone.
  • the CPP/LNP based delivery systems disclosed herein have several advantages over the presently used LNP mediated therapy alone.
  • CPP Cell penetrating peptides
  • CPP Cell-penetrating peptides
  • PTD protein/peptide transduction domains
  • Trojan peptides are based on several seminal findings which challenged the laws of cellular biochemistry and contested the traditional dogma that the cell plasma membrane was impermeable to proteins and peptides, see (Langel, 2019).
  • CPPs enabled to overcome this basic dogma by understanding the rules and pathways of chemistry in plasma membrane translocation, including the complex interactions between positively charged CPPs and negatively charged phospholipid membranes.
  • CPPs are currently used for trans-barrier delivery of most of the cargo types with biological relevance : small molecule drugs, peptides, proteins, all types of oligonucleotides incl. plasmids etc.
  • antisense ONs ASO
  • RNAi RNA interference
  • PMOs phosphorodiamidate morpholino oligomers
  • PSs phosphorothioates
  • Non-covalent strategy ON complexing to CPPs by often yields stable NPs, likely by combination of electrostatic interactions (between the positively charged CPPs and negatively charged ONs) and available hydrophobic interactions (from hydrophobic amino acids or inserted fatty acid chains), sometimes associated with an amphipathic feature of CPPs or specific structures of the complexes (Morris et al., 1997, Wyman et al., 1997, Futaki et al., 2001 , Morris et al., 2001 , Simeoni et al., 2003, Crombez et al., 2009, Eguchi et al., 2009, Michiue et al., 2009).
  • CPPs are actively used in different gene therapeutic platforms such as plasmid delivery or modifications of the genomic DNA (Taylor and Zahid, 2020).
  • the gene-editing technologies in the form of CRISPR)/CRISP-Cas (Shalaby et al., 2020) has been considerably improved by the use of CPPs, e.g. PepFect14 (Falato et al., 2022) in different cell types for therapeutic and research purposes are reviewed in (Langel, 2019).
  • Oligonucleotide-functionalized transportan and its analogs have been widely used for oligonucleotide delivery, reviewed in (Langel, 2021).
  • Transportan was designed by us as a bifunctional bioactive peptide with the ability to interact with plasma membranes. Intensive structure-activity studies of transportan yielded in cargo delivery vectors with improve chemical properties for delivery of different cargos. The role of transportan in drug delivery in general and nucleic acid delivery in particular is currently growing rapidly.
  • the cell membrane has an amphiphilic nature; to enhance CPP/membrane interactions, several CPPs, incl.
  • PepFects and NickFects have been designed to have both hydrophilic and hydrophobic regions and/or moieties in their sequence.
  • the membrane may be an artificial membrane such as an artificially constructed complex membrane formed of, for example, lipids, phospholipids or molecules having both hydrophilic and hydrophobic compounds or structures, biological membrane that is a double-layer of phospholipids and other lipids like cholesterol and proteins inserted. Most biological membranes are negatively charged and most biological uptake is by selective transporters and energy dependent processes.
  • the membrane may be a biological membrane including but not limited to eukaryotic cell membranes, prokaryotic cell membranes and epithelial membranes, e.g. the mucous membranes, serous membranes, cutaneous membranes, synovial membranes and the blood-brain barrier.
  • the membrane may be a lipid bilayer or phospholipid bilayer.
  • the membrane may be a lipid membrane of a phospholipid membrane.
  • the membrane may be a plasma membrane.
  • Eukaryotic cells membranes include, but are not limited to, membranes of immune cells such as white blood cells, red blood cells, monocytes, macrophages, neutrophils, T cells, B cells or dendritic cells, epithelial cells, endothelial cells, keratinocytes, muscle cells, skin cells, nerve cells and fat cells.
  • immune cells such as white blood cells, red blood cells, monocytes, macrophages, neutrophils, T cells, B cells or dendritic cells, epithelial cells, endothelial cells, keratinocytes, muscle cells, skin cells, nerve cells and fat cells.
  • Transportan showed the translocation to the cell cytosol (Pooga et al., 1998a), and was able to translocate the covalently attached ON cargo (Pooga et al., 1998b). This fueled the systematic structure-activity studies of its structure, first enabling the shorter transportan 10 (Soomets et al., 2000) and, later, the efficient series of PepFects and (branched) NickFects (Langel, 2021):
  • Transportan 10 TP10: AGYLLGKINLKALAALAKKIL
  • HSP70 protein has been transfected in Bomirsky Hamster Cells (BHM) in complex with PepFecet14 (Gestin et al., 2022).
  • BHM Bomirsky Hamster Cells
  • PepFect14 was able to form a complex with HSP70 and to deliver it inside cells in the same fashion with oligonucleotide delivery.
  • the delivered HSP70 showed an effect in the cell regulation indicating that the protein was biologically available in the cytoplasm and the interactions with PepFect14 did not impeach its active sites once the plasma barrier crossed (Gestin et al., 2022). This finding enables to include the delivery of bioactive proteins through the biobarriers as well.
  • a membrane-permeable construct is a construct that is biomembrane (membrane)-permeable, i.e. capable of passing across or through a membrane into the cell or intracellular environment, into the organelle (when targeted) as well as passing in vivo bio-barriers for organelle delivery in vivo.
  • the membranes and bio-barriers may be single or multiple layer structures.
  • the membrane may be an artificial membrane such as an artificially constructed complex membrane formed of, for example, lipids, phospholipids or molecules having both hydrophilic and hydrophobic compounds or structures.
  • the membrane may be a biological membrane including but not limited to eukaryotic cell membranes and prokaryotic cell membranes.
  • the membrane-permeable constructs of the present disclosure comprise a covalent or noncovalent complex of a lipid nanoparticle (LNP) of any type and a cell penetrating peptide (CPP).
  • LNP lipid nanoparticle
  • CPP cell penetrating peptide
  • the membrane-permeable construct further comprises a cargo that is contained within, embedded in, encaptured by, associated with or complex bound to the membrane-permeable construct.
  • the membrane-permeable constructs of the present disclosure may comprise a CPP and a cargo that is contained within, embedded in, encaptured by, associated with or complex bound to the membrane-permeable construct.
  • the membrane permeable construct may consist essentially of a CPP and a cargo.
  • the membrane-permeable construct may consist essentially of a CPP solution mixed with a cargo solution.
  • the membrane-permeable construct may consist essentially of a CPP and a cargo that is contained within, embedded in, encaptured by, associated with or complex bound to the membrane-permeable construct.
  • a membrane- permeable construct for transport of cargo across a lipid membrane and subsequent delivery of cargo into cells is a membrane-permeable construct comprising a lipid nanoparticle (LNP), one or more cell penetrating peptides (CPP), and optionally, a cargo.
  • LNP lipid nanoparticle
  • CPP cell penetrating peptides
  • the LNP, and the one or more cell penetrating peptides (CPP) of the membrane-permeable construct form a complex through chemical interactions.
  • the complex is formed either through chemical linkage via covalent bonds or through ionic bonds, ionic interactions, non-covalent bonds, or non-covalent interactions.
  • the LNP and the one or more cell penetrating peptides (CPP) of the membrane-permeable construct form a complex through non-covalent bonds.
  • the membrane-permeable construct comprises a cargo.
  • the cargo of the membrane- permeable construct forms a complex with the LNP and/or CPP components of the membrane- permeable construct.
  • the complex formed by the cargo and the LNP and/or CPP components of the membrane- permeable construct occur either through chemical linkage via covalent bonds or through ionic bonds, ionic interactions, non-covalent bonds, or non-covalent interactions.
  • the complex formed between the cargo and the LNP and/or CPP components of the membrane-permeable construct is formed through non-covalent interactions. In one or more exemplary embodiments of the present disclosure, the complex formed between the cargo and the LNP and/or CPP components of the membrane-permeable construct is formed through ionic interactions or ionic bonds.
  • the one or more CPPs is selected from the list consisting of Transportan (TP), Transportan 10 (TP10), PepFect 14 (PF14), NickFect 51 (NF51 ), NickFect 55 (NF55), NF554, NF70, NF71 , PF132, PF141 , PF142, PF143, GP1 , GP2, GP3 and GP4.
  • the one or more CPPs is selected from the list consisting of NickFect 51 (NF51 ), NickFect 55 (NF55), NF554, NF70, NF71 , PF132, PF141 , PF142, PF143, GP1 , GP2, GP3 and GP4.
  • the one or more CPPs is selected from the list consisting of PepFect 14 (PF14), NickFect 51 (NF51 ), NickFect 55 (NF55), NF71 , GP1. GP2, GP3 and GP4.
  • the one or more CPPs is selected from the list consisting of PepFect 14 (PF14), NickFect 51 (NF51 ), NickFect 55 (NF55), and NF71.
  • the one or more CPPs is selected from the list consisting of NickFect 51 (NF51 ), NickFect 55 (NF55), NF554, NF 70, NF71 , GP1 , GP2, GP3 and GP4.
  • the one or more CPPs is selected from the list consisting of PF132, PF141 , PF142, PF143, GP1 , GP2, GP3 and GP4.
  • the one or more CPPs is selected from the group consisting of GP3 and GP4.
  • the CPP is selected as Transportan (TP).
  • the CPP is selected as Transportan 10 (TP10).
  • the CPP is selected as PepFect 14 (PF14).
  • the CPP is selected as NickFect 51 (NF51).
  • the CPP is selected as NickFect 55 (NF55).
  • the CPP is selected as NF554. In one or more exemplary embodiments of the present disclosure, the CPP is selected as NF70. In one or more exemplary embodiments of the present disclosure, the CPP is selected as NF71. In one or more exemplary embodiments of the present disclosure, the CPP is selected as PF132. In one or more exemplary embodiments of the present disclosure, the CPP is selected as PF141. In one or more exemplary embodiments of the present disclosure, the CPP is selected as PF142. In one or more exemplary embodiments of the present disclosure, the CPP is selected as PF143. In one or more exemplary embodiments of the present disclosure, the CPP is selected as GP1.
  • the CPP is selected as GP2. In one or more exemplary embodiments of the present disclosure, the CPP is selected as GP3. In one or more exemplary embodiments of the present disclosure, the CPP is selected as GP4.
  • the membrane-permeable construct comprises an LNP and a CPP in a molar ratio of about 5:1 , such as 6:1 , 7:1 , 8:1 , 9:1 or about 10:1.
  • the membrane-permeable construct comprises an LNP and a CPP in a V/V ratio of about 5:1 , such as 6:1 , 7:1 , 8:1 , 9:1 or about 10:1.
  • the membrane-permeable construct comprises an LNP and a CPP in a W/W ratio of about 5:1 , such as 6:1 , 7:1 , 8:1 , 9:1 or about 10:1.
  • the membrane-permeable construct comprises an LNP in a concentration of 50 mM and a CPP in a concentration of 1 mM, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 mM. In one or more exemplary embodiments of the present disclosure, the membrane-permeable construct comprises an LNP in a concentration of 50 mM and a CPP in a concentration of 5 mM.
  • the membrane-permeable construct comprises an LNP in a concentration of 50 mM and a CPP in a concentration of 10 mM.
  • the membrane-permeable construct comprises an LNP in a concentration of 30-70 mM, such as 35-65, 40-60, 45-55 or 50 mM and a CPP in a concentration of 3-12 mM, such as 4-11 , 5-10, 3-7, 4-6, 5, 8-12, 9-11 , or 10 mM.
  • the membrane-permeable construct is about 50 nm in diameter, such as 50, 48-52, 45-55 or 40-60 nm in diameter.
  • the membrane-permeable construct is about 50 nm in diameter.
  • the membrane-permeable construct is a nanoparticle.
  • the present disclosure relates to a composition comprising the membrane-permeable construct as defined herein.
  • Lipid nanoparticles Lipid nanoparticles
  • LNPs are particles that are made up of lipids and are typically globular in shape. Lipids can generally be described as amphiphilic molecules that contain three domains: a polar head group, a hydrophobic tail region and a linker between the two domains. In aqueous solutions and depending on the lipid composition, the LNP can form one or more lipid bilayer(s) with one or more hydrophilic core space(s) inside of the lipid bilayer(s) (e.g. liposomes and vesicles) or the LNP may form a particle with a hydrophobic core composed of the hydrophobic tails of the lipids (e.g. micelles). In a non-limiting example LNPs are composed of ionizable lipids, cationic lipids, phospholipids, cholesterol and polyethylene glycol (PEG)-lipids.
  • PEG polyethylene glycol
  • LNPs composed of the lipids discussed herein generate particles with a diameter of about 50 nm with a narrow size distribution and this size distribution is crucial to allowing these particles to pass through the fenestrated liver vasculature.
  • LNPs can be functionalized by the association of other molecules to the surface of the LNP or by embedding of molecules into lipid layer(s) of the LNP.
  • a non-limiting example of such functionalization is the association or embedding of CPPs into or onto the LNP.
  • Other non-limiting examples of functionalization include association or embedding of dyes, tracers, radionuclides, phosphorophores and fluorophores with the LNPs.
  • association or embedding of other molecule into or onto the LNP can for example occur through chemical linkage via covalent bonds or through ionic bonds, ionic interactions, non- covalent bonds, or non-covalent interactions.
  • the CPPs defined in the present disclosure form a complex by chemical linkage via covalent bonds or through ionic bonds, ionic interactions, non-covalent bonds, or non-covalent interactions with the LNP.
  • LNPs can also be used to contain or carry a cargo for delivery to a molecular target, such as a cell of an animal or human.
  • a cargo can be associated with the LNP surface, embedded in the lipid layer of the LNP or contained within the LNP through chemical linkage via covalent bonds or through ionic bonds, ionic interactions, non-covalent bonds, or non-covalent interactions.
  • the CPPs defined in the present disclosure form a complex by chemical linkage via covalent bonds or through ionic bonds, ionic interactions, non-covalent bonds, or non-covalent interactions with the LNP.
  • the LNPs are selected as one or more of the group consisting of micelles, vesicles and liposomes. In one or more exemplary embodiments of the present disclosure, the LNPs are liposomes.
  • the liposomes comprise DPPC, Cholesterol and DSPE.
  • the DSPE is PEGylated, i.e., the DSPE is DSPE-PEG.
  • the liposome is composed of DPPC, cholesterol and DSPE-PEG.
  • the liposome is composed of DPPC, cholesterol and DSPE-PEG in a ratio of 16:11 :3.
  • the liposome is composed of DOTAP, DPPC and cholesterol.
  • the liposome is composed of DOTAP, DPPC and cholesterol in a ratio of 8:8:1 )
  • the LNP is composed of SM- 102, DSDPC, cholesterol and PEG200-DMG in a ratio of 50:10:38.5:1.5.
  • the LNP is composed of SM- 102, DSDPC, cholesterol and PEG200-DMG in a ratio of 40:20:37:3.
  • An ionizable lipid as discussed herein relates to lipids that are protonized at low pH, which makes them positively charged. However, they remain neutral at physiological pH and can for example chosen from the non-exhaustive list consisting of (2S)-2,5-bis(3- aminopropylamino)-N-[2- (dioctadecylamino)acetyl] pentanamide (DOGS; Transfectam), N1-[2-((1S)-1-[(3- aminopropyl)amino]-4-[di(3-aminopropyl)amino] butylcarboxamido)ethyl]-3,4-di[oleyloxy]- benzamide (MVL5), DC-Cholesterol and N4 -cholesterylspermine (GL67).
  • Other ionizable lipids that can be used in the formation of LNPs as defined herein are well known to the skilled person.
  • a cationic lipid as discussed herein relates to lipids containing a a head group with a permanent positive charge.
  • cationic lipids can be selected from the non-exhaustive list consisting of 1 ,2- di-O-octadecenyl-3-trimethylammonium-propane (DOTMA), 1 ,2-dioleoyl-sn- glycero-3-phosphoethanolamine (DOPE) and 1 ,2-dioleoyl-3-trimethylammonium-propane (DOTAP).
  • DOTMA 1,2- di-O-octadecenyl-3-trimethylammonium-propane
  • DOPE 1 ,2-dioleoyl-sn- glycero-3-phosphoethanolamine
  • DOTAP 1,2-dioleoyl-3-trimethylammonium-propane
  • lipids include phospholipids such as for example phosphatidylcholine and phosphatidylethanolamine, cholesterol or polyethylene glycol (PEG)-functionalized lipids (PEG- lipids).
  • PEG polyethylene glycol
  • Other phospholipids and PEG-functionalized lipids in general that can be used in teh formation of LNPs as defined herein are well known to the skilled person.
  • lipids that can be used in the formation of LNPs as defined herein arepreented in table 1.
  • Other lipids suitable for use in the formation of LNPs are well known to the skilled person.
  • Table 1 Examples of lipids suitable for use in the formation of LNPs.
  • the LNPs of the present disclosure is formed by dissolution of a selection of one or more lipids in desired concentrations in organic solvent, removal of the organic solvent by e.g. rotary evaporation to form a lipid film, hydration of the lipid film in a dispersion medium by agitation, and thereby forming the desired LNPs.
  • LNPs with a specific size or diameter for example, by extrusion of the LNPs through a membrane filter having a specific pore size.
  • the organic solvent is chosen as chloroform.
  • other organic solvents that can be used for the formation of LNPs are generally known to the skilled person.
  • An example of a suitable dispersion medium is HBS buffer and other suitable buffers are well known to the skilled person.
  • the LNP can further also comprise a cargo intended for delivery across a membrane.
  • the methods for producing a LNP comprises loading of a cargo.
  • loading of a lipophilic cargo is achieved during LNP formation for example by dissolution of the lipophilic cargo in the organic solvent together with the lipids that make up the LNP.
  • the lipophilic cargo is then associated with or embedded in the lipophilic parts of the LNP already during the formation of the lipid film.
  • loading of a hydrophilic cargo into the hydrophilic core of a LNP is achieved during LNP formation for example by dissolution of the hydrophilic cargo in the dispersion medium prior to hydration of the lipid film.
  • the hydrophilic cargo is then loaded into the hydrophilic core, when the lipid film is hydrated and agitated with the dispersion medium containing a hydrophilic cargo during the LNP formation.
  • the cargo is loaded in a concentration of 1 mol% of the total lipid content of the LNP.
  • the cargo is loaded in a concentration of 1 , such as 2, 3, 4 or 5 mol% of the total lipid content of LNP.
  • Cell penetrating amino acid sequences also referred to as cell penetrating peptides (CPPs) are short amino acid sequences that transport different types of cargo molecules across a lipid membrane and facilitate cellular uptake of the cargo molecules.
  • a property of cell penetrating amino acids is their ability to translocate the lipid membrane and facilitate the delivery of various molecular cargoes to the cytoplasm or to an organelle of a cell or to an intracellular cell surface or an in vivo barrier.
  • the cell penetrating amino acid sequence comprises the following known CPP sequences exemplified below:
  • Transportan 10 TP10: AGYLLGKINLKALAALAKKIL (Soomets et al., 2000)
  • NF55 O(Nb-stearoyl-AGYLLG)-INLKALAALAKAIL amide (Freimann et al., 2016)
  • NF554 K(NE-stearoyl-AGYLLG)-INLKALAALAKAIL amide (Freimann et al., 2016)
  • the C-terminal of the CPPs listed above is further amidated.
  • the cargo molecule is associated (embedded) by the CPP/LNP complex either through chemical linkage via covalent bonds or through non-covalent bond or ionic bonds or non-covalent interactions or ionic interactions.
  • the embedded cargo may be transported from outside of the cell across the membrane of the cell and enter into the cell or through the bio-barriers.
  • Cargo includes nucleic acids (e.g. RNA, miRNA, DNA, siRNA, shRNA, antisense oligonucleotides, decoy DNA, plasmid DNA), but even the small molecule drugs, imaging agents (e.g. fluorophore), radioactive tracers, metal chelates.
  • the peptide may be selected from a group consisting of, but not limited to, a cell or tumour targeting peptide, an aptamer, a receptor ligand, a peptide ligand, a cytotoxic peptide, a bioactive peptide, an antibody, and a diagnostic agent.
  • the nucleic acid may comprise one or more nucleic acids where each one encodes one peptide or polypeptide.
  • the cargo molecule may be a combination of a protein, a lipid, and/or a polysaccharide including lipoproteins and glycolipids.
  • the cargo may be selected from a group consisting of, but not limited to, oligonucleotides including single-stranded oligonucleotides (e.g. DNA, RNA, PNA, LNA and their analogues), double-stranded oligonucleotides (e.g. siRNA, shRNA and decoyDNA) and cyclic DNA (e.g. plasmids).
  • the membrane-permeable construct comprises a LNP/CPP/cargo complex according to the present invention wherein a cargo non-covalently interacting therewith, for example via ionic interactions.
  • cargo suitably is ionic and carries negative charges.
  • the components of the LNP/CPP/cargo complex are present, in a ratio of 0-100% or 100-0%.
  • Nanoparticles are generally understood to be small particles that have a diameter of between 10 and 100 nm.
  • a nanoparticle is to be understood as a particle comprises a membrane-permeable construct as defined herein that has a diameter between 10-100 nm.
  • the membrane-permeable construct complex forms a nanoparticle.
  • the nanoparticle consists of a membrane-permeable construct as defined herein that has a diameter between 20-100 nm.
  • the nanoparticle of the present invention may for example be 20 to 100 nm in diameter, such as about 30-90, 40-80, 50-70, 55-65, 20-30, 30-40, 40-50, 60-70, 70-80, 80-90, 90-100, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95 or such as about 100 nm.
  • a nanoparticle of a desired size is obtained by extrusion of the nanoparticle through a membrane filter, where the membrane filter comprises pores wherein the pore size and/or pore diameter determines the diameter of the nanoparticles obtained after the extrusion process.
  • the membrane-permeable construct of the present disclosure is produced in several consecutive steps that include the steps of providing a LNP (lipid nanoparticle), providing a cell penetrating peptide (CPP), and mixing the LNP together with the CPP.
  • LNP lipid nanoparticle
  • CPP cell penetrating peptide
  • a general method for producing the membrane-permeable construct as defined herein comprises the steps providing a lipid nanoparticle (LNP), providing a cell penetrating peptide (CPP), mixing the lipid nanoparticle with the cell penetrating peptide (CPP),
  • the membrane-permeable constructs can further also optionally comprise a cargo intended for delivery across a membrane.
  • the methods for producing a membrane-permeable construct further comprises the steps providing a cargo, and loading the cargo onto or into the membrane-permeable construct.
  • the loading of the cargo onto or into the membrane-permeable construct is achieved by mixing the membrane-permeable membrane with the cargo.
  • the lipid nanoparticle (LNP) used for producing the membrane-permeable construct comprises the cargo.
  • the lipid nanoparticle (LNP) used for producing the membrane-permeable construct contains the cargo.
  • the cargo is embedded in, associated with, connected to, or loaded onto or into the lipid nanoparticle (LNP) used for producing the membrane-permeable construct.
  • the cargo is loaded in a concentration of 1 mol% of the total lipid content of the membrane-permeable construct.
  • the cargo is loaded in a concentration of 1 , such as 2, 3, 4 or 5 mol% of the total lipid content of the membrane-permeable construct.
  • the methods for producing a membrane-permeable construct comprises mixing an LNP and a CPP in a molar ratio of about 5:1 , such as 6:1 , 7:1 , 8:1 , 9:1 or about 10:1.
  • the methods for producing a membrane-permeable construct comprises mixing an LNP and a CPP in a V/V ratio of about 5:1 , such as 6:1 , 7:1 , 8:1 , 9:1 or about 10:1.
  • the methods for producing a membrane-permeable construct comprises mixing an LNP and a CPP in a w/w ratio of about 5:1 , such as 6:1 , 7:1 , 8:1 , 9:1 or about 10:1.
  • the methods for producing a membrane-permeable construct comprises mixing an LNP in a concentration of 50 mM and a CPP in a concentration of 1 mM, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 mM.
  • the methods for producing a membrane-permeable construct comprises mixing an LNP in a concentration of 50 mM and a CPP in a concentration of 5 mM.
  • the methods for producing a membrane-permeable construct comprises mixing an LNP in a concentration of 50 mM and a CPP in a concentration of 10 mM. In one or more exemplary embodiments of the present disclosure, the methods for producing a membrane-permeable construct comprises mixing an LNP in a concentration of 30-70 mM, such as 35-65, 40-60, 45-55 or 50 mM and a CPP in a concentration of 3-12 mM, such as 4-11 , 5-10, 3-7, 4-6, 5, 8-12, 9-11 , or 10 mM.
  • the methods for producing a membrane-permeable construct comprises mixing a LNP and a CPP in a charge ratio (CR) of 1 :1.5-3, 1 :2-3, 1 ;1 ,5, 1 :2 or 1 :3.
  • the methods for producing a membrane-permeable construct comprises mixing a LNP and a CPP in a charge ratio (CR) of 1 :1.5-3.
  • the methods for producing a membrane-permeable construct comprises mixing a LNP and a CPP in a charge ratio (CR) of 1 :2-3.
  • the methods for producing a membrane-permeable construct comprises mixing a LNP and a CPP in a charge ratio (CR) of 1 :1.5. In one or more exemplary embodiments of the present disclosure, the methods for producing a membrane-permeable construct comprises mixing a LNP and a CPP in a charge ratio (CR) of 1 :2. In one or more exemplary embodiments of the present disclosure, the methods for producing a membrane-permeable construct comprises mixing a LNP and a CPP in a charge ratio (CR) of 1 :3.
  • the methods for loading a cargo onto a membrane-permeable construct comprises mixing the membrane-permeable construct with a cargo in a charge ratio (CR) of the cargo to the CPP comprised by the membrane-permeable construct of 1 :1.5-3, 1 :2-3, 1 ;1 ,5, 1 :2 or 1 :3.
  • the methods for loading a cargo onto a membrane-permeable construct comprises mixing the membrane-permeable construct with a cargo in a charge ratio (CR) of the cargo to the CPP comprised by the membrane-permeable construct of 1 :1.5-3.
  • the methods for loading a cargo onto a membrane- permeable construct comprises mixing the membrane-permeable construct with a cargo in a charge ratio (CR) of the cargo to the CPP comprised by the membrane-permeable construct of 1 :2- 3. In one or more exemplary embodiments of the present disclosure, the methods for loading a cargo onto a membrane-permeable construct comprises mixing the membrane-permeable construct with a cargo in a charge ratio (CR) of the cargo to the CPP comprised by the membrane- permeable construct of 1 :1.5.
  • the methods for loading a cargo onto a membrane-permeable construct comprises mixing the membrane-permeable construct with a cargo in a charge ratio (CR) of the cargo to the CPP comprised by the membrane-permeable construct of 1 :2. In one or more exemplary embodiments of the present disclosure, the methods for loading a cargo onto a membrane-permeable construct comprises mixing the membrane-permeable construct with a cargo in a charge ratio (CR) of the cargo to the CPP comprised by the membrane-permeable construct of 1 :3.
  • the LNP is selected as one of the group consisting of a liposome, a vesicle and micelle.
  • the LNP is selected as a liposome.
  • the LNP used in the methods for producing a membrane-permeable construct as disclosed herein is a liposome and the cargo is selected as one or more of a DNA molecule, a RNA molecule, an oligonucleotide, a protein, a peptide and a small molecule.
  • the LNP used in the methods for producing a membrane- permeable construct as disclosed herein is a liposome containing a cargo, where the cargo is selected as one or more of a DNA molecule, a RNA molecule, an oligonucleotide, a protein, a peptide and a small molecule, wherein the cargo is dissolved in HBS buffer and contacted with the lipid film during hydration of the lipid film during formation of the liposome.
  • the LNP used in the methods for producing a membrane- permeable construct as disclosed herein is selected as a liposome composed of DPPC, DSPE- PEG and cholesterol in a 16:11 :3 ratio.
  • the LNP used in the methods for producing a membrane- permeable construct as disclosed herein is selected as a liposome composed of DOTAP, DPPC and cholesterol in a 8:8:1 ratio.
  • the lipid composition ratio of the liposome is a V/V percent ratio or w/w percent ratio.
  • the membrane-construct is subjected to size separation in order to produce a membrane-permeable construct that is uniform in size.
  • the membrane-permeable construct is formed by addition of one or more CPPs as defined herein to the LNPs before extrusion of the LNPs through a membrane filter.
  • the membrane-permeable construct is formed by addition of one or more CPPs as defined herein to the LNPs after extrusion of the LNPs through a membrane filter.
  • the membrane-permeable construct is formed by addition of one or more CPPs as defined herein to LNPs loaded with a lipophilic cargo before or after extrusion of the LNPs through a membrane filter.
  • the membrane-permeable construct is formed by addition of one or more CPPs as defined herein to LNPs loaded with a hydrophilic cargo before or after extrusion of the LNPs through a membrane filter. In one or more exemplary methods for producing the membrane-permeable construct of the present disclosure, the membrane-permeable construct is formed by dissolution of the one or more CPPs in an organic solvent together with the lipid(s) chosen to form the LNP.
  • the membrane-permeable construct is formed by dissolution of the one or more CPPs into the dispersion medium prior to hydration of the lipid film.
  • the membrane-permeable construct is formed by addition of the one or more CPPs to a composition comprising an LNP that has been extruded through a membrane filter.
  • the method for producing a membrane-permeable construct includes addition of the one or more CPPs to a composition comprising an LNP before extrusion through a membrane filter.
  • membrane-permeable constructs of the present disclosure that comprises a CPP component and a cargo
  • these membrane-permeable constructs can be produced by
  • a membrane-permeable construct produced in this way may consist essentially of a CPP and a cargo. It is desirable that a CPP used in this fashion is a lipidated CPP, such as for example stearoylated or arachidoylated CPP.
  • the CPP and the cargo may be mixed in a molar ratio (MR) of 15-30:1.
  • the CPP and the cargo may be mixed in a molar ratio of about 15:1.
  • the CPP and the cargo may be mixed in a molar ratio of about 30:1.
  • the CPP and the cargo may be mixed in a charge ratio (CR) of 1.5-3:1 , 2-3:1 , 1.5:1 , 2:1 , or 3:1 .
  • the CPP and the cargo may be mixed in a charge ratio (CR) of 1.5-3:1.
  • the CPP and the cargo may be mixed in a charge ratio (CR) of 2-3:1.
  • the CPP and the cargo may be mixed in a charge ratio (CR) of 1.5:1. In one or more exemplary embodiments, the CPP and the cargo may be mixed in a charge ratio (CR) of 2:1. In one or more exemplary embodiments, the CPP and the cargo may be mixed in a charge ratio (CR) of 3:1.
  • the one or more CPPs used in a method for producing a membrane-permeable construct is selected as one or more lipidated CPPs.
  • the one or more CPPs used in a method for producing for producing a membrane-permeable construct is selected as one or more stearylated CPPs.
  • the one or more CPPs used in a method for producing for producing a membrane-permeable construct is selected as one or more arachidoylated CPPs.
  • the one or more CPPs used in a method for producing for producing a membrane-permeable construct is selected as one or more from the list consisting of Transportan (TP), Transportan 10 (TP10), PepFect 14 (PF14), NickFect 51 (NF51), NickFect 55 (NF55), NF554, NF70, NF71 , PF132, PF141 , PF142, PF143, GP1 , GP2, GP3 and GP4.
  • the one or more CPPs used in a method for producing for producing a membrane-permeable construct is selected as one or more from the list consisting of PepFect 14 (PF14), NickFect 51 (NF51), NickFect 55 (NF55), NF554, NF70, NF71 , PF132, PF141 , PF142, PF143, GP1 , GP2, GP3 and GP4.
  • the one or more CPPs used in a method for producing for producing a membrane-permeable construct is selected as one or more from the list consisting of NickFect 51 (NF51), NickFect 55 (NF55), NF554, NF70, NF71 , PF132, PF141 , PF142, PF143, GP1 , GP2, GP3 and GP4.
  • the membrane-permeable constructs, nanoparticles or complexes of the invention may be formulated for delivery in pharmaceutical compositions.
  • the pharmaceutical composition will normally be sterile and will typically include a pharmaceutically acceptable carrier and/or adjuvant.
  • a pharmaceutical composition of the present invention may additionally comprise a pharmaceutically acceptable adjuvant and/or carrier.
  • Compositions of the invention suitably comprise a construct or complex of the invention together with a pharmaceutically acceptable carrier.
  • the present invention provides a pharmaceutical composition for use in medicine.
  • the present invention further provides a pharmaceutical composition for use in a therapeutic use, e.g RNA vaccination or gene therapy, e.g. in treatment or preventing cancer.
  • the cancer may be any cancer or tumour that is a solid cancer or tumour.
  • the solid cancer or tumour may bone, bladder, brain, breast, colon, oesophagus, gastrointestinal tract, genito-urinary tract, kidney, liver, lung, nervous system, ovary, pancreas, prostate, retina, skin, stomach, testicular and/or uterus cancer.
  • the present disclosure relates to a membrane- permeable construct, nanoparticle or pharmaceutical composition as defined herein for use a as a medicament.
  • the membrane-permeable construct, nanoparticle or pharmaceutical composition as defined herein is intended for use in a vaccine.
  • membrane-permeable construct, nanoparticle or pharmaceutical composition as defined herein is intended for use in the treatment or prevention of cancer.
  • membrane-permeable construct, nanoparticle or pharmaceutical composition as defined herein is intended for use in the treatment of solid tumors.
  • the membrane-permeable construct, nanoparticle or pharmaceutical composition as defined herein is intended for use in a method of treating cancer in an individual, the method comprising administering an effective amount of the membrane-permeable construct, the nanoparticle, or pharmaceutical composition to the individual.
  • the membrane-permeable construct, nanoparticle or pharmaceutical composition as defined herein is intended for use in a method of gene silencing in an individual, the method comprising administering an effective amount of the membrane-permeable construct, the nanoparticle, or pharmaceutical composition to the individual.
  • the membrane-permeable construct, nanoparticle or pharmaceutical composition as defined herein is intended for use in a method of vaccination in an individual, the method comprising administering an effective amount of the membrane-permeable construct, the nanoparticle, or pharmaceutical composition to the individual.
  • a membrane-permeable construct for transport of cargo across a lipid membrane and subsequent into cells comprising a cell penetrating peptide (CPP).
  • CPP cell penetrating peptide
  • the membrane-permeable construct according to item 1 comprising a lipid nanoparticle (LNP), and a cell penetrating peptide (CPP).
  • LNP lipid nanoparticle
  • CPP cell penetrating peptide
  • CPP cell penetrating peptide
  • TP Transportan
  • TP10 Transportan 10
  • PF14 PepFect 14
  • NF51 NickFect 51
  • NF55 NickFect 55
  • NF554 NF70, NF71 , PF132, PF141 , PF142, PF143, GP1 , GP2, GP3 and GP4.
  • lipid nanoparticle is composed of ionizable cationic lipids, phospholipids, cholesterol and polyethylene glycol (PEG)-lipids.
  • lipid nanoparticle is a liposome, vesicle, or micelle.
  • lipid nanoparticle is a liposome.
  • liposome comprises DPPC, Cholesterol and DSPE.
  • DSPE is PEGylated
  • the liposome is composed of DPPC, Cholesterol and PEGylated DSPE (DSPE-PEG) in a ratio of 16:11 :3 DPPC:Cholesterol:Pegylated DSPE (DSPE-PEG).
  • membrane-permeable construct according to any one of the preceding items, wherein the membrane-permeable construct further comprises a cargo.
  • the cargo is selected as one or more of a peptide or protein, a non-peptide pharmaceutical, a nucleic acid, a single stranded or double stranded oligonucleotide, imaging agents, metal chelates, and small molecule drugs.
  • the membrane-permeable construct according to item 18, wherein the peptide or protein cargo is selected from the group consisting of a cell targeting peptide or tumour targeting peptide, an aptamer, a receptor ligand, a peptide ligand, a cytotoxic peptide, a bioactive peptide, an antibody, and a diagnostic agent.
  • the membrane-permeable construct according to item 18, wherein the nucleic acid is selected from the group consisting of an RNA, mRNA, miRNA, DNA, siRNA, shRNA, antisense oligonucleotides, decoy DNA, a plasmid and a cyclic DNA molecule.
  • the membrane-permeable construct according to any one of the preceding items wherein the membrane-permeable construct is a complex formed by a lipid nanoparticle (LNP) as defined in any one of items 6-13 and a cell penetrating peptide (CPP) as defined in any one of items 2-5, or wherein the membrane-permeable construct is a complex formed by a lipid nanoparticle (LNP) as defined in any one of items 6-13, a cell penetrating peptide (CPP) as defined in any one of items 2-5 and a cargo as defined in any one of items 14-21 .
  • LNP lipid nanoparticle
  • CPP cell penetrating peptide
  • 25. The membrane-permeable construct according to any one of the preceding items, wherein the membrane-permeable construct consists essentially of cell penetrating peptide (CPP).
  • membrane-permeable construct according to any one of the preceding items, wherein the membrane-permeable construct consists essentially of cell penetrating peptide and a cargo.
  • the membrane-permeable construct according to any one of the preceding items comprising CPP to cargo in a charge ratio (CR) of 1.5-3:1 , 2-3:1 , 1.5:1 , 2:1 or 3:1 .
  • membrane-permeable construct according to any one of the preceding items, wherein the membrane permeable construct is a nanoparticle.
  • T he nanoparticle according to item 39 wherein the nanoparticle has a diameter of about 20-100 nm, such as about 30-90, 40-80, 50-70, 55-65, 20-30, 30-40, 40-50, 60-70, 70-80, 80-90, 90-100, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95 or such as about 100 nm.
  • a method for producing a membrane-permeable construct comprising the steps providing a cell penetrating peptide (CPP)
  • the method for producing a membrane-permeable construct according to item 41 comprising the steps providing a lipid nanoparticle (LNP), providing a cell penetrating peptide (CPP), and mixing the lipid nanoparticle (LNP) with the cell penetrating peptide (CPP), wherein the mixing of the lipid nanoparticle (LNP) and the cell penetrating peptide produces the membrane-permeable construct.
  • the cell penetrating peptide (CPP) comprises a fatty acid modification.
  • CPP cell penetrating peptide
  • lipid nanoparticle is composed of ionizable cationic lipids, phospholipids, cholesterol and polyethylene glycol (PEG)-iipids.
  • lipid nanoparticle is a liposome, vesicle, or micelle.
  • the liposome is composed of DPPC, Cholesterol and DSPE(PEG) in a ratio of 16:11 :3.
  • the method according to item 55, wherein the loading of the cargo onto or into the membrane-permeable construct is achieved by mixing the membrane-permeable membrane with the cargo.
  • the lipid nanoparticle (LNP) comprises a cargo.
  • the method comprises loading of a cargo into or onto the lipid nanoparticle (LNP).
  • the cargo is selected as one or more of a peptide or protein, a non-peptide pharmaceutical, a nucleic acid, a single stranded or double stranded oligonucleotide, imaging agents, metal chelates, and small molecule drugs. 60.
  • the cargo is a peptide or protein selected from the group consisting of a cell targeting peptide or tumour targeting peptide, an aptamer, a receptor ligand, a peptide ligand, a cytotoxic peptide, a bioactive peptide, an antibody, and a diagnostic agent.
  • the cargo is a nucleic acid selected from the group consisting of an RNA, mRNA, miRNA, DNA, siRNA, shRNA, antisense oligonucleotides, decoy DNA, a plasmid and a (cyclic) DNA molecule.
  • the cargo is a single stranded oligonucleotides selected as one or more from the group consisting of DNA, RNA and their analogues, such as PNA and LNA, and the double stranded oligonucleotide is selected as one or more from the group consisting of siRNA, shRNA, decoyDNA, plasmids and cyclic DNA.
  • the membrane-permeable construct is produced by mixing CPP and LNP in a charge ratio (CR) of 3:1 .
  • the method for producing a membrane-permeable construct consists essentially of providing a cell penetrating peptide (CPP), and mixing the CPP with a cargo, thereby providing a membrane permeable construct consisting essentially of a cell penetrating peptide and a cargo.
  • CPP cell penetrating peptide
  • a composition comprising the membrane-permeable construct according to any one of items 1-38, the nanoparticle of any one of the items 39-40, or the membrane-permeable construct as produced by the method as defined in any one of items 41-76.
  • a pharmaceutical composition comprising the membrane-permeable construct according to any one of items 1-38, the nanoparticle of any one of the items 39-40, or the membrane- permeable construct as produced by the method as defined in any one of items 41-76, and a pharmaceutically acceptable carrier.
  • CPP selected from the list of 1-16, Transportan (TP), Transportan 10 (TP10), PepFect 14 (PF14), NickFect 51 (NF51 ), NickFect 55 (NF55), NF554, NF70, NF71 , PF132, PF141 , PF142, PF143, GP1 , GP2, GP3 and GP4 in concentration of 1 and 5 mM in the transfection mixture will be added.
  • HEK-293 cells are used for the transfection experiments. Cells are grown in Dulbecco’s modified Eagles medium (DMEM) supplemented with glutamax, 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate, 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 pg/ml streptomycin. 5.0 x 104 HEK 293 cells are seeded into 24-well plates 24 h prior to experiments.
  • DMEM Dulbecco’s modified Eagles medium
  • FBS fetal bovine serum
  • the cell medium in the wells was replaced with 450 pl serum-free medium.
  • the cells are treated with complexes in charge ratios ranging from 1 :1 to 1 :5 for 4 h. 55 pl fetal bovine serum is added and the cells are incubated for an additional 20 h. After incubation the cells are lysed with cell lysis buffer. Luciferase activity is measured using a Glomax luminometer. Readouts are normalized to protein content, and measured using Bradford protein assay. We expect that the most efficient LNP/CPP mixture for plasmid transfection will be selected based on these optimization results. The efficacy of transfection is decided in comparison with the only LNP mixture as well as the selected CPP at the same concentration.
  • HEK-293 cells are used for the transfection experiments. Cells are grown in Dulbecco’s modified Eagles medium (DMEM) supplemented with glutamax, 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate, 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 pg/ml streptomycin. 5.0 x 104 HEK 293 cells are seeded into 24-well Plates 24 h prior to experiments.
  • DMEM Dulbecco’s modified Eagles medium
  • FBS fetal bovine serum
  • the cell medium in the wells was replaced with 450 pl serum-free medium.
  • the cells are treated with complexes in charge ratios ranging from 1 :1 to 1 :5 for 4 h. 55 pl fetal bovine serum is added and the cells are incubated for an additional 20 h. After incubation the cells are lysed with cell lysis buffer. Luciferase activity is measured using a Glomax luminometer. Readouts are normalized to protein content, and measured using Bradford protein assay. We expect that the improvement of the efficacy of the plasmid transfection will be achieved and will be selected based on these optimization results. The efficacy of transfection is decided in comparison with the only LNP mixture as well as the selected CPP at the same concentration.
  • Optical microscopy 96-well plates are seeded with 7,000 BHM pLuc expressing cells per well in 100 pL DMEM supplemented with 10% FBS and incubated at 37 °C for one day.
  • the complex of optimized LNP mixture with CPP:HSP70-Alexa 568 (2 pM:200 nM) is formed in milliQ water for 30 min at room temperature before addition to the cells for 1 or 3 h.
  • the wells are emptied and wash thrice with 200 pL Gibco Opti-MEM before a final addition of 100pL of Gibco FluoroBrite.
  • the plate is then imaged for the cargo uptake using an epi-fuorescence microscope.
  • the protein cargo delivery by optimized LNP/CPP mixture will be applied for delivery of other negatively charged, therapeutically relevant proteins among enzymes, transcription factors, genome editing proteins, Cas9 proteins, TALEs, TALENs, nucleases, binding proteins (e.g., ligands, receptors, antibodies, antibody fragments; nucleic acid binding proteins, etc.), structural proteins, and therapeutic proteins (e.g., tumor suppressor proteins, therapeutic enzymes, growth factors, growth factor receptors, transcription factors, proteases etc.
  • binding proteins e.g., ligands, receptors, antibodies, antibody fragments; nucleic acid binding proteins, etc.
  • therapeutic proteins e.g., tumor suppressor proteins, therapeutic enzymes, growth factors, growth factor receptors, transcription factors, proteases etc.
  • the cells are treated with the complexes at different MRs and siRNA concentrations and then incubated for 24 h at 37 °C.
  • the cells are lysed using 100 pl of cell lysis buffer (Promega, Sweden) for 30 min at RT.
  • Luciferase activity is measured on a GlomaxTM 96 microplate luminometer (Promega, Sweden) using Promega’s luciferase assay system to determine gene-silencing efficiency. Relative light units (RLU) were obtained and normalized to the control (untreated cells).
  • the positive control RNAiMAX was utilized according to the manufacturer’s instruction.
  • Stealth RNAiTM siRNA negative control was used as a negative control.
  • the most efficient LNP/CPP/Cargo mixtures will be used to show the efficacy of transfection in vivo.
  • the optimized, 100 pl of most efficient mixtures of LNP/CPP/Cargo plasmid in MQ-water/10% glucose or 10.8% mannitol solution will be injected into the tail-vein of mice.
  • Hydrodynamic infusions in tail vein of mice will be carried out using 2 ml saline buffer injected with high pressure. Hydrodynamic mean diameter of the particles will be determined.
  • the efficacy of in vivo transfection is decided in comparison with the only LNP mixture as well as the selected CPP at the same concentration.
  • the CPPs with the alternative names GP11 , GP12, GP13, GPM, GP15 and GP16 used in the following examples X-Y correspond to the following CPPs and CPP sequences:
  • GP11 corresponds to PepFect14, PF14: stearoyl-AGYLLGKLLOOLAAAALOOLL amide.
  • GP15 corresponds to PepFect132, PF132: stearoyl-LHLLHHINLKALAALAKKIL amide.
  • GP12 corresponds to NickFect55, NF55: O(Nb-stearoyl-AGYLLG)-INLKALAALAKAIL amide.
  • GP13 corresponds to NickFect554, NF554: K(NE-stearoyl-AGYLLG)-INLKALAALAKAIL amide.
  • GP16 corresponds to NickFect71 , NF71 : O(Nb-stearoyl-HHYHHG)-ILLKALKALAKAIL amide.
  • GPM corresponds to GP2: stearoyl-HHYHHGINLKALAALAKAIL amide.
  • All peptides have fatty acid residue in the N-terminus and all peptides are C-terminally amidated.
  • mRNA refers to a reporter mRNA encoding firefly luciferase (CleanCap FLuc mRNA, Trilink)
  • plasmid refers to a reporter plasmid encoding firefly luciferase (pGL3 plasmid with the size 4818 bp from Promega)
  • siRNA refers to firefly luciferase targeting small interfering RNA.
  • Sense and antisense strands were annealed in ultrapure water. Sequence of the siRNA can be found in POROSK, L. et al., Biomater Sci. 2019 Sep. Lipids, materials and components for formulation of LNPs and CPP complexes
  • Absolute ethanol ( 96%), ultrapure water, molecular biology grade glucose (Fisher Scientific), 1 M HEPES buffer solution (Sigma), Low bind tubes (Axigen Maxymum Recovery 1.5 ml tubes), PCR grade 0.2 ml tubes (Nerbe Plus), Lipid Nanoparticle Exploration Kit (Cayman chemicals), Dialysis chambers 3.5K MWCO (Thermo Scientific), Fluorescent dyes: PicoGreen and RiboGreen (Thermo Fisher Scientific).
  • Vortex tabletop centrifuge (up to 16,000 x g), freeze-dryer, -80°C freezer, pipettes and pipet tips with filters.
  • DMEM media supplemented with non-essential amino acids, sodium pyruvate, Penicillin, Streptomycin, foetal bovine serum (Thermo), HEPES.
  • trypsin-EDTA was used for detaching cells from the culture plate incubation with 0.25% trypsin-EDTA.
  • Reporter protein detection expressed firefly luciferase was detected from the cell lysate.
  • 0.1% Triton-X 100 solution on 1 x PBS was used and 30 pl of lysis solution was incubated with transfected cells in 96-well plate well for 20 min.
  • For detection 20 pl of cell lysate was transferred to a white 96-well plate and luciferase detection mix containing luciferin was added to the lysate and luminescence was detected with GLOMAX plate reader.
  • CPP and oligonucleotide were mixed in ultrapure water at charge ratios (CR) based on the positive charges in the CPP and the negative charges in the oligonucleotide backbone.
  • the different mixing method used include
  • siRNA stock solution Dilute siRNA stock solution to 2 pM working solution in ultrapure water. Dilute 1 mM CPP stock solution to 50 pM working solution. Add water to the tube and dilute the nucleic acid working solution. Mix well. Add CPP working solution and mix by pipetting. Incubate at room temperature for 30 minutes. The complexes are now ready and can be added to the cells in 90 microliters of media.
  • Example 1.3 Charge ratio and charge ratio calculations
  • the charge ratio (CR) as refered to in this disclosure generally relates to the ratio of electrical charges of the CPP to the charges of either the cargo, mRNA or plasmids, or the LNPs disclosed herein.
  • each amino acid contributes 1 negative charge (-1 ) and each base pair of a plasmid 2 negative charges (-2).
  • the CPP concentration used needs to correspond to an amount of positive net charges that is three times the number of negative charges provided by the concentration of DNA or plasmid used together with the CPP.
  • the number of positive charges provided by the CPP needs to match the negative charges of a plasmid.
  • the total negative charge is 21000.
  • an average molecular weight of per base pair of 6993000 Da, and a plasmid dose for use in transfection of 0,1 pg a total of 1 ,43x10 -14 mol plasmid is used.
  • the amount of CPP required to reach a CR of 1 :1 when mixed with 1 ,43x10 -14 mol plasmid can then be calculated as the total negative charge (21000) divided by the positive net charge of the CPP (+3) times the mol plasmid used (1.43x10 -14 ), which equals 1 ,001x10 -1 °. Examples of solutions and concentrations used to reach a given charge ratio is provided in the tables of examples 1.3.1 and 1.3.2 below.
  • Table 6 One way to mix a CPP/plasmid complex having a charge ratio (OR) of 2:1 , wherein the CPP has a net charge of +5.
  • Table 7 Example of ingredients used to mix a CPP/plasmid complex having a charge ratio (CR) of 2:1 , wherein the CPP has a net charge of +3.
  • CR charge ratio
  • Table 8 Example of ingredients used to mix a CPP/mRNA complex having a charge ratio (CR) of 3:1 , wherein the CPP has a net charge of +3.
  • Table 9 Example of ingredients used to mix a CPP/mRNA complex having a charge ratio (CR) of 3:1 , wherein the CPP has a net charge of +5.
  • CR charge ratio
  • Table 10 Example of ingredients used to mix a CPP/mRNA complex having a charge ratio (CR) of 3:1 , wherein the CPP has a net charge of +3.
  • CR charge ratio
  • compositions of LNPs were prepared according to the LNP mix 1 approach or the LNP mix 2 approach.
  • LNP mix 1 consisting of SM-102, 1 ,2-DSPC, Cholesterol, and DMG-PEG(2000), was mixed in following molar ratios: 50:10:38.5:1.5
  • SM-102 100 mg/ml
  • 1 ,2-DSPC 25 mg/ml
  • Cholesterol 5 mg/ml
  • DMG-PEG(2000) 1 mg/ml
  • a 1 ml of ethanolic lipid mixture can be prepared by mixing -35 pl SM-102 100 mg/ml, -32 pl 1 ,2- DSPC 25 mg/ml, -296 pl Cholesterol 5 mg/ml, -378 pl DMG-PEG(2000) 1 mg/ml, and 259 pl absolute ethanol.
  • Oligonucleotide solution was prepared in aqueous solution. For this 620 pg of nucleic acid was adjusted to 3 ml with 50 mM sodium acetate, pH 5.0. Ethanolic lipid mixture was mixed with oligonucleotide solution in 1 :3 ratio, with oligonucleotide aqueous solution in excess.
  • Solvent-injection mixing ethanolic lipid mixture was injected to aqueous acidic oligo solution with a syringe. Needle placed in the centre of the aqueous solution tube. Mixing at 400 rpm, for 30 min, T>55°C (1 ,2-DSPC transition temperature) Mixing by pipetting: Mixed via pipette by rapidly transferring the ethanolic lipid mixture into the acidic aqueous nucleic acid solution. Mixed for >15 sec by repeated vigorous pipetting. Left undisturbed for 10 min.
  • Dialysis of the LNP mix against neutral buffer (HEPES buffered glucose (10 mM HEPES, 5% glucose) or HEPES (10 mM) pH 7.4) was performed in a Dialysis chamber with a MWCO of 2,400, followed by filter sterilisation of the solutions with 0.22 urn filter.
  • neutral buffer HEPES buffered glucose (10 mM HEPES, 5% glucose
  • Nucleic acid dose was determined with nucleic acid intercalating dye (depending on the nucleic acid, PicoGreen, RiboGreen or EvaGreen against standards of known concentrations).
  • the ionizable lipid (SM-102) was replaced with DLin-MC3-DMA, thus the ethanolic lipid mixture of the LNP mix 2 was instead:
  • the LNP mix was otherwise prepared as described in example 2.1 : LNP mix 1.
  • LNP/CPP/oligonucleotide complexes were prepared by addition of CPP to a lipid mixture in ethanol. Per 1 pg of nucleic acid a dose of 0.15 - 0.7 pl of 4 mM CPP solution in ethanol was added to the ethanolic lipid mixture to yield the following mixture:
  • Example 3.2 Preparation of LN P and plasmid/mRNA mix followed by addition of CPP
  • LNP mix 1 For complexes consisting of LNP where CPP was added after nucleic acid was mixed with the LNP, the complexation was performed by preparing the LNP mix 1 as previously described. Oligonucleotide solution was prepared in aqueous solution. For this 620 pg of nucleic acid was adjusted to 3 ml with 50 mM sodium acetate, pH 5.0 or the LNP was mixed in HEPES or HEPES buffered glucose. LNP mix 1 was then mixed with oligonucleotide solution in 1 :3 ratio, with oligonucleotide aqueous solution in excess. The LNP prepared in sodium acetate was then dialyzed against HEPES or HEPES buffered saline.
  • the loading was determined with fluorescent nucleic acid intercalating dye and CPP was added according to the nucleic acid dose.
  • CPP charge ratios
  • the LNP were prepared as described above, but the CPP was added according to molar ratio (CPP to siRNA, with CPP in excess).
  • the molar ratios tested ranged from MR5:1 to 40:1 based on CPP to nucleic acid ratio.
  • the LNP mix 1 was used to form LNPs (35 pl SM-102 100 mg/ml; 32 pl 1 ,2-DSPC 25 mg/ml; 296 pl Cholesterol 5 mg/ml; 378 pl DMG-PEG(2000) 1 mg/ml; 259 pl absolute ethanol).
  • 30 pl of 6.2 pg of firefly luciferase targeting siRNA in aqueous solution was mixed with 10 pl of ethanolic lipid mixture in a 3:1 ratio with siRNA solution in excess.
  • the LNP was then submitted to dialyzis and loading determined from the dialyzed LNPs.
  • the CPP/siRNA complexes were formed in the ultrapure water or buffer, with no significant difference between the two groups. Molar ratio CPP to siRNA 30:1 was used with CPP in excess. The same molar ratio of CPP to siRNA was used when LNP was included.
  • the LNP/CPP mix were formed a) by adding the CPP to the ethanolic phase with lipids and then mixing with the aqueous solution of the siRNA or b) preparing the LNPs consisting of the lipids and siRNA, dialyzed, dose matched and then the CPP solution was added to the LNP/siRNA in buffer.
  • the membrane permeable constructs prepared above were added to wells of microtiter plates containing U87 cells expressing firefly luciferase.
  • the concentration of siRNa was 14 nm per well. 24 hours after treatment, the cells were lysed and luminescence was measured. Successful siRNA inhibition of luciferase by the membrane permeable constructs was detected as a reduction in the luminescence measured for the treated cells compared to untreated (UT) cells (see figure 1 and 2).
  • Both membrane-permeable constructs obtained by addition of CPP to the ethanolic lipid mix and addition of CPP to the pre-formed LNP/siRNA showed an increase in downregulation when compared to LNP/siRNA without CPPs.
  • the addition of CPP to already formed and dose adjusted LNP/siRNA gave more consistent outcome.
  • the membrane-permeable constructs successfully deliver their cargo to the U87 cells and the siRNA successfully inhibited luciferase expression.
  • CPPs to either ethanolic lipid mixture or to pre-formed LNP/siRNA leads to an increase in down-regulation of luciferase.
  • the degree of down-regulation depends on the siRNA dose and used CPP ratio.
  • CPP/mRNA Similar approach was used as used for LNP mix+CPP/siRNA.
  • the CPP was added to the ethanolic lipid mixture (group LNP/CPP+mRNA) or added to already formed LNP/mRNA (group LNP/mRNA + CPP).
  • the mRNA was in 1 mM Sodium Citrate pH 6.4, therefore this buffer was attested additionally to HBS pH 7.4, HEPES pH 7.4 and sodium actetate pH 6 and pH 3.
  • Experimental conditions 96-well plate format with 30 ng of mRNA per well. Serum containing media. Reporter protein (luciferase) levels were determined from total cell lysate 24 posttransfection.
  • CR refers to charge ratio of charges from the CPP to the negative charges of nucleic acid or to the negative charges of the LNP.
  • a value such as CR2 should be understood as a charge ratio of CPP positive charges to negative charges of either LNP, nucleic acid or other cargo of 2:1 .
  • Example 6 Change of mixing method of LNP and reduced mRNA dose
  • LNP mix 1 and LNP mix 2 were used in this experimental set.
  • similar mixing of LNP was used as referred in Liu 2021 with slight modifications.
  • the LNP and mRNA are mixed in an acidic buffer at concentrated conditions, and after formulation, the acidity is neutralized with the addition of buffer at physiologic pH.
  • PBS was used.
  • HEPES buffer was used instead. No dialysis was performed. Due to the lack of dialysis, the NA doses were not adjusted, as the input was according to the used doses.
  • LNP1 LNP mixture with SM-102 100 mg/ml
  • LNP2 LNP mixture with DLin-MC3-DMA 100 mg/ml
  • LNP1/CPP and LNP2/CPP refer to conditions where the CPP is mixed with the lipid mixture in the ethanolic phase, and then mRNA in an aqueous buffer is added.
  • LNP/CPP For LNP/CPP mix where NA was added after mixing (LNP/CPP), the ethanolic mixture of LNP/CPP (0.05 ul of SM-102 100 mg/ml (working solution concentration), 0.05 ul of 1 ,2-DSPC 25 mg/ml, 0.24 ul of cholesterol 10 mg/ml, 0.61 ul of DMG-PEG(2000) 1 mg/ml, 0.15-0.7 ul CPP in ethanol 4 mM, and extra ethanol if needed to have the same final volume for all tested mixes).
  • the ethanolic mixture or LNP/CPP was mixed with 1 ug of mRNA in an aqueous solution (water and acetate buffer; as we compare the two methods, the mix processing was the same). After incubation, an HEPES buffer was used to dilute the particles to a suitable concentration with 80 ng of mRNA per 10 ul of solution.
  • LNP1/mRNA + CPP and LNP2/mRNA + CPP refers to conditions where the CPP is added after the LNP/mRNA is already formed, and diluted in HEPES.
  • the ethanolic mixture was prepared (consisting of 35 ul of SM-102 100 mg/ml, 32 ul of 1 ,2-DSPC 25 mg/ml, 296 ul of cholesterol 10 mg/ml, 378 ul of DMG-PEG(2000) 1 mg/ml).
  • An oligonucleotide solution was added (620 ul of mRNA adjusted to 1 ml with 50 mM sodium acetate, pH 5.0). After mixing, to the LNPs ethanolic solution of CPP solution was added and after short incubation HEPES was added. The volume adjusted, so after adding the (4 mM working solution diluted accordingly) CPP, there would be 80 ng of mRNA per 10 ul of solution.
  • LNP1/CPP + mRNA leads to similar levels of the reporter as 50 ng of mRNA.
  • Transfection experiments were performed in CHO-K1 cells (figure 7) and HeLa cells (figure 8) to test the ability of compositions of membrane-permeable constructs comprising the CPPs GP1 , GP2, GP4, PF14, NF51 , NF71 together with plasmid cargo in figure 7 and compositions of membrane-permeable constructs comprising the CPPs GP11 , GP12, GP13, GPM, GP15, and GP16 together with LNPs and plasmids in figure 8, to transfect cells with plasmids expressing luciferase.
  • CHO-K1 cells were transfected with membrane-permeable constructs comprising the CPPs GP1 , GP2, GP4, PF14, NF51 , NF71 in a 96 well setup.
  • the CPP/pDNA complexes achieve high expression levels without the addition of LNP mixes.
  • CPP/pDNA complexes were formed at indicated charge rations (CR) with CPP in excess.
  • the CPP was diluted in water prior addition to plasmid in water.
  • Per 96 well plate well 0.1 ug of plasmid dose was used in the 10 ul of complex solution and final volume (complexes and media) of 100 ul per well.
  • Negative controls include CHO-K1 cells transfected using plasmid without CPP and an untreated sample.
  • the membrane-permeable construct was prepared in several concentrations based on the charge ratio (CR) of a positive charge excess contributed by the CPP to the negative charge contributed by the plasmid phosphate backbone.
  • the efficacy of the transfection was measured by means of detecting the luminescence signal (RLU) for lysed cells after 24 hours of incubation.
  • LNP with SM-102 nor DLin-MC3-DMA achieved very high efficacies when plasmid DNA was used as the nucleic acid cargo.
  • the LNP/CPP/pDNA was prepared as follows.
  • the ethanolic mixture of LNP/CPP (0.05 ul of DLin-MC3-DMA 100 mg/ml (working solution concentration), 0.05 ul of 1 ,2-DSPC 25 mg/ml, 0.24 ul of cholesterol 10 mg/ml, 0.61 ul of DMG-PEG(2000) 1 mg/ml, 0.15-0.7 ul CPP in ethanol 4 mM, and extra ethanol if needed to have the same final volume for all tested mixes).
  • the ethanolic mixture or LNP/CPP was mixed with 1 ug of plasmid in an aqueous solution (water and acetate buffer; as we compare the two methods, the mix processing was the same). After incubation, the particles were dialyzed against HEPES buffer and diluted to a suitable concentration with 80 ng of NA per 10 ul of solution.
  • the ethanolic mixture was prepared (consisting of 35 ul of DLin-MC3-DMA 100 mg/ml, 32 ul of 1 ,2-DSPC 25 mg/ml, 296 ul of cholesterol 10 mg/ml, 378 ul of DMG-PEG(2000) 1 mg/ml and 259 ul of absolute ethanol).
  • An oligonucleotide solution was added (620 ul of NA adjusted to 3 ml with 50 mM sodium acetate, pH 5.0). After mixing, the LNPs were dialysed against HEPES. Then, the NA concentration was determined and volume adjusted, so after adding the (4 mM working solution diluted accordingly) CPP, there would be 80 ng of NA per 10 ul of solution.
  • the CPPs tested herein were highly efficient in driving delivery of cargo such as plasmids, mRNA and siRNA when used both on their own and when used together with LNPs as defined herein.
  • siRNA where the molar ratio 15:1 is better than 30:1 if the LNP is involved to the LNP/CPP/siRNA complexes. Whereas for CPP/siRNA the MR30:1 would be preferred.
  • Figure 1 shows the luminescence signal detected from firefly luciferase expressing cells (U87 cells) treated with various membrane permeable constructs loaded with siRNA as cargo and control samples, where UT is untreated cells and siRNA is siRNA treated cells without membrane permeable construct. The luminescence signal was measured for each sample is normalized onto the luminescence signal measured for untreated cells.
  • Figure 2 shows the luminescence signal detected from firefly luciferase expressing cells (U87 cells) treated with various membrane permeable constructs loaded with siRNA as cargo and control samples, where UT is untreated cells and siRNA is siRNA treated cells without membrane permeable construct.
  • the membrane permeable constructs comprise the CPPs PF14, NF55 and NF71 respectively in molar ratios (MR) of 15:1 and 30:1 to the concentration of siRNA.
  • Fig. 3 shows the luminescence signal detected from HaCaT cells transfected with mRNA encoding luciferase using a CPP-mRNA mixture with a charge ratio (CR) of 2:1 , a mixture where mRNA was added to a LNP/CPP preparation, a mixture where a CPP was added to a LNP/mRNA preparation, with controls being an untreated sample, a sample treated with mRNA and a sample treated with LNP without mRNA.
  • CR charge ratio
  • Figure 4 shows the luminescence signal detected from HeLa cells transfected with 80 ng of mRNA encoding luciferase in 10% FBS.
  • the transfection was carried using mixtures of CPP/mRNA, LNP1/mRNA solutions to which CPP was added in various charge ratios (CR), or mixtures of LNP2/mRNA solutions to which a CPP was added in various charge ratios (CR).
  • Controls comprising LNP1 mixture, LNP2 mixture and mRNA were included.
  • Fig. 5 shows HeLa cells transfected with complexes comprising GP16 and 50 or 20 ng mRNA expressing luciferase in various charge ratios (CR). LNPs were formulated in acidic buffer and neutralised with HEPES after complexion.
  • Fig. 5 shows HeLa cells transfected with complexes comprising GP14 and 50 or 20 ng mRNA expressing luciferase in various charge ratios (CR).
  • LNPs were formulated in acidic buffer and neutralised with HEPES after complexion.
  • Fig. 7 shows transfection efficiency as result of a luminescence signal from CHO-K1 cells transfected using mixtures comprising CPP (GP1 , GP2, GP4, PF14, NF51 , and NF71) and cargo plasmid expressing firefly luciferase.
  • Controls include a sample with plasmid (pDNA) without CPP and an untreated sample.
  • Fig. 8 shows transfection efficiency as result of a luminescence signal from HeLa cells transfected using mixtures comprising LNP, CPP (GP11 , GP12, GP13, GP14, GP15, and GP16) and cargo plasmid expressing firefly luciferase.
  • Controls include an untreated sample, a sample with plasmid (pDNA) without CPP and a sample comprising LNP without mRNA.
  • DaxibotulinumtoxinA for Injection for the Treatment of Glabellar Lines results from Each of Two Multicenter, Randomized, Double-Blind, Placebo-Controlled, Phase 3 Studies (SAKURA 1 and SAKURA 2). Plast Reconstr Surg, 145, 45- 58.
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Abstract

The present invention provides for a membrane-permeable construct for transport of cargo across a lipid membrane and subsequent delivery of cargo into cells as well as in vivo, wherein the construct comprises a Lipid nanoparticle (LNP) complexed with known or proprietary cell penetrating peptide (CPP) embedding the cargo molecule.

Description

MIXTURES OF LIPID NANOPARTICLES AND CELL PENETRATING PEPTIDES
FIELD
The present invention relates to a biobarrier-permeable constructs for transport of cargo such as oligonucleotides across a lipid membrane and subsequent delivery of cargo into cells as well through the in vivo epithelial barriers such as blood-brain-barrier, using the nanoparticle mixtures of lipid nanoparticles and cell penetrating peptides embedding the cargo. The present invention also relates to therapeutic uses of such a construct, for example in the vaccination and in the treatment of cancer.
BACKGROUND
Nucleic acid delivery issues are many since most therapies require the transmembrane delivery of relatively large amounts of oligonucleotides since the RNA or mRNA are supposed to cause the cellular immune response. To name the main obstacles where the novel chemical approaches are inevitable: packaging/conjugation of nanostructures between the nucleic acid payloads and the carrier system, protection of nucleic acids from enzymatic degradation, promoting cellular entry or trans-barrier delivery, and targeting the delivery to specific tissue types and sites within the cell in order to be used for gene therapy and gene editing purposes. To overcome these issues, it has been necessary to obtain a fundamental understanding of the nucleic acid interactions with the delivery systems as well as with the biological environment in order to understand the mechanisms of toxicity and minimize it.
Another area of chemical modification of nucleic acid therapies, especially in case of vaccination, is the reduction of undesired immunogenicity to avoid the cytokine storm. E.g. the severe COVID-19 is characterized by dysregulated cytokine release profile, dysfunctional immune responses, and hypercoagulation with a high risk of progression to multi-organ failure and death (Premeaux et al., 2022).
Numerous scientific studies and applications of nucleic acid delivery systems are available today, based on viral or non-viral methods. Non-viral delivery systems offer improved biosafety and flexibility, and have been tremendously advanced during the recent year’s development. One can find a plethora of non-viral nucleic acid delivery systems, including e.g. protamines, PEI, GalNAc, carbohydrate-based cationic glycopolymers, cholesterol, folic acid, antibodies, exosomes, lipid nanoparticles, cell penetrating peptides etc. (Oyama et al., 2021 ), all developed in search of non- toxic and highly efficient therapeutic delivery systems. However, there is still a need for optimized efficient therapeutic delivery systems that are both non-toxic and highly efficient.
SUMMARY
The Inventors have shown that the membrane-permeable constructs of the present invention, the mixtures of LNP and CPPs of the PepFect and NickFect types, with varying ratios in range of 0-100 % or 100-0 %, respectively, have improved and are more efficient at delivery of cargo, several types of oligonucleotides (ON) : antisense ON, siRNA, miRNA, plasmid and mRNA into the intracellular compartment as well as through in vivo biobarriers. In particular, the constructs synergistically increase the cargo transduction in vitro and in vivo, exposing significantly less toxic effects.
It is expected that the membrane-permeable constructs may be used for vaccination and targeted gene therapy in e.g. tumours. Further, the constructs of the present invention are more effective for cell cargo delivery, preferably for oligonucleotide delivery. The constructs are more effective at all ON delivery than existing CPPs or LNPs on their own.
The membrane permeable constructs of the present invention have low toxicity and less side effects due to the decreased amounts of LNP and CPP and are expected to be cheaper to synthesise compared to existing CPPs or LNPs.
In an initial aspect of the present invention, there is provided a membrane-permeable construct for transport of ON cargo across a lipid membrane and subsequent delivery of cargo into cells, wherein the construct comprises a known cell penetrating amino acid sequence of the PepFect or NickFect types (See above).
Thus, in a first aspect of the present invention, there is provided a membrane-permeable construct for transport of ON cargo across a lipid membrane and subsequent delivery of cargo into cells, wherein the construct comprises a known cell penetrating amino acid sequence of the PepFect or NickFect types (see above) and the known LNP described in Yue-Xuan Li et al. 2022 . In a second aspect, the present invention relates to a membrane-permeable construct for transport of cargo across a lipid membrane and subsequent delivery of cargo into cells, the membrane permeable construct comprising a lipid nanoparticle (LNP), and a cell penetrating peptide (CPP).
In a third aspect, the present invention relates to a method for producing a membrane-permeable construct, the method comprising the steps of providing a lipid nanoparticle (LNP), providing a cell penetrating peptide (CPP), and mixing the lipid nanoparticle (LNP) with the cell penetrating peptide (CPP), wherein the mixing of the lipid nanoparticle (LNP) and the cell penetrating peptide produces the membrane-permeable construct.
In another aspect of the present invention, there is provided a complex comprising the construct of the invention and a ON cargo non-covalently interacting therewith (e.g. interacting via ionic interactions).
In yet a further aspect of the present invention, there if provided a method of vaccination and of treating cancer in an individual comprising administering an effective amount of the pharmaceutical composition of the invention to an individual.
In another aspect of the present invention, there are provided the methods of gene expression and gene silencing in an individual comprising administering an effective amount of the construct of the invention to the individual. DETAILED DESCRIPITON
Among the multiple non-viral delivery systems for oligonucleotides, two systems seem to be outstanding for their originality, chemical flexibility, low toxicity and high therapeutic potential - lipid nanoparticles (LNP) and cell penetrating peptides (CPP).
Here, we propose the connection of these two delivery systems: LNP and a type of CPP, PepFects and NickFects, for the more efficient delivery systems for oligonucleotides in purpose for vaccination and any other type of therapy.
LNPs are composed of ionizable cationic lipids, phospholipids, cholesterol and polyethylene glycol (PEG)-lipids. LNPs generate ~50 nm particles with narrow size distributions, this size is crucial to allowing these particles to pass through the fenestrated liver vasculature. Critical chemical issues for clinical translation have been ionizable cationic lipid optimization, diffusible PEG-lipid design and a robust, scalable manufacturing process. LNP technology is now facilitating mRNA applications for protein replacement therapy and vaccine development (Kulkarni et al., 2021 ) against influenza virus vaccine, Zika virus in mice and non-human primates, P. falciparum, personalized vaccines for cancer immunotherapy, and SARS-CoV-2.
There are several known advantages of using CPPs over delivery using other similar delivery methods. For example the mRNA/LNP therapeutic design used with the COVID vaccines have several side effects, including, inflammatory responses, immunogenicity and allergic reactions and developers are trying to optimize these delivery systems to reduce mRNA ending up in the liver and reduce the inflammatory side effects.
The CPP based delivery system disclosed herein is advantageous over known LNP based delivery systems in that it has increased efficiency with respect to cargo delivery, is non-inflammatory and can therefore be used for inflammatory diseases, such as, but not limited to immune diseases and neurodegenerative disease treatments without exacerbating disease symptoms. The CPP based delivery system also does not cause unexpected immunogenecity and are robust to produce and stable for everyday use. Thus, there is a need for ways to deliver therapeutics to patients without triggering indesirable side effects and symptoms of disease. It is well known that while all known LNPs activate the Toll receptors, which causes both an inflammatory response and cellular immune responses, the CPP disclosed herein are known not to be ligands of the Toll receptors. This means they do not trigger the same inflammatory and immune responses when used as delivery system without LNPs. Furthermore, when the CPPs disclosed herein are used together with LNPs, the inflammatory response and immune response in the receiver is reduced compared to the response seen when using delivery systems including LNPs without CPPs.
As such CPP based delivery systems are not just an alternative to existing delivery systems, but represent an improvement in delivery of therapeutic molecules to patients in general and with a particular advantage when the delivery of therapeutics is related to diseases or disorders with inflammatory components or symptoms.
The use of CPPs in combination with LNPs can also alleviate the side effects often associated with LNP mediated delivery of therapeutics alone. Thus, the CPP/LNP based delivery systems disclosed herein have several advantages over the presently used LNP mediated therapy alone.
Cell penetrating peptides (CPP)
Cell-penetrating peptides (CPP; also known as protein/peptide transduction domains, PTD, or Trojan peptides) are based on several seminal findings which challenged the laws of cellular biochemistry and contested the traditional dogma that the cell plasma membrane was impermeable to proteins and peptides, see (Langel, 2019). CPPs enabled to overcome this basic dogma by understanding the rules and pathways of chemistry in plasma membrane translocation, including the complex interactions between positively charged CPPs and negatively charged phospholipid membranes. CPPs are currently used for trans-barrier delivery of most of the cargo types with biological relevance : small molecule drugs, peptides, proteins, all types of oligonucleotides incl. plasmids etc.
As a major breakthrough, recently (September 2022), Revance Inc., USA, announced FDA apporoval of DAXXIFY (daxibotulinumtoxinA-lanm) for injection, called Peptide Exchange Technology, and temporay treatment of glabellar lines in adults, a “first and only peptide- formulated neuromodulator with long-lasting results” (revance.com), containing RTP004 (35 aa) as a CPP, highly purified daxibotulinumtoxinA (RTT150, a 150-kDa botulinum toxin type A) and other excipients including polysorbate-20 (a surfactant), buffers, sugar. RTP004 forms a strong electrostatic bond with daxibotulinumtoxinA, enabling “the product to be formulated without human serum albumin and to be stable at room temperature before reconstitution” (Carruthers et al., 2020).
CPP-assisted functional and efficient delivery of all types of nucleic acid based therapeutic molecules to cells and in vivo has been demonstrated. Both, the covalent and non-covalent strategies for conjugation have been successfully applied, see Fig.4., making this chemical approach especially valuable. The innovation has been introduced at several levels : design and choice of the chemical design of the CPPs and nucleic acids, their attachment to each other, introduction of the chemical methods to decrease the toxicity of the system, targeting of the system to specific cells and barriers, endosomal escape, resistance to nucleases, the in vitro and in vivo modification of the efficacy of the transfection etc.
The methods for gene silencing (or inhibiting specific genes) using antisense ONs (ASO) and RNA interference (RNAi) with CPPs conjugates have often been reported. Classified by the antisense mechanisms and the character of the ONs, the antisense technologies include the use of classical single-strand antisense ON, antigene ON, splice-correcting antisense ON (SCO), doublestrand small interfering RNA (siRNA), microRNA (miRNA) and anti-miRNA (antimiR), including promoting the degradation of the targeted RNA or modulating RNA function without degradation. Two major backbones for ASO platforms today are the phosphorodiamidate morpholino oligomers (PMOs) and the phosphorothioates (PSs) (Shadid et al., 2021). CPPs have been attached for such ON delivery in both, covalent and non-covalent (usually forming nanoparticles) manner.
Non-covalent strategy. ON complexing to CPPs by often yields stable NPs, likely by combination of electrostatic interactions (between the positively charged CPPs and negatively charged ONs) and available hydrophobic interactions (from hydrophobic amino acids or inserted fatty acid chains), sometimes associated with an amphipathic feature of CPPs or specific structures of the complexes (Morris et al., 1997, Wyman et al., 1997, Futaki et al., 2001 , Morris et al., 2001 , Simeoni et al., 2003, Crombez et al., 2009, Eguchi et al., 2009, Michiue et al., 2009). CPPs are actively used in different gene therapeutic platforms such as plasmid delivery or modifications of the genomic DNA (Taylor and Zahid, 2020). The gene-editing technologies in the form of CRISPR)/CRISP-Cas (Shalaby et al., 2020) has been considerably improved by the use of CPPs, e.g. PepFect14 (Falato et al., 2022) in different cell types for therapeutic and research purposes are reviewed in (Langel, 2019).
Oligonucleotide-functionalized transportan and its analogs have been widely used for oligonucleotide delivery, reviewed in (Langel, 2021). Transportan was designed by us as a bifunctional bioactive peptide with the ability to interact with plasma membranes. Intensive structure-activity studies of transportan yielded in cargo delivery vectors with improve chemical properties for delivery of different cargos. The role of transportan in drug delivery in general and nucleic acid delivery in particular is currently growing rapidly.
We have introduced several highly efficient transportan-based ON delivery vectors (multiple PepFects and NickFects), enabling non-covalent simple formulation nanocomplex technology of antisense, siRNA, miRNA, plasmid and mRNA delivery, see International Patent Application No. PCT/EP2020/050524, 2019, and (Langel, 2021). We have shown that these nanoparticles that are taken up by ceils largely through scavenger receptor type A mediated endocytosis. PepFect14 delivery of the complexed pDNA encoding for CRISPR-Cas9 showed the targeted knock-down of the luciferase gene in the cell line expressing GFP (Falato et al., 2022).
The cell membrane has an amphiphilic nature; to enhance CPP/membrane interactions, several CPPs, incl. PepFects and NickFects, have been designed to have both hydrophilic and hydrophobic regions and/or moieties in their sequence. The membrane may be an artificial membrane such as an artificially constructed complex membrane formed of, for example, lipids, phospholipids or molecules having both hydrophilic and hydrophobic compounds or structures, biological membrane that is a double-layer of phospholipids and other lipids like cholesterol and proteins inserted. Most biological membranes are negatively charged and most biological uptake is by selective transporters and energy dependent processes.
The membrane may be a biological membrane including but not limited to eukaryotic cell membranes, prokaryotic cell membranes and epithelial membranes, e.g. the mucous membranes, serous membranes, cutaneous membranes, synovial membranes and the blood-brain barrier. The membrane may be a lipid bilayer or phospholipid bilayer. The membrane may be a lipid membrane of a phospholipid membrane. The membrane may be a plasma membrane. Eukaryotic cells membranes include, but are not limited to, membranes of immune cells such as white blood cells, red blood cells, monocytes, macrophages, neutrophils, T cells, B cells or dendritic cells, epithelial cells, endothelial cells, keratinocytes, muscle cells, skin cells, nerve cells and fat cells.
Transportan showed the translocation to the cell cytosol (Pooga et al., 1998a), and was able to translocate the covalently attached ON cargo (Pooga et al., 1998b). This fueled the systematic structure-activity studies of its structure, first enabling the shorter transportan 10 (Soomets et al., 2000) and, later, the efficient series of PepFects and (branched) NickFects (Langel, 2021):
Transportan, TP: GWTLNSAGYLLGKINLKALAALAKKIL
Transportan 10, TP10: AGYLLGKINLKALAALAKKIL
PepFect 14, PF14: stearoyl-AGYLLGKLLOOLAAAALOOLL
NickFect 51, NF51 : O(Nb-stearoyl-AGYLLG)-INLKALAALAKKIL
NickFect 55, NF55 : O(Nb-stearoyl-AGYLLG)-INLKALAALAKAIL amide
It has been suggested that we get more mRNAs into the cells in a translatable form with PepFects as compared with LNPs, since more expression is observed with the same amount of mRNA when we compare PepFect to lipid transfection. Also, PF14 yielded a more homogenous response across the cell population than LipoMM, likely due to the by induction of endocytosis (van Asbeck et al., 2021). PF14/mRNA nanoparticles greatly outperformed a lipid-based transfection agent in vivo, leading to expression in various cell types of tumor associated tissue in the context of ovarian cancer (van den Brand et al., 2019).
Additionally, HSP70 protein has been transfected in Bomirsky Hamster Cells (BHM) in complex with PepFecet14 (Gestin et al., 2022). PepFect14 was able to form a complex with HSP70 and to deliver it inside cells in the same fashion with oligonucleotide delivery. The delivered HSP70 showed an effect in the cell regulation indicating that the protein was biologically available in the cytoplasm and the interactions with PepFect14 did not impeach its active sites once the plasma barrier crossed (Gestin et al., 2022). This finding enables to include the delivery of bioactive proteins through the biobarriers as well.
LNP/CPP mixtures
Recent studies on a few cationic CPPs have revealed that they can stimulate the cellular uptake of nanoparticles (NPs) simply via co-administration (bystander manner), which bypasses the requirement of chemical modification. In this study, we investigated the other classes of CPPs and discovered that transportan (TP) peptide, as well as the PepFect and NickFect peptides, also exhibited such bystander activities. When simply co-administered, TP peptide enabled the cells to engulf a variety of NPs, as well as common solute tracers, while these payloads had little or no ability to enter the cells by themselves. This result was validated in vitro and ex vivo, and CPPs showed no physical interaction with co-administered NPs (bystander cargo). Together, these findings improve the understanding of CPP-assisted cell entry, and open up a new avenue to apply this peptide for nanomaterial delivery (Li et al., 2022).
Membrane-permeable construct
A membrane-permeable construct is a construct that is biomembrane (membrane)-permeable, i.e. capable of passing across or through a membrane into the cell or intracellular environment, into the organelle (when targeted) as well as passing in vivo bio-barriers for organelle delivery in vivo. The membranes and bio-barriers may be single or multiple layer structures. The membrane may be an artificial membrane such as an artificially constructed complex membrane formed of, for example, lipids, phospholipids or molecules having both hydrophilic and hydrophobic compounds or structures. The membrane may be a biological membrane including but not limited to eukaryotic cell membranes and prokaryotic cell membranes.
The membrane-permeable constructs of the present disclosure comprise a covalent or noncovalent complex of a lipid nanoparticle (LNP) of any type and a cell penetrating peptide (CPP). In some cases the membrane-permeable construct further comprises a cargo that is contained within, embedded in, encaptured by, associated with or complex bound to the membrane-permeable construct.
Additionally, the membrane-permeable constructs of the present disclosure may comprise a CPP and a cargo that is contained within, embedded in, encaptured by, associated with or complex bound to the membrane-permeable construct. The membrane permeable construct may consist essentially of a CPP and a cargo. The membrane-permeable construct may consist essentially of a CPP solution mixed with a cargo solution. The membrane-permeable construct may consist essentially of a CPP and a cargo that is contained within, embedded in, encaptured by, associated with or complex bound to the membrane-permeable construct.
Therefore, in or one or more exemplary embodiments of the present disclosure, a membrane- permeable construct for transport of cargo across a lipid membrane and subsequent delivery of cargo into cells as defined within the present context is a membrane-permeable construct comprising a lipid nanoparticle (LNP), one or more cell penetrating peptides (CPP), and optionally, a cargo.
In one or more exemplary embodiments of the present disclosure, the LNP, and the one or more cell penetrating peptides (CPP) of the membrane-permeable construct form a complex through chemical interactions. The complex is formed either through chemical linkage via covalent bonds or through ionic bonds, ionic interactions, non-covalent bonds, or non-covalent interactions.
In one or more exemplary embodiments of the present disclosure, the LNP and the one or more cell penetrating peptides (CPP) of the membrane-permeable construct, form a complex through non-covalent bonds.
In or one or more exemplary embodiments of the present disclosure, the membrane-permeable construct comprises a cargo. In one or more exemplary embodiments, the cargo of the membrane- permeable construct forms a complex with the LNP and/or CPP components of the membrane- permeable construct.
The complex formed by the cargo and the LNP and/or CPP components of the membrane- permeable construct occur either through chemical linkage via covalent bonds or through ionic bonds, ionic interactions, non-covalent bonds, or non-covalent interactions.
In one or more exemplary embodiments of the present disclosure, the complex formed between the cargo and the LNP and/or CPP components of the membrane-permeable construct is formed through non-covalent interactions. In one or more exemplary embodiments of the present disclosure, the complex formed between the cargo and the LNP and/or CPP components of the membrane-permeable construct is formed through ionic interactions or ionic bonds.
In one or more exemplary embodiments of the present disclosure, the one or more CPPs is selected from the list consisting of Transportan (TP), Transportan 10 (TP10), PepFect 14 (PF14), NickFect 51 (NF51 ), NickFect 55 (NF55), NF554, NF70, NF71 , PF132, PF141 , PF142, PF143, GP1 , GP2, GP3 and GP4.
In one or more exemplary embodiments of the present disclosure, the one or more CPPs is selected from the list consisting of NickFect 51 (NF51 ), NickFect 55 (NF55), NF554, NF70, NF71 , PF132, PF141 , PF142, PF143, GP1 , GP2, GP3 and GP4.
In one or more exemplary embodiments of the present disclosure, the one or more CPPs is selected from the list consisting of PepFect 14 (PF14), NickFect 51 (NF51 ), NickFect 55 (NF55), NF71 , GP1. GP2, GP3 and GP4.
In one or more exemplary embodiments of the present disclosure, the one or more CPPs is selected from the list consisting of PepFect 14 (PF14), NickFect 51 (NF51 ), NickFect 55 (NF55), and NF71.
In one or more exemplary embodiments of the present disclosure, the one or more CPPs is selected from the list consisting of NickFect 51 (NF51 ), NickFect 55 (NF55), NF554, NF 70, NF71 , GP1 , GP2, GP3 and GP4.
In one or more exemplary embodiments of the present disclosure, the one or more CPPs is selected from the list consisting of PF132, PF141 , PF142, PF143, GP1 , GP2, GP3 and GP4.
In one or more exemplary embodiments of the present disclosure, the one or more CPPs is selected from the group consisting of GP3 and GP4. In one or more exemplary embodiments of the present disclosure, the CPP is selected as Transportan (TP). In one or more exemplary embodiments of the present disclosure, the CPP is selected as Transportan 10 (TP10). In one or more exemplary embodiments of the present disclosure, the CPP is selected as PepFect 14 (PF14). In one or more exemplary embodiments of the present disclosure, the CPP is selected as NickFect 51 (NF51). In one or more exemplary embodiments of the present disclosure, the CPP is selected as NickFect 55 (NF55). In one or more exemplary embodiments of the present disclosure, the CPP is selected as NF554. In one or more exemplary embodiments of the present disclosure, the CPP is selected as NF70. In one or more exemplary embodiments of the present disclosure, the CPP is selected as NF71. In one or more exemplary embodiments of the present disclosure, the CPP is selected as PF132. In one or more exemplary embodiments of the present disclosure, the CPP is selected as PF141. In one or more exemplary embodiments of the present disclosure, the CPP is selected as PF142. In one or more exemplary embodiments of the present disclosure, the CPP is selected as PF143. In one or more exemplary embodiments of the present disclosure, the CPP is selected as GP1. In one or more exemplary embodiments of the present disclosure, the CPP is selected as GP2. In one or more exemplary embodiments of the present disclosure, the CPP is selected as GP3. In one or more exemplary embodiments of the present disclosure, the CPP is selected as GP4.
In one or more exemplary embodiments of the present disclosure, the membrane-permeable construct comprises an LNP and a CPP in a molar ratio of about 5:1 , such as 6:1 , 7:1 , 8:1 , 9:1 or about 10:1.
In one or more exemplary embodiments of the present disclosure, the membrane-permeable construct comprises an LNP and a CPP in a V/V ratio of about 5:1 , such as 6:1 , 7:1 , 8:1 , 9:1 or about 10:1.
In one or more exemplary embodiments of the present disclosure, the membrane-permeable construct comprises an LNP and a CPP in a W/W ratio of about 5:1 , such as 6:1 , 7:1 , 8:1 , 9:1 or about 10:1.
In one or more exemplary embodiments of the present disclosure, the membrane-permeable construct comprises an LNP in a concentration of 50 mM and a CPP in a concentration of 1 mM, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 mM. In one or more exemplary embodiments of the present disclosure, the membrane-permeable construct comprises an LNP in a concentration of 50 mM and a CPP in a concentration of 5 mM.
In one or more exemplary embodiments of the present disclosure, the membrane-permeable construct comprises an LNP in a concentration of 50 mM and a CPP in a concentration of 10 mM.
In one or more exemplary embodiments of the present disclosure, the membrane-permeable construct comprises an LNP in a concentration of 30-70 mM, such as 35-65, 40-60, 45-55 or 50 mM and a CPP in a concentration of 3-12 mM, such as 4-11 , 5-10, 3-7, 4-6, 5, 8-12, 9-11 , or 10 mM.
In or one or more exemplary embodiments of the present disclosure, the membrane-permeable construct is about 50 nm in diameter, such as 50, 48-52, 45-55 or 40-60 nm in diameter.
In or one or more exemplary embodiments of the present disclosure, the membrane-permeable construct is about 50 nm in diameter.
In or one or more exemplary embodiments of the present disclosure, the membrane-permeable construct is a nanoparticle.
In or more exemplary embodiments, the present disclosure relates to a composition comprising the membrane-permeable construct as defined herein.
Lipid nanoparticles (LNPs)
LNPs are particles that are made up of lipids and are typically globular in shape. Lipids can generally be described as amphiphilic molecules that contain three domains: a polar head group, a hydrophobic tail region and a linker between the two domains. In aqueous solutions and depending on the lipid composition, the LNP can form one or more lipid bilayer(s) with one or more hydrophilic core space(s) inside of the lipid bilayer(s) (e.g. liposomes and vesicles) or the LNP may form a particle with a hydrophobic core composed of the hydrophobic tails of the lipids (e.g. micelles). In a non-limiting example LNPs are composed of ionizable lipids, cationic lipids, phospholipids, cholesterol and polyethylene glycol (PEG)-lipids.
LNPs composed of the lipids discussed herein generate particles with a diameter of about 50 nm with a narrow size distribution and this size distribution is crucial to allowing these particles to pass through the fenestrated liver vasculature.
LNPs can be functionalized by the association of other molecules to the surface of the LNP or by embedding of molecules into lipid layer(s) of the LNP. A non-limiting example of such functionalization is the association or embedding of CPPs into or onto the LNP. Other non-limiting examples of functionalization include association or embedding of dyes, tracers, radionuclides, phosphorophores and fluorophores with the LNPs.
The association or embedding of other molecule into or onto the LNP can for example occur through chemical linkage via covalent bonds or through ionic bonds, ionic interactions, non- covalent bonds, or non-covalent interactions.
In one or more exemplary embodiments of the present disclosure, the CPPs defined in the present disclosure form a complex by chemical linkage via covalent bonds or through ionic bonds, ionic interactions, non-covalent bonds, or non-covalent interactions with the LNP.
LNPs can also be used to contain or carry a cargo for delivery to a molecular target, such as a cell of an animal or human. A cargo can be associated with the LNP surface, embedded in the lipid layer of the LNP or contained within the LNP through chemical linkage via covalent bonds or through ionic bonds, ionic interactions, non-covalent bonds, or non-covalent interactions.
In one or more exemplary embodiments of the present disclosure, the CPPs defined in the present disclosure form a complex by chemical linkage via covalent bonds or through ionic bonds, ionic interactions, non-covalent bonds, or non-covalent interactions with the LNP.
In one or more exemplary embodiments, the LNPs are selected as one or more of the group consisting of micelles, vesicles and liposomes. In one or more exemplary embodiments of the present disclosure, the LNPs are liposomes.
In one or more exemplary embodiments of the present disclosure, the liposomes comprise DPPC, Cholesterol and DSPE.
In one or more exemplary embodiments of the present disclosure, the DSPE is PEGylated, i.e., the DSPE is DSPE-PEG.
In one or more exemplary embodiments of the present disclosure, the liposome is composed of DPPC, cholesterol and DSPE-PEG.
In one or more exemplary embodiments of the present disclosure, the liposome is composed of DPPC, cholesterol and DSPE-PEG in a ratio of 16:11 :3.
In one or more exemplary embodiments of the present disclosure, the liposome is composed of DOTAP, DPPC and cholesterol.
In one or more exemplary embodiments of the present disclosure, the liposome is composed of DOTAP, DPPC and cholesterol in a ratio of 8:8:1 )
In one or more exemplary embodiments of the present disclosure, the LNP is composed of SM- 102, DSDPC, cholesterol and PEG200-DMG in a ratio of 50:10:38.5:1.5.
In one or more exemplary embodiments of the present disclosure, the LNP is composed of SM- 102, DSDPC, cholesterol and PEG200-DMG in a ratio of 40:20:37:3.
Ionizable lipids
An ionizable lipid as discussed herein relates to lipids that are protonized at low pH, which makes them positively charged. However, they remain neutral at physiological pH and can for example chosen from the non-exhaustive list consisting of (2S)-2,5-bis(3- aminopropylamino)-N-[2- (dioctadecylamino)acetyl] pentanamide (DOGS; Transfectam), N1-[2-((1S)-1-[(3- aminopropyl)amino]-4-[di(3-aminopropyl)amino] butylcarboxamido)ethyl]-3,4-di[oleyloxy]- benzamide (MVL5), DC-Cholesterol and N4 -cholesterylspermine (GL67). Other ionizable lipids that can be used in the formation of LNPs as defined herein are well known to the skilled person.
Cationic lipids
A cationic lipid as discussed herein relates to lipids containing a a head group with a permanent positive charge. Examples of such cationic lipids can be selected from the non-exhaustive list consisting of 1 ,2- di-O-octadecenyl-3-trimethylammonium-propane (DOTMA), 1 ,2-dioleoyl-sn- glycero-3-phosphoethanolamine (DOPE) and 1 ,2-dioleoyl-3-trimethylammonium-propane (DOTAP). Other cationic lipids that can be used in the formation of LNPs as defined herein are well known to the skilled person.
Other lipids
Other types of lipids include phospholipids such as for example phosphatidylcholine and phosphatidylethanolamine, cholesterol or polyethylene glycol (PEG)-functionalized lipids (PEG- lipids). Other phospholipids and PEG-functionalized lipids in general that can be used in teh formation of LNPs as defined herein are well known to the skilled person.
A non-exhaustive list of lipids that can be used in the formation of LNPs as defined herein arepreented in table 1. Other lipids suitable for use in the formation of LNPs are well known to the skilled person.
Table 1: Examples of lipids suitable for use in the formation of LNPs.
Figure imgf000018_0001
Figure imgf000019_0001
Figure imgf000020_0001
Methods for production and loading of LNPs
Several methods for providing LNPs are known in the prior art. A non-limiting example of the film hydration method for providing LNPs is included below and it is contemplated that when using the film hydration method, the LNPs of the present disclosure is formed by dissolution of a selection of one or more lipids in desired concentrations in organic solvent, removal of the organic solvent by e.g. rotary evaporation to form a lipid film, hydration of the lipid film in a dispersion medium by agitation, and thereby forming the desired LNPs.
It is possible to obtain LNPs with a specific size or diameter, for example, by extrusion of the LNPs through a membrane filter having a specific pore size.
In one or more exemplary embodiments of the present disclosure, the organic solvent is chosen as chloroform. However, other organic solvents that can be used for the formation of LNPs are generally known to the skilled person. An example of a suitable dispersion medium is HBS buffer and other suitable buffers are well known to the skilled person.
The LNP can further also comprise a cargo intended for delivery across a membrane. Thus, in one or more exemplary embodiments, the methods for producing a LNP comprises loading of a cargo. In one or more exemplary embodiments of the present disclosure, loading of a lipophilic cargo is achieved during LNP formation for example by dissolution of the lipophilic cargo in the organic solvent together with the lipids that make up the LNP. The lipophilic cargo is then associated with or embedded in the lipophilic parts of the LNP already during the formation of the lipid film.
In one or more exemplary embodiments of the present disclosure, loading of a hydrophilic cargo into the hydrophilic core of a LNP is achieved during LNP formation for example by dissolution of the hydrophilic cargo in the dispersion medium prior to hydration of the lipid film. The hydrophilic cargo is then loaded into the hydrophilic core, when the lipid film is hydrated and agitated with the dispersion medium containing a hydrophilic cargo during the LNP formation.
In one or more exemplary embodiments of the present disclosure, the cargo is loaded in a concentration of 1 mol% of the total lipid content of the LNP.
In one or more exemplary embodiments of the present disclosure, the cargo is loaded in a concentration of 1 , such as 2, 3, 4 or 5 mol% of the total lipid content of LNP.
Cell penetrating amino acid sequence
Cell penetrating amino acid sequences, also referred to as cell penetrating peptides (CPPs), are short amino acid sequences that transport different types of cargo molecules across a lipid membrane and facilitate cellular uptake of the cargo molecules. A property of cell penetrating amino acids is their ability to translocate the lipid membrane and facilitate the delivery of various molecular cargoes to the cytoplasm or to an organelle of a cell or to an intracellular cell surface or an in vivo barrier.
In an embodiment of the present invention, the cell penetrating amino acid sequence comprises the following known CPP sequences exemplified below:
Transportan : GWTLNSAGYLLGKINLKALAALAKKIL (Pooga et al., 1998a)
Transportan 10, TP10: AGYLLGKINLKALAALAKKIL (Soomets et al., 2000)
PF14 : stearoyl-AGYLLGKLLOOLAAAALOOLL amide (Ezzat et al., 2011)
NF51 : O(Nb-stearoyl-AGYLLG)-INLKALAALAKKIL amide (Arukuusk et al., 2013)
NF55 : O(Nb-stearoyl-AGYLLG)-INLKALAALAKAIL amide (Freimann et al., 2016) NF554: K(NE-stearoyl-AGYLLG)-INLKALAALAKAIL amide (Freimann et al., 2016)
NF 70 : O(Nb-arachidoyl-HHHHYHHG)-ILLKALKALAKAIL amide (Porosk et al., 2019)
NF 71 : O(Nb-stearoyl-HHYHHG)-ILLKALKALAKAIL amide (Porosk et al., 2019)
PF132 : stearoyl-LHLLHHKINLKALAALAKKIL amide (Regberg et al., 2016)
PF141 : stearoyl-HLHHLLKLLOOLAAAALOOLL amide (Regberg et al., 2016)
PF142 : stearoyl-LHLLHHKLLOOLAAAALOOLL amide (Regberg et al., 2016)
PF143 : stearoyl-HHHHHHKLLOOLAAAALOOLL amide (Regberg et al., 2016) or proprietary (Arthinity) sequences:
GP1 stearoyl-HHYHHGLLOOLAAAALOOLL amide
GP2 stearoyl-HHYHHGINLKALAALAKAIL amide
GP3 O(Nb-stearoyl-AGYLLG)-LLOOLAAAALOOLL amide
GP4 O(Nb-stearoyl-HHYHHG)-LLOOLAAAALOOLL amide where : O = ornithine. K(NE-stearoyl-XXXX) or O(Nb-stearoyl-XXXX) indicates that the branched peptide continues from the side chain amino group of lysine or ornithine, respectively, and not from the a-amino group :
Stearoyl-AGYLLG Stearoyl-AGYLLG
NFS1
■'O-INLKALAALAKML amide NF5S4
Figure imgf000022_0001
K-INLK.ALAALAK.AIL amide
Additionally, in one more exemplary embodiments of the present disclosure, the C-terminal of the CPPs listed above is further amidated.
The sequences listed above are included in table 2 are mapped out in table 2 with reference to their component sequences and references to the relevant SEQ ID NOs of the sequence listing. Table 2: CPP sequence to reference SEQ ID NOs of sequence listing
Figure imgf000023_0001
Figure imgf000024_0001
Figure imgf000025_0001
Figure imgf000026_0001
Cargo
The cargo molecule is associated (embedded) by the CPP/LNP complex either through chemical linkage via covalent bonds or through non-covalent bond or ionic bonds or non-covalent interactions or ionic interactions. The embedded cargo may be transported from outside of the cell across the membrane of the cell and enter into the cell or through the bio-barriers.
Cargo includes nucleic acids (e.g. RNA, miRNA, DNA, siRNA, shRNA, antisense oligonucleotides, decoy DNA, plasmid DNA), but even the small molecule drugs, imaging agents (e.g. fluorophore), radioactive tracers, metal chelates. The peptide may be selected from a group consisting of, but not limited to, a cell or tumour targeting peptide, an aptamer, a receptor ligand, a peptide ligand, a cytotoxic peptide, a bioactive peptide, an antibody, and a diagnostic agent. When the cargo molecule is a nucleic acid, the nucleic acid may comprise one or more nucleic acids where each one encodes one peptide or polypeptide. The cargo molecule may be a combination of a protein, a lipid, and/or a polysaccharide including lipoproteins and glycolipids. The cargo may be selected from a group consisting of, but not limited to, oligonucleotides including single-stranded oligonucleotides (e.g. DNA, RNA, PNA, LNA and their analogues), double-stranded oligonucleotides (e.g. siRNA, shRNA and decoyDNA) and cyclic DNA (e.g. plasmids).
In a further embodiment of the present invention, the membrane-permeable construct comprises a LNP/CPP/cargo complex according to the present invention wherein a cargo non-covalently interacting therewith, for example via ionic interactions. Such cargo suitably is ionic and carries negative charges. In one or more additional embodiments of the present dislosure, the components of the LNP/CPP/cargo complex are present, in a ratio of 0-100% or 100-0%.
Nanoparticle
Nanoparticles are generally understood to be small particles that have a diameter of between 10 and 100 nm. In the present context a nanoparticle is to be understood as a particle comprises a membrane-permeable construct as defined herein that has a diameter between 10-100 nm.
Thus, in a specific embodiment of the present invention, the membrane-permeable construct complex forms a nanoparticle.
In one or more exemplary embodiments, the nanoparticle consists of a membrane-permeable construct as defined herein that has a diameter between 20-100 nm.
In one or more exemplary embodiments of the present disclosure, the nanoparticle of the present invention may for example be 20 to 100 nm in diameter, such as about 30-90, 40-80, 50-70, 55-65, 20-30, 30-40, 40-50, 60-70, 70-80, 80-90, 90-100, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95 or such as about 100 nm.
Several methods for size separation of particles and nanoparticles are known in the prior art and the skilled person is capable of selecting a suitable method for size separation of the nanoparticles as disclosed herein. However, in an example that is intended to be non-limiting, a nanoparticle of a desired size is obtained by extrusion of the nanoparticle through a membrane filter, where the membrane filter comprises pores wherein the pore size and/or pore diameter determines the diameter of the nanoparticles obtained after the extrusion process. Methods for producing a membrane-permeable construct as defined herein
The membrane-permeable construct of the present disclosure is produced in several consecutive steps that include the steps of providing a LNP (lipid nanoparticle), providing a cell penetrating peptide (CPP), and mixing the LNP together with the CPP.
Thus, a general method for producing the membrane-permeable construct as defined herein comprises the steps providing a lipid nanoparticle (LNP), providing a cell penetrating peptide (CPP), mixing the lipid nanoparticle with the cell penetrating peptide (CPP),
The membrane-permeable constructs can further also optionally comprise a cargo intended for delivery across a membrane. Thus, in one or more exemplary embodiments, the methods for producing a membrane-permeable construct further comprises the steps providing a cargo, and loading the cargo onto or into the membrane-permeable construct.
In one or more exemplary methods for producing the membrane-permeable construct of the present disclosure, the loading of the cargo onto or into the membrane-permeable construct is achieved by mixing the membrane-permeable membrane with the cargo.
In one or more exemplary methods for producing the membrane-permeable construct of the present disclosure, the lipid nanoparticle (LNP) used for producing the membrane-permeable construct comprises the cargo.
In one or more exemplary methods for producing the membrane-permeable construct of the present disclosure, the lipid nanoparticle (LNP) used for producing the membrane-permeable construct contains the cargo.
In one or more exemplary methods for producing the membrane-permeable construct of the present disclosure, the cargo is embedded in, associated with, connected to, or loaded onto or into the lipid nanoparticle (LNP) used for producing the membrane-permeable construct. In one or more exemplary methods for producing the membrane-permeable construct of the present disclosure, the cargo is loaded in a concentration of 1 mol% of the total lipid content of the membrane-permeable construct.
In one or more exemplary methods for producing the membrane-permeable construct of the present disclosure, the cargo is loaded in a concentration of 1 , such as 2, 3, 4 or 5 mol% of the total lipid content of the membrane-permeable construct.
In one or more exemplary embodiments of the present disclosure, the methods for producing a membrane-permeable construct comprises mixing an LNP and a CPP in a molar ratio of about 5:1 , such as 6:1 , 7:1 , 8:1 , 9:1 or about 10:1.
In one or more exemplary embodiments of the present disclosure, the methods for producing a membrane-permeable construct comprises mixing an LNP and a CPP in a V/V ratio of about 5:1 , such as 6:1 , 7:1 , 8:1 , 9:1 or about 10:1.
In one or more exemplary embodiments of the present disclosure, the methods for producing a membrane-permeable construct comprises mixing an LNP and a CPP in a w/w ratio of about 5:1 , such as 6:1 , 7:1 , 8:1 , 9:1 or about 10:1.
In one or more exemplary embodiments of the present disclosure, the methods for producing a membrane-permeable construct comprises mixing an LNP in a concentration of 50 mM and a CPP in a concentration of 1 mM, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 mM.
In one or more exemplary embodiments of the present disclosure, the methods for producing a membrane-permeable construct comprises mixing an LNP in a concentration of 50 mM and a CPP in a concentration of 5 mM.
In one or more exemplary embodiments of the present disclosure, the methods for producing a membrane-permeable construct comprises mixing an LNP in a concentration of 50 mM and a CPP in a concentration of 10 mM. In one or more exemplary embodiments of the present disclosure, the methods for producing a membrane-permeable construct comprises mixing an LNP in a concentration of 30-70 mM, such as 35-65, 40-60, 45-55 or 50 mM and a CPP in a concentration of 3-12 mM, such as 4-11 , 5-10, 3-7, 4-6, 5, 8-12, 9-11 , or 10 mM.
In one or more exemplary embodiments of the present disclosure, the methods for producing a membrane-permeable construct comprises mixing a LNP and a CPP in a charge ratio (CR) of 1 :1.5-3, 1 :2-3, 1 ;1 ,5, 1 :2 or 1 :3. In one or more exemplary embodiments of the present disclosure, the methods for producing a membrane-permeable construct comprises mixing a LNP and a CPP in a charge ratio (CR) of 1 :1.5-3. In one or more exemplary embodiments of the present disclosure, the methods for producing a membrane-permeable construct comprises mixing a LNP and a CPP in a charge ratio (CR) of 1 :2-3. In one or more exemplary embodiments of the present disclosure, the methods for producing a membrane-permeable construct comprises mixing a LNP and a CPP in a charge ratio (CR) of 1 :1.5. In one or more exemplary embodiments of the present disclosure, the methods for producing a membrane-permeable construct comprises mixing a LNP and a CPP in a charge ratio (CR) of 1 :2. In one or more exemplary embodiments of the present disclosure, the methods for producing a membrane-permeable construct comprises mixing a LNP and a CPP in a charge ratio (CR) of 1 :3.
In one or more exemplary embodiments of the present disclosure, the methods for loading a cargo onto a membrane-permeable construct comprises mixing the membrane-permeable construct with a cargo in a charge ratio (CR) of the cargo to the CPP comprised by the membrane-permeable construct of 1 :1.5-3, 1 :2-3, 1 ;1 ,5, 1 :2 or 1 :3. In one or more exemplary embodiments of the present disclosure, the methods for loading a cargo onto a membrane-permeable construct comprises mixing the membrane-permeable construct with a cargo in a charge ratio (CR) of the cargo to the CPP comprised by the membrane-permeable construct of 1 :1.5-3. In one or more exemplary embodiments of the present disclosure, the methods for loading a cargo onto a membrane- permeable construct comprises mixing the membrane-permeable construct with a cargo in a charge ratio (CR) of the cargo to the CPP comprised by the membrane-permeable construct of 1 :2- 3. In one or more exemplary embodiments of the present disclosure, the methods for loading a cargo onto a membrane-permeable construct comprises mixing the membrane-permeable construct with a cargo in a charge ratio (CR) of the cargo to the CPP comprised by the membrane- permeable construct of 1 :1.5. In one or more exemplary embodiments of the present disclosure, the methods for loading a cargo onto a membrane-permeable construct comprises mixing the membrane-permeable construct with a cargo in a charge ratio (CR) of the cargo to the CPP comprised by the membrane-permeable construct of 1 :2. In one or more exemplary embodiments of the present disclosure, the methods for loading a cargo onto a membrane-permeable construct comprises mixing the membrane-permeable construct with a cargo in a charge ratio (CR) of the cargo to the CPP comprised by the membrane-permeable construct of 1 :3.
In one or more exemplary methods for producing the membrane-permeable construct of the present disclosure, the LNP is selected as one of the group consisting of a liposome, a vesicle and micelle.
In one or more exemplary methods for producing the membrane-permeable construct of the present disclosure, the LNP is selected as a liposome.
Thus, in one or more exemplary embodiments, the LNP used in the methods for producing a membrane-permeable construct as disclosed herein is a liposome and the cargo is selected as one or more of a DNA molecule, a RNA molecule, an oligonucleotide, a protein, a peptide and a small molecule.
In one or more exemplary embodiments, the LNP used in the methods for producing a membrane- permeable construct as disclosed herein is a liposome containing a cargo, where the cargo is selected as one or more of a DNA molecule, a RNA molecule, an oligonucleotide, a protein, a peptide and a small molecule, wherein the cargo is dissolved in HBS buffer and contacted with the lipid film during hydration of the lipid film during formation of the liposome.
In one or more exemplary embodiments, the LNP used in the methods for producing a membrane- permeable construct as disclosed herein is selected as a liposome composed of DPPC, DSPE- PEG and cholesterol in a 16:11 :3 ratio.
In one or more exemplary embodiments, the LNP used in the methods for producing a membrane- permeable construct as disclosed herein is selected as a liposome composed of DOTAP, DPPC and cholesterol in a 8:8:1 ratio. In one or more embodiments, the lipid composition ratio of the liposome is a V/V percent ratio or w/w percent ratio.
In one or more exemplary methods for producing the membrane-permeable construct of the present disclosure, the membrane-construct is subjected to size separation in order to produce a membrane-permeable construct that is uniform in size.
One example of such a size separation technique is extrusion of the membrane-permeable membrane through a membrane filter with a specific pore size. However, other size separation methods are known in the prior art and the skilled person will know how to select a suitable size separation technique in accordance with the circumstances.
In one or more exemplary methods for producing the membrane-permeable construct of the present disclosure, the membrane-permeable construct is formed by addition of one or more CPPs as defined herein to the LNPs before extrusion of the LNPs through a membrane filter.
In one or more exemplary methods for producing the membrane-permeable construct of the present disclosure, the membrane-permeable construct is formed by addition of one or more CPPs as defined herein to the LNPs after extrusion of the LNPs through a membrane filter.
In one or more exemplary methods for producing the membrane-permeable construct of the present disclosure, the membrane-permeable construct is formed by addition of one or more CPPs as defined herein to LNPs loaded with a lipophilic cargo before or after extrusion of the LNPs through a membrane filter.
In one or more exemplary methods for producing the membrane-permeable construct of the present disclosure, the membrane-permeable construct is formed by addition of one or more CPPs as defined herein to LNPs loaded with a hydrophilic cargo before or after extrusion of the LNPs through a membrane filter. In one or more exemplary methods for producing the membrane-permeable construct of the present disclosure, the membrane-permeable construct is formed by dissolution of the one or more CPPs in an organic solvent together with the lipid(s) chosen to form the LNP.
In one or more exemplary methods for producing the membrane-permeable construct of the present disclosure, the membrane-permeable construct is formed by dissolution of the one or more CPPs into the dispersion medium prior to hydration of the lipid film.
In one or more exemplary methods for producing the membrane-permeable construct of the present disclosure, the membrane-permeable construct is formed by addition of the one or more CPPs to a composition comprising an LNP that has been extruded through a membrane filter.
In one or more exemplary embodiments of the present disclosure, the method for producing a membrane-permeable construct includes addition of the one or more CPPs to a composition comprising an LNP before extrusion through a membrane filter.
In the case of membrane-permeable constructs of the present disclosure that comprises a CPP component and a cargo, these membrane-permeable constructs can be produced by
Providing a solution comprising a CPP,
Providing a solution comprising a cargo, and
Mixing the solution comprising a CPP with the solution comprising the cargo.
A membrane-permeable construct produced in this way may consist essentially of a CPP and a cargo. It is desirable that a CPP used in this fashion is a lipidated CPP, such as for example stearoylated or arachidoylated CPP.
The CPP and the cargo may be mixed in a molar ratio (MR) of 15-30:1. The CPP and the cargo may be mixed in a molar ratio of about 15:1. The CPP and the cargo may be mixed in a molar ratio of about 30:1. Alternatively, the CPP and the cargo may be mixed in a charge ratio (CR) of 1.5-3:1 , 2-3:1 , 1.5:1 , 2:1 , or 3:1 . In one or more exemplary embodiments, the CPP and the cargo may be mixed in a charge ratio (CR) of 1.5-3:1. In one or more exemplary embodiments, the CPP and the cargo may be mixed in a charge ratio (CR) of 2-3:1. In one or more exemplary embodiments, the CPP and the cargo may be mixed in a charge ratio (CR) of 1.5:1. In one or more exemplary embodiments, the CPP and the cargo may be mixed in a charge ratio (CR) of 2:1. In one or more exemplary embodiments, the CPP and the cargo may be mixed in a charge ratio (CR) of 3:1.
In one or more exemplary methods for producing the membrane-permeable construct of the present disclosure, the one or more CPPs used in a method for producing a membrane-permeable construct is selected as one or more lipidated CPPs.
In one or more exemplary methods for producing the membrane-permeable construct of the present disclosure, the one or more CPPs used in a method for producing for producing a membrane-permeable construct is selected as one or more stearylated CPPs.
In one or more exemplary methods for producing the membrane-permeable construct of the present disclosure, the one or more CPPs used in a method for producing for producing a membrane-permeable construct is selected as one or more arachidoylated CPPs.
In one or more exemplary methods for producing the membrane-permeable construct of the present disclosure, the one or more CPPs used in a method for producing for producing a membrane-permeable construct is selected as one or more from the list consisting of Transportan (TP), Transportan 10 (TP10), PepFect 14 (PF14), NickFect 51 (NF51), NickFect 55 (NF55), NF554, NF70, NF71 , PF132, PF141 , PF142, PF143, GP1 , GP2, GP3 and GP4.
In one or more exemplary methods for producing the membrane-permeable construct of the present disclosure, the one or more CPPs used in a method for producing for producing a membrane-permeable construct is selected as one or more from the list consisting of PepFect 14 (PF14), NickFect 51 (NF51), NickFect 55 (NF55), NF554, NF70, NF71 , PF132, PF141 , PF142, PF143, GP1 , GP2, GP3 and GP4. In one or more exemplary methods for producing the membrane-permeable construct of the present disclosure, the one or more CPPs used in a method for producing for producing a membrane-permeable construct is selected as one or more from the list consisting of NickFect 51 (NF51), NickFect 55 (NF55), NF554, NF70, NF71 , PF132, PF141 , PF142, PF143, GP1 , GP2, GP3 and GP4.
Pharmaceutical Compositions
The membrane-permeable constructs, nanoparticles or complexes of the invention may be formulated for delivery in pharmaceutical compositions. The pharmaceutical composition will normally be sterile and will typically include a pharmaceutically acceptable carrier and/or adjuvant. A pharmaceutical composition of the present invention may additionally comprise a pharmaceutically acceptable adjuvant and/or carrier. Compositions of the invention suitably comprise a construct or complex of the invention together with a pharmaceutically acceptable carrier.
Therapeutic use
The present invention provides a pharmaceutical composition for use in medicine. The present invention further provides a pharmaceutical composition for use in a therapeutic use, e.g RNA vaccination or gene therapy, e.g. in treatment or preventing cancer. The cancer may be any cancer or tumour that is a solid cancer or tumour. The solid cancer or tumour may bone, bladder, brain, breast, colon, oesophagus, gastrointestinal tract, genito-urinary tract, kidney, liver, lung, nervous system, ovary, pancreas, prostate, retina, skin, stomach, testicular and/or uterus cancer.
Thus, in one or more exemplary embodiments, the present disclosure relates to a membrane- permeable construct, nanoparticle or pharmaceutical composition as defined herein for use a as a medicament.
For example, the membrane-permeable construct, nanoparticle or pharmaceutical composition as defined herein is intended for use in a vaccine.
Additionally, the membrane-permeable construct, nanoparticle or pharmaceutical composition as defined herein is intended for use in the treatment or prevention of cancer. Moreover, the membrane-permeable construct, nanoparticle or pharmaceutical composition as defined herein is intended for use in the treatment of solid tumors.
Thus, the membrane-permeable construct, nanoparticle or pharmaceutical composition as defined herein is intended for use in a method of treating cancer in an individual, the method comprising administering an effective amount of the membrane-permeable construct, the nanoparticle, or pharmaceutical composition to the individual.
Also, the membrane-permeable construct, nanoparticle or pharmaceutical composition as defined herein is intended for use in a method of gene silencing in an individual, the method comprising administering an effective amount of the membrane-permeable construct, the nanoparticle, or pharmaceutical composition to the individual.
Furthermore, the membrane-permeable construct, nanoparticle or pharmaceutical composition as defined herein is intended for use in a method of vaccination in an individual, the method comprising administering an effective amount of the membrane-permeable construct, the nanoparticle, or pharmaceutical composition to the individual.
General
It is to be understood that different applications of the disclosed products and methods may be tailored to the specific needs in the art.
It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only and is not intended to be limiting.
All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. Items
1. A membrane-permeable construct for transport of cargo across a lipid membrane and subsequent into cells, the membrane construct comprising a cell penetrating peptide (CPP).
2. The membrane-permeable construct according to item 1 comprising a lipid nanoparticle (LNP), and a cell penetrating peptide (CPP).
3. The membrane-permeable construct according to any one of items 1-2, wherein the cell penetrating peptide (CPP) comprises a fatty acid modification.
4. The membrane-permeable construct according to any one of items 1-3, wherein the cell penetrating peptide (CPP) is lipidated.
5. The membrane permeable construct according to any one of the preceding items, wherein the cell penetrating peptide (CPP) is stearylated.
6. The membrane-permeable construct according to any one of the preceding items, wherein the cell penetrating peptide (CPP) is selected as one or more from the list consisting of Transportan (TP), Transportan 10 (TP10), PepFect 14 (PF14), NickFect 51 (NF51), NickFect 55 (NF55), NF554, NF70, NF71 , PF132, PF141 , PF142, PF143, GP1 , GP2, GP3 and GP4.
7. The membrane-permeable construct according to any one of the preceding items, wherein the lipid nanoparticle (LNP) is composed of ionizable cationic lipids, phospholipids, cholesterol and polyethylene glycol (PEG)-lipids.
8. The membrane-permeable construct according to any one of the preceding items, wherein the lipid nanoparticle (LNP) is a liposome, vesicle, or micelle.
9. The membrane-permeable construct according to any one of the preceding items, wherein the lipid nanoparticle (LNP) is a liposome. 10. The membrane permeable construct according to any one of the preceding items, wherein the liposome comprises DPPC, Cholesterol and DSPE.
11. The membrane-permeable construct according to any one of the preceding items, wherein DSPE is PEGylated (DSPE-PEG).
12. The membrane-permeable construct according to any one of the preceding items, wherein the liposome is composed of DPPC, Cholesterol and PEGylated DSPE (DSPE-PEG) in a ratio of 16:11 :3 DPPC:Cholesterol:Pegylated DSPE (DSPE-PEG).
13. The membrane-permeable construct according to any one of the preceding items, wherein the liposome comprises DOTAP, DPPC and cholesterol.
14. The membrane-permeable construct according to any one of the preceding items, wherein the liposome is composed of DOTAP, DPPC and cholesterol in a ratio of 8:8:1.
15. The membrane-permeable construct according to any one of the preceding items, wherein the membrane-permeable construct further comprises a cargo.
16. The membrane-permeable construct according to any one of the preceding items, wherein the cargo is loaded into or onto the membrane-permeable construct.
17. The membrane-permeable construct according to any one of the preceding items, wherein the cargo is loaded into or onto the lipid nanoparticle (LNP).
18. The membrane-permeable construct according to any one of the preceding items, wherein the cargo is selected as one or more of a peptide or protein, a non-peptide pharmaceutical, a nucleic acid, a single stranded or double stranded oligonucleotide, imaging agents, metal chelates, and small molecule drugs. The membrane-permeable construct according to item 18, wherein the peptide or protein cargo is selected from the group consisting of a cell targeting peptide or tumour targeting peptide, an aptamer, a receptor ligand, a peptide ligand, a cytotoxic peptide, a bioactive peptide, an antibody, and a diagnostic agent. The membrane-permeable construct according to item 18, wherein the nucleic acid is selected from the group consisting of an RNA, mRNA, miRNA, DNA, siRNA, shRNA, antisense oligonucleotides, decoy DNA, a plasmid and a cyclic DNA molecule. The membrane-permeable construct according to item 18, wherein the single stranded oligonucleotides is selected as one or more from the group consisting of DNA, RNA and their analogues, such as PNA and LNA, and the double stranded oligonucleotide is selected as one or more from the group consisting of siRNA, shRNA, decoyDNA, cyclic DNA and plasmids. The membrane-permeable construct according to item 18, wherein the imaging agents are selected from the group consisting of fluorophores and radioactive tracers. The membrane-permeable construct according to any one of the preceding items wherein the membrane-permeable construct is a complex formed by a lipid nanoparticle (LNP) as defined in any one of items 6-13 and a cell penetrating peptide (CPP) as defined in any one of items 2-5, or wherein the membrane-permeable construct is a complex formed by a lipid nanoparticle (LNP) as defined in any one of items 6-13, a cell penetrating peptide (CPP) as defined in any one of items 2-5 and a cargo as defined in any one of items 14-21 . The membrane-permeable construct according to item 23, wherein the complex is formed either through chemical linkage via covalent bonds or through ionic bonds, ionic interactions, non-covalent bonds, or non-covalent interactions. 25. The membrane-permeable construct according to any one of the preceding items, wherein the membrane-permeable construct consists essentially of cell penetrating peptide (CPP).
26. The membrane-permeable construct according to any one of the preceding items, wherein the membrane-permeable construct consists essentially of cell penetrating peptide and a cargo.
27. The membrane-permeable construct according to any one of the preceding items comprising CPP to LNP in a charge ratio (CR) of 1.5-3:1 , 2-3:1 , 1 .5:1 , 2:1 or 3:1.
28. The membrane-permeable construct according to any one of the preceding items comprising CPP to LNP in a charge ratio (CR) of 1.5-3:1.
29. The membrane-permeable construct according to any one of the preceding items comprising CPP to LNP in a charge ratio (CR) of 2-3:1.
30. The membrane-permeable construct according to any one of the preceding items comprising CPP to LNP in a charge ratio (CR) of 1.5:1.
31. The membrane-permeable construct according to any one of the preceding items comprising CPP to LNP in a charge ratio (CR) of 2:1.
32. The membrane-permeable construct according to any one of the preceding items comprising CPP to LNP in a charge ratio (CR) of 3:1.
33. The membrane-permeable construct according to any one of the preceding items comprising CPP to cargo in a charge ratio (CR) of 1.5-3:1 , 2-3:1 , 1.5:1 , 2:1 or 3:1 .
34. The membrane-permeable construct according to any one of the preceding items comprising CPP to cargo in a charge ratio (CR) of 1.5-3:1.
35. The membrane-permeable construct according to any one of the preceding items comprising CPP to cargo in a charge ratio (CR) of 2-3:1 . 36. The membrane-permeable construct according to any one of the preceding items comprising CPP to cargo in a charge ratio (CR) of 1.5:1.
37. The membrane-permeable construct according to any one of the preceding items comprising CPP to cargo in a charge ratio (CR) of 2:1 .
38. The membrane-permeable construct according to any one of the preceding items comprising CPP to cargo in a charge ratio (CR) of 3:1 .
39. The membrane-permeable construct according to any one of the preceding items, wherein the membrane permeable construct is a nanoparticle.
40. T he nanoparticle according to item 39, wherein the nanoparticle has a diameter of about 20-100 nm, such as about 30-90, 40-80, 50-70, 55-65, 20-30, 30-40, 40-50, 60-70, 70-80, 80-90, 90-100, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95 or such as about 100 nm.
41. A method for producing a membrane-permeable construct comprising the steps providing a cell penetrating peptide (CPP)
42. The method for producing a membrane-permeable construct according to item 41 comprising the steps providing a lipid nanoparticle (LNP), providing a cell penetrating peptide (CPP), and mixing the lipid nanoparticle (LNP) with the cell penetrating peptide (CPP), wherein the mixing of the lipid nanoparticle (LNP) and the cell penetrating peptide produces the membrane-permeable construct. 43. The method according to item 41-42, wherein the cell penetrating peptide (CPP) comprises a fatty acid modification.
44. The method according to any one of items 41-43, wherein the cell penetrating peptide (CPP) is lipidated.
45. The method according to any one of items 41-44, wherein the cell penetrating peptide (CPP) is stearylated.
46. The method according to any one of items 41-45, wherein the cell penetrating peptide (CPP) is selected as one or more from the list consisting of PepFect 14 (PF14), NickFect 51 (NF51), NickFect 55 (NF55), NF554, NF70, NF71 , PF132, PF141 , PF142, PF143, GP1 , GP2, GP3 and GP4.
47. The method according to any one of items 41-46, wherein the lipid nanoparticle (LNP) is composed of ionizable cationic lipids, phospholipids, cholesterol and polyethylene glycol (PEG)-iipids.
48. The method according to any of items 41-47, wherein the lipid nanoparticle (LNP) is a liposome, vesicle, or micelle.
49. The method according to any of items 41-48, wherein the lipid nanoparticle (LNP) is a liposome.
50. The method according to item 49, wherein the liposome comprises DPPC, Cholesterol and DSPE.
51. The method according to item 50, wherein DSPE is DSPE is PEGylated (DSPE-PEG).
52. The method according to any one of items 49-51 , wherein the liposome is composed of DPPC, Cholesterol and DSPE(PEG) in a ratio of 16:11 :3. The method according to item 49, wherein the liposome comprises DOTAP, DPPC and cholesterol. The method according to item 53, wherein the liposome is composed of DOTAP, DPPC and cholesterol in a ratio of 8:8:1. A method according to any one of 41-45, wherein the method further comprises the steps providing a cargo, and loading the cargo onto or into the membrane-permeable construct. The method according to item 55, wherein the loading of the cargo onto or into the membrane-permeable construct is achieved by mixing the membrane-permeable membrane with the cargo. The method according to any one of items 41-56, wherein the lipid nanoparticle (LNP) comprises a cargo. The method according to any one of items 41-57, wherein the method comprises loading of a cargo into or onto the lipid nanoparticle (LNP). The method according to any of items 55-58, wherein the cargo is selected as one or more of a peptide or protein, a non-peptide pharmaceutical, a nucleic acid, a single stranded or double stranded oligonucleotide, imaging agents, metal chelates, and small molecule drugs. 60. The method according to item 59, wherein the cargo is a peptide or protein selected from the group consisting of a cell targeting peptide or tumour targeting peptide, an aptamer, a receptor ligand, a peptide ligand, a cytotoxic peptide, a bioactive peptide, an antibody, and a diagnostic agent.
61. The method according to item 59, wherein the cargo is a nucleic acid selected from the group consisting of an RNA, mRNA, miRNA, DNA, siRNA, shRNA, antisense oligonucleotides, decoy DNA, a plasmid and a (cyclic) DNA molecule.
62. The method according to item 59, wherein the cargo is a single stranded oligonucleotides selected as one or more from the group consisting of DNA, RNA and their analogues, such as PNA and LNA, and the double stranded oligonucleotide is selected as one or more from the group consisting of siRNA, shRNA, decoyDNA, plasmids and cyclic DNA.
63. The method according to item 59, wherein the cargo is an imaging agent selected from the group consisting of fluorophores and radioactive tracers.
64. The method according to any one of items 41-63, wherein the membrane-permeable construct is produced by mixing CPP and LNP in a charge ratio (CR) of 1.5-3:1 , 2-3:1 , 1.5:1 , 2:1 or 3:1.
65. The method according to any one of items 41-64, wherein the membrane-permeable construct is produced by mixing CPP and LNP in a charge ratio (CR) of 1.5-3:1.
66. The method according to any one of items 41-65, wherein the membrane-permeable construct is produced by mixing CPP and LNP in a charge ratio (CR) of 2-3:1.
67. The method according to any one of items 41-66, wherein the membrane-permeable construct is produced by mixing CPP and LNP in a charge ratio (CR) of 1.5:1.
68. The method according to any one of items 41-67, wherein the membrane-permeable construct is produced by mixing CPP and LNP in a charge ratio (CR) of 2:1 .
69. The method according to any one of items 41-68, wherein the membrane-permeable construct is produced by mixing CPP and LNP in a charge ratio (CR) of 3:1 . 70. The method according to any one of items 41-69, wherein the method for producing a membrane-permeable construct consists essentially of providing a cell penetrating peptide (CPP), and mixing the CPP with a cargo, thereby providing a membrane permeable construct consisting essentially of a cell penetrating peptide and a cargo.
71. The method according to any one of items 41-70, wherein the membrane-permeable construct is produced by mixing CPP to cargo in a charge ratio (CR) of 1.5-3:1 , 2-3:1 , 1.5:1 , 2:1 or 3:1.
72. The method according to any one of items 41-71 , wherein the membrane-permeable construct is produced by mixing CPP to cargo in a charge ratio (CR) of 1.5-3:1.
73. The method according to any one of items 41-72, wherein the membrane-permeable construct is produced by mixing CPP to cargo in a charge ratio (CR) of 2-3:1.
74. The method according to any one of items 41-73, wherein the membrane-permeable construct is produced by mixing CPP to cargo in a charge ratio (CR) of 1.5:1.
75. The method according to any one of items 41-74, wherein the membrane-permeable construct is produced by mixing CPP to cargo in a charge ratio (CR) of 2:1.
76. The method according to any one of items 41-75, wherein the membrane-permeable construct is produced by mixing CPP to cargo in a charge ratio (CR) of 3:1.
77. A composition comprising the membrane-permeable construct according to any one of items 1-38, the nanoparticle of any one of the items 39-40, or the membrane-permeable construct as produced by the method as defined in any one of items 41-76. 78. A pharmaceutical composition comprising the membrane-permeable construct according to any one of items 1-38, the nanoparticle of any one of the items 39-40, or the membrane- permeable construct as produced by the method as defined in any one of items 41-76, and a pharmaceutically acceptable carrier.
79. A membrane-permeable construct according to any one of items 1-38, the nanoparticle according to any one of items 39-40, the composition according to item 77 or a pharmaceutical composition according to item 78 for use as a medicament.
80. A membrane-permeable construct according to any one of items 1-38, the nanoparticle according to any one of items 39-40, the composition according to item 77 or a pharmaceutical composition according to item 78 for use in a vaccine.
81. A membrane-permeable construct according to any one of items 1-38, the nanoparticle according to any one of items 39-40, the composition according to item 77 or a pharmaceutical composition according to item 78 for use in the treatment or prevention of cancer.
82. A membrane-permeable construct according to any one of items 1-38, the nanoparticle according to any one of items 39-40, the composition according to item 77 or a pharmaceutical composition according to item 78 for use in the treatment of solid tumors.
83. A membrane-permeable construct according to any one of items 1-38, the nanoparticle according to any one of items 39-40, the composition according to item 77 or a pharmaceutical composition according to item 78 for use in a method of treating cancer in an individual, the method comprising administering an effective amount of the membrane- permeable construct, the nanoparticle, the composition, or the pharmaceutical composition to the individual.
84. A membrane-permeable construct according to any one of items 1-38, the nanoparticle according to any one of items 39-40, the composition according to item 77 or a pharmaceutical composition according to item 78 for use in a method of gene silencing in an individual, the method comprising administering an effective amount of the membrane- permeable construct, the nanoparticle, the composition, or pharmaceutical composition to the individual. 85. A membrane-permeable construct according to any one of items 1-38, the nanoparticle according to any one of items 39-40, the composition according to item 77 or a pharmaceutical composition according to item 78 for use in a method of vaccination in an individual, the method comprising administering an effective amount of the membrane- permeable construct, the nanoparticle, the composition, or the pharmaceutical composition to the individual.
PROPHETIC EXAMPLES
Prophetic example 1
Initial optimization of the mixture constituents will be carried out based on the data available on the clinical applications of the LNP for mRNA delivery. The mixture of LNP in delivery of mRNA-1273 (Moderna) is used as a starting point : 50:10:38.5:1.5 (SM-102 : DSDPC : Cholesterol : PEG200- DMG) (Yang et al., 2022).
To test the most efficient CPP for the mixture, one (at a time) CPP selected from the list of 1-16, Transportan (TP), Transportan 10 (TP10), PepFect 14 (PF14), NickFect 51 (NF51 ), NickFect 55 (NF55), NF554, NF70, NF71 , PF132, PF141 , PF142, PF143, GP1 , GP2, GP3 and GP4 in concentration of 1 and 5 mM in the transfection mixture will be added.
The cargo, pDNA encoding pGL3, luciferase expressing plasmid, is added to the formed mixture of the lipids and CPP. HEK-293 cells are used for the transfection experiments. Cells are grown in Dulbecco’s modified Eagles medium (DMEM) supplemented with glutamax, 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate, 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 pg/ml streptomycin. 5.0 x 104 HEK 293 cells are seeded into 24-well plates 24 h prior to experiments. During the LNP/CPP-plasmid complex formation the cell medium in the wells was replaced with 450 pl serum-free medium. The cells are treated with complexes in charge ratios ranging from 1 :1 to 1 :5 for 4 h. 55 pl fetal bovine serum is added and the cells are incubated for an additional 20 h. After incubation the cells are lysed with cell lysis buffer. Luciferase activity is measured using a Glomax luminometer. Readouts are normalized to protein content, and measured using Bradford protein assay. We expect that the most efficient LNP/CPP mixture for plasmid transfection will be selected based on these optimization results. The efficacy of transfection is decided in comparison with the only LNP mixture as well as the selected CPP at the same concentration. Prophetic example 2
Further optimization of the mixture components will be carried out in order to define the additional ration of the LNP and CPP with the cargo plasmid. The initial ration of LNP 50:10:38.5:1.5 (SM-102 : DSDPC : Cholesterol : PEG200-DMG) will be changed to 40:20:37:3 (SM-102 : DSDPC : Cholesterol : PEG200-DMG) and the most efficient CPP (List 1-16, Transportan (TP), Transportan 10 (TP10), PepFect 14 (PF14), NickFect 51 (NF51), NickFect 55 (NF55), NF554, NF70, NF71 , PF132, PF141 , PF142, PF143, GP1 , GP2, GP3 and GP4, selected from the initial optimization experiment) in different concentrations, 0.1 ; 1 and 10 mM will be tested.
The cargo, pDNA encoding pGL3, luciferase expressing plasmid, is added to the formed mixture of the lipids and CPP. HEK-293 cells are used for the transfection experiments. Cells are grown in Dulbecco’s modified Eagles medium (DMEM) supplemented with glutamax, 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate, 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 pg/ml streptomycin. 5.0 x 104 HEK 293 cells are seeded into 24-well Plates 24 h prior to experiments. During the LNP/CPP-plasmid complex formation the cell medium in the wells was replaced with 450 pl serum-free medium. The cells are treated with complexes in charge ratios ranging from 1 :1 to 1 :5 for 4 h. 55 pl fetal bovine serum is added and the cells are incubated for an additional 20 h. After incubation the cells are lysed with cell lysis buffer. Luciferase activity is measured using a Glomax luminometer. Readouts are normalized to protein content, and measured using Bradford protein assay. We expect that the improvement of the efficacy of the plasmid transfection will be achieved and will be selected based on these optimization results. The efficacy of transfection is decided in comparison with the only LNP mixture as well as the selected CPP at the same concentration.
Prophetic example 2A
Optimization of the mixture components will be carried out in order to define the additional ration of the LNP and CPP with the cargo protein, heat shock protein, HSP70. The initial ration of LNP 50:10:38.5:1.5 (SM-102 : DSDPC : Cholesterol : PEG200-DMG) will be changed to 40:20:37:3 (SM-102 : DSDPC : Cholesterol : PEG200-DMG) and the most efficient CPP (List 1-16, Transportan (TP), Transportan 10 (TP10), PepFect 14 (PF14), NickFect 51 (NF51), NickFect 55 (NF55), NF554, NF70, NF71 , PF132, PF141 , PF142, PF143, GP1 , GP2, GP3 and GP4, selected from the initial optimization experiment) in different concentrations, 0.1 ; 1 and 10 mM will be tested.
Optical microscopy 96-well plates are seeded with 7,000 BHM pLuc expressing cells per well in 100 pL DMEM supplemented with 10% FBS and incubated at 37 °C for one day. The complex of optimized LNP mixture with CPP:HSP70-Alexa 568 (2 pM:200 nM) is formed in milliQ water for 30 min at room temperature before addition to the cells for 1 or 3 h. The wells are emptied and wash thrice with 200 pL Gibco Opti-MEM before a final addition of 100pL of Gibco FluoroBrite. The plate is then imaged for the cargo uptake using an epi-fuorescence microscope.
We expect that the improvement of the efficacy of the protein transfection will be achieved and will be selected based on these optimization results. The efficacy of transfection is decided in comparison with the only LNP mixture as well as the selected CPP at the same concentration.
The protein cargo delivery by optimized LNP/CPP mixture will be applied for delivery of other negatively charged, therapeutically relevant proteins among enzymes, transcription factors, genome editing proteins, Cas9 proteins, TALEs, TALENs, nucleases, binding proteins (e.g., ligands, receptors, antibodies, antibody fragments; nucleic acid binding proteins, etc.), structural proteins, and therapeutic proteins (e.g., tumor suppressor proteins, therapeutic enzymes, growth factors, growth factor receptors, transcription factors, proteases etc.
Prophetic example 2B
Optimization of the mixture components will be carried out in order to define the additional ration of the LNP and CPP with the cargo siRNA to knock-down luciferase enzyme. The initial ration of LNP 50:10:38.5:1.5 (SM-102 : DSDPC : Cholesterol : PEG200-DMG) will be changed to 40:20:37:3 (SM-102 : DSDPC : Cholesterol : PEG200-DMG) and the most efficient CPP (List 1-16, Transportan (TP), Transportan 10 (TP10), PepFect 14 (PF14), NickFect 51 (NF51), NickFect 55 (NF55), NF554, NF70, NF71 , PF132, PF141 , PF142, PF143, GP1 , GP2, GP3 and GP4, selected from the initial optimization experiment) in different concentrations, 0.1 ; 1 and 10 mM will be tested.
In the down-regulation experiments, the optimized LNP/CPP mixture at the CPP to siRNA(Luc) molar ratio 30 : 1 HeLa-luc cell lines (5 x 104) are seeded into 24-well plate, or the cells were seeded (1 x 104) in 96-well-plate 24 h before the experiments. The cells are treated with the complexes at different MRs and siRNA concentrations and then incubated for 24 h at 37 °C. The cells are lysed using 100 pl of cell lysis buffer (Promega, Sweden) for 30 min at RT. Luciferase activity is measured on a Glomax™ 96 microplate luminometer (Promega, Sweden) using Promega’s luciferase assay system to determine gene-silencing efficiency. Relative light units (RLU) were obtained and normalized to the control (untreated cells). The positive control RNAiMAX was utilized according to the manufacturer’s instruction. Stealth RNAi™ siRNA negative control was used as a negative control.
We expect that the improvement of the efficacy of the siRNA transfection will be achieved and will be selected based on these optimization results. The efficacy of transfection is decided in comparison with the only LNP mixture as well as the selected CPP at the same concentration. Prophetic example 3
The most efficient LNP/CPP/Cargo mixtures will be used to show the efficacy of transfection in vivo. For in vivo treatment trials, the optimized, 100 pl of most efficient mixtures of LNP/CPP/Cargo plasmid in MQ-water/10% glucose or 10.8% mannitol solution will be injected into the tail-vein of mice. Hydrodynamic infusions in tail vein of mice will be carried out using 2 ml saline buffer injected with high pressure. Hydrodynamic mean diameter of the particles will be determined. We expect that the efficient plasmid transfection in vivo will be achieved and will be selected based on these optimization results. The efficacy of in vivo transfection is decided in comparison with the only LNP mixture as well as the selected CPP at the same concentration.
Materials & Methods
Ceil penetrating peptides used in examples X-Y
The CPPs with the alternative names GP11 , GP12, GP13, GPM, GP15 and GP16 used in the following examples X-Y correspond to the following CPPs and CPP sequences:
GP11 corresponds to PepFect14, PF14: stearoyl-AGYLLGKLLOOLAAAALOOLL amide.
GP15 corresponds to PepFect132, PF132: stearoyl-LHLLHHINLKALAALAKKIL amide.
GP12 corresponds to NickFect55, NF55: O(Nb-stearoyl-AGYLLG)-INLKALAALAKAIL amide.
GP13 corresponds to NickFect554, NF554: K(NE-stearoyl-AGYLLG)-INLKALAALAKAIL amide.
GP16 corresponds to NickFect71 , NF71 : O(Nb-stearoyl-HHYHHG)-ILLKALKALAKAIL amide.
GPM corresponds to GP2: stearoyl-HHYHHGINLKALAALAKAIL amide.
All peptides have fatty acid residue in the N-terminus and all peptides are C-terminally amidated.
Nucleic acid molecules used in examples X-Y
In the following examples X-Y different Nucleic acid molecules were referred to as mRNA, plasmid and siRNA respectively. In the context of examples X-Y, the term mRNA refers to a reporter mRNA encoding firefly luciferase (CleanCap FLuc mRNA, Trilink), the term plasmid refers to a reporter plasmid encoding firefly luciferase (pGL3 plasmid with the size 4818 bp from Promega) and siRNA refers to firefly luciferase targeting small interfering RNA. Sense and antisense strands were annealed in ultrapure water. Sequence of the siRNA can be found in POROSK, L. et al., Biomater Sci. 2019 Sep. Lipids, materials and components for formulation of LNPs and CPP complexes
Absolute ethanol ( 96%), ultrapure water, molecular biology grade glucose (Fisher Scientific), 1 M HEPES buffer solution (Sigma), Low bind tubes (Axigen Maxymum Recovery 1.5 ml tubes), PCR grade 0.2 ml tubes (Nerbe Plus), Lipid Nanoparticle Exploration Kit (Cayman chemicals), Dialysis chambers 3.5K MWCO (Thermo Scientific), Fluorescent dyes: PicoGreen and RiboGreen (Thermo Fisher Scientific).
Equipment
Vortex, tabletop centrifuge (up to 16,000 x g), freeze-dryer, -80°C freezer, pipettes and pipet tips with filters.
Cell culture work
Cell lines used: HaCaT, HeLa, U87-luc
Media and media components: DMEM media supplemented with non-essential amino acids, sodium pyruvate, Penicillin, Streptomycin, foetal bovine serum (Thermo), HEPES. For detaching cells from the culture plate incubation with 0.25% trypsin-EDTA was used.
All plates and single use plastic was cell-culture grade, individually packed and sterile prior to use. 96-well plates, 10 ml and 5 ml serological pipets with compatible pipettor.
Reporter protein detection: expressed firefly luciferase was detected from the cell lysate. For cell lysis 0.1% Triton-X 100 solution on 1 x PBS was used and 30 pl of lysis solution was incubated with transfected cells in 96-well plate well for 20 min. For detection 20 pl of cell lysate was transferred to a white 96-well plate and luciferase detection mix containing luciferin was added to the lysate and luminescence was detected with GLOMAX plate reader.
EXAMPLES
Example 1: Preparation of membrane-permeable constructs
Example 1.1: Preparation of CPP complexes
For complexes consisting of CPP and oligonucleotide different mixing approaches were used. CPP and oligonucleotide was mixed in ultrapure water at charge ratios (CR) based on the positive charges in the CPP and the negative charges in the oligonucleotide backbone. The different mixing method used include
-Mixing 0.6 - 3 pl of 1 mM CPP in ultrapure water (depending on the CPP and used charge ratio) with 1 pg of oligonucleotide in ultrapure water/buffer and adding ultrapure water to a final volume of 50 - 100 pl.
-Mixing CPP diluted in water and oligonucleotide diluted in water in HEPES (10 mM, pH 7.4) or HEPES buffered glucose (10 mM HEPES; 5% glucose, pH 7.4).
-Mixing CPP diluted in ethanolic phase and oligonucleotide diluted in ultrapure water.
-Mixing CPP diluted in ethanolic phase and oligonucleotide diluted in HEPES or HEPES buffered glucose.
Example 1.2: Preparation of CPP/siRNA complex at molar ratio (MR) 30:1
For achieving 15 nM siRNA concentration per 96 well plate well in 100 pl final volume per well, where CPP/siRNA complex mix is 1/10th of the final volume (complexes are 10 pl per well added to 90 pl of media on cells in the well). Dilute siRNA stock solution to 2 pM working solution in ultrapure water. Dilute 1 mM CPP stock solution to 50 pM working solution. Add water to the tube and dilute the nucleic acid working solution. Mix well. Add CPP working solution and mix by pipetting. Incubate at room temperature for 30 minutes. The complexes are now ready and can be added to the cells in 90 microliters of media.
Figure imgf000052_0001
Table 3: CPP:siRNA molar ratio of 30:1
For mixing the same complexes formed in buffer, add accordingly the buffer to the water and continue as stated above.
Figure imgf000052_0002
Table 4: CPP:siRNA molar ratio of 30:1
Example 1.3: Charge ratio and charge ratio calculations The charge ratio (CR) as refered to in this disclosure generally relates to the ratio of electrical charges of the CPP to the charges of either the cargo, mRNA or plasmids, or the LNPs disclosed herein.
For CPPs the charge contribution of each amino acid is added up to yield a net charge of the CPP. For example GP12/NF55 and GP13/NF554 has a net charge of 3 positive charges (+3). In case of nucleic acids, each nucleic acid contributes 1 negative charge (-1 ) and each base pair of a plasmid 2 negative charges (-2).
Thus, to achieve a composition with a CR of 3:1 of CPP to DNA/plasmid, the CPP concentration used needs to correspond to an amount of positive net charges that is three times the number of negative charges provided by the concentration of DNA or plasmid used together with the CPP.
To arrive at a CR of 1 : 1 , the number of positive charges provided by the CPP needs to match the negative charges of a plasmid. For a plasmid containing 10500 bp, the total negative charge is 21000. With an average molecular weight of per base pair of 6993000 Da, and a plasmid dose for use in transfection of 0,1 pg, a total of 1 ,43x10-14 mol plasmid is used.
The amount of CPP required to reach a CR of 1 :1 when mixed with 1 ,43x10-14 mol plasmid can then be calculated as the total negative charge (21000) divided by the positive net charge of the CPP (+3) times the mol plasmid used (1.43x10-14), which equals 1 ,001x10-1°. Examples of solutions and concentrations used to reach a given charge ratio is provided in the tables of examples 1.3.1 and 1.3.2 below.
Example 1.3.1: Preparation of CPP/plasmid complex at charge ratio(CR) 2:1
For achieving 100 nM plasmid concentration per 96-well plate well in 100 pl final volume per well, where CPP/plasmid complex mix is 1/10th of the final volume.
Prepare CPP working solution (100 pM) and plasmid working solution (0.05 mg/ml) in ultrapure water. Mix according to the tables XY1 , XY2 or XY3, where water and plasmid is mixed, and the CPP then added. Mix by pipetting and incubate at room temperature for 30 minutes.
Following incubation for 30 minutes the CPP/plasmid complex prior is ready to be added to the media on the cells. For complexes with buffer, mix water with buffer, and then follow the abovementioned protocol.
Figure imgf000053_0001
Table 5: One way to mix a CPP/plasmid complex having a charge ratio (OR) of 2:1 , wherein the CPP has a net charge of +3.
Figure imgf000054_0001
Table 6: One way to mix a CPP/plasmid complex having a charge ratio (OR) of 2:1 , wherein the CPP has a net charge of +5.
Figure imgf000054_0002
Table 7: Example of ingredients used to mix a CPP/plasmid complex having a charge ratio (CR) of 2:1 , wherein the CPP has a net charge of +3.
Example 1.3.2: Preparation of CPP/mRNA complex at a charge ratio (CR) 3:1
For achieving 100 nM mRNA concentration per 96-well plate well in 100 pl final volume per well, where CPP/plasmid complex mix is 1/10th of the final volume.
Prepare CPP working solution (100 pM) and mRNA working solution (0.05 mg/ml) in ultrapure water. Mix according to the tables XX1 , XX2 or XX3, where water and plasmid is mixed, and the CPP then added. Mix by pipetting and incubate at room temperature for 30 minutes prior to addition to the media on the cells. For complexes with buffer, mix water with buffer, and then follow the abovementioned protocol.
Figure imgf000054_0003
Table 8: Example of ingredients used to mix a CPP/mRNA complex having a charge ratio (CR) of 3:1 , wherein the CPP has a net charge of +3.
Figure imgf000054_0004
Figure imgf000055_0001
Table 9: Example of ingredients used to mix a CPP/mRNA complex having a charge ratio (CR) of 3:1 , wherein the CPP has a net charge of +5.
Figure imgf000055_0002
Table 10: Example of ingredients used to mix a CPP/mRNA complex having a charge ratio (CR) of 3:1 , wherein the CPP has a net charge of +3.
Example 2: Preparation of LNPs
Specific compositions of LNPs were prepared according to the LNP mix 1 approach or the LNP mix 2 approach.
Example 2.1: Preparation of LNP mix 1
LNP mix 1 consisting of SM-102, 1 ,2-DSPC, Cholesterol, and DMG-PEG(2000), was mixed in following molar ratios: 50:10:38.5:1.5
Components were mixed according to manufacturer’s instructions at shown molar ratios. For each component stock solutions were prepared in absolute ethanol to yield stocks of SM-102: 100 mg/ml; 1 ,2-DSPC: 25 mg/ml; Cholesterol: 5 mg/ml; and DMG-PEG(2000): 1 mg/ml).
A 1 ml of ethanolic lipid mixture can be prepared by mixing -35 pl SM-102 100 mg/ml, -32 pl 1 ,2- DSPC 25 mg/ml, -296 pl Cholesterol 5 mg/ml, -378 pl DMG-PEG(2000) 1 mg/ml, and 259 pl absolute ethanol.
An Oligonucleotide solution was prepared in aqueous solution. For this 620 pg of nucleic acid was adjusted to 3 ml with 50 mM sodium acetate, pH 5.0. Ethanolic lipid mixture was mixed with oligonucleotide solution in 1 :3 ratio, with oligonucleotide aqueous solution in excess.
Two mixing approaches were tested:
Solvent-injection mixing: ethanolic lipid mixture was injected to aqueous acidic oligo solution with a syringe. Needle placed in the centre of the aqueous solution tube. Mixing at 400 rpm, for 30 min, T>55°C (1 ,2-DSPC transition temperature) Mixing by pipetting: Mixed via pipette by rapidly transferring the ethanolic lipid mixture into the acidic aqueous nucleic acid solution. Mixed for >15 sec by repeated vigorous pipetting. Left undisturbed for 10 min.
Final preparations:
Dialysis of the LNP mix against neutral buffer (HEPES buffered glucose (10 mM HEPES, 5% glucose) or HEPES (10 mM) pH 7.4) was performed in a Dialysis chamber with a MWCO of 2,400, followed by filter sterilisation of the solutions with 0.22 urn filter.
Nucleic acid dose was determined with nucleic acid intercalating dye (depending on the nucleic acid, PicoGreen, RiboGreen or EvaGreen against standards of known concentrations).
Example 2.2: Preparation of LNP mix 2
For plasmid delivery, the ionizable lipid (SM-102) was replaced with DLin-MC3-DMA, thus the ethanolic lipid mixture of the LNP mix 2 was instead:
-35 pl DLin-MC3-DMA 100 mg/ml, -32 pl 1 ,2-DSPC 25 mg/ml, -296 pl Cholesterol 5 mg/ml, -378 pl DMG-PEG(2000) 1 mg/ml and -259 pl absolute ethanol.
The LNP mix was otherwise prepared as described in example 2.1 : LNP mix 1.
Example 3: Preparation of LNP/CPP/plasmid or LNP/CPP/mRNA complexes
Example 3.1 : Preparation of LNP/CPP mix followed by addition of oligonucleotide
LNP/CPP/oligonucleotide complexes were prepared by addition of CPP to a lipid mixture in ethanol. Per 1 pg of nucleic acid a dose of 0.15 - 0.7 pl of 4 mM CPP solution in ethanol was added to the ethanolic lipid mixture to yield the following mixture:
0.05 pl SM-102 or DLin-MC3-DMA 100 mg/ml,
0.05 pl 1 ,2-DSPC 25 mg/ml,
0.24 pl Cholesterol 10 mg/ml,
0.61 pl DMG-PEG(2000) 1 mg/ml,
0-0.5 pl absolute ethanol (added ethanol depends on the volume of CPP), 0.15-0.7 pl 4 mM CPP, depending on the CPP and its molecular weight 9-10.5 mg/ml (added CPP depends on the tested charge ratio).
Subsequently plasmid or mRNA in acidic aqueous solution, HEPES buffer or HEPES buffered glucose was mixed with the ethanolic lipid mixture. This mixture was then either dialyzed or incubated for a short period of time before dilution to suitable concentration. Example 3.2: Preparation of LN P and plasmid/mRNA mix followed by addition of CPP
For complexes consisting of LNP where CPP was added after nucleic acid was mixed with the LNP, the complexation was performed by preparing the LNP mix 1 as previously described. Oligonucleotide solution was prepared in aqueous solution. For this 620 pg of nucleic acid was adjusted to 3 ml with 50 mM sodium acetate, pH 5.0 or the LNP was mixed in HEPES or HEPES buffered glucose. LNP mix 1 was then mixed with oligonucleotide solution in 1 :3 ratio, with oligonucleotide aqueous solution in excess. The LNP prepared in sodium acetate was then dialyzed against HEPES or HEPES buffered saline. After collection of LNPs, the loading was determined with fluorescent nucleic acid intercalating dye and CPP was added according to the nucleic acid dose. For the LNP prepared in HEPES buffer of HEPES buffered glucose, the CPP solution was added at different charge ratios (CPP to nucleic acid).
In the case of siRNA as nucleic acid, the LNP were prepared as described above, but the CPP was added according to molar ratio (CPP to siRNA, with CPP in excess). The molar ratios tested ranged from MR5:1 to 40:1 based on CPP to nucleic acid ratio.
These methods for preparation of LNP, CPP and mRNA/plasmid/siRNA also apply for situations where LNP mix 2 was used instead.
Example 4: Delivery of siRNA and enhancement of downregulation
The LNP mix 1 was used to form LNPs (35 pl SM-102 100 mg/ml; 32 pl 1 ,2-DSPC 25 mg/ml; 296 pl Cholesterol 5 mg/ml; 378 pl DMG-PEG(2000) 1 mg/ml; 259 pl absolute ethanol). In 30 pl of 6.2 pg of firefly luciferase targeting siRNA in aqueous solution was mixed with 10 pl of ethanolic lipid mixture in a 3:1 ratio with siRNA solution in excess. The LNP was then submitted to dialyzis and loading determined from the dialyzed LNPs.
The CPP/siRNA complexes were formed in the ultrapure water or buffer, with no significant difference between the two groups. Molar ratio CPP to siRNA 30:1 was used with CPP in excess. The same molar ratio of CPP to siRNA was used when LNP was included.
The LNP/CPP mix were formed a) by adding the CPP to the ethanolic phase with lipids and then mixing with the aqueous solution of the siRNA or b) preparing the LNPs consisting of the lipids and siRNA, dialyzed, dose matched and then the CPP solution was added to the LNP/siRNA in buffer.
The membrane permeable constructs prepared above were added to wells of microtiter plates containing U87 cells expressing firefly luciferase. The concentration of siRNa was 14 nm per well. 24 hours after treatment, the cells were lysed and luminescence was measured. Successful siRNA inhibition of luciferase by the membrane permeable constructs was detected as a reduction in the luminescence measured for the treated cells compared to untreated (UT) cells (see figure 1 and 2).
Both membrane-permeable constructs obtained by addition of CPP to the ethanolic lipid mix and addition of CPP to the pre-formed LNP/siRNA showed an increase in downregulation when compared to LNP/siRNA without CPPs. The addition of CPP to already formed and dose adjusted LNP/siRNA gave more consistent outcome. Thus, the membrane-permeable constructs successfully deliver their cargo to the U87 cells and the siRNA successfully inhibited luciferase expression.
Furthermore, addition of CPPs to either ethanolic lipid mixture or to pre-formed LNP/siRNA leads to an increase in down-regulation of luciferase. The degree of down-regulation depends on the siRNA dose and used CPP ratio.
Example 5: Delivery ofmRNA encoding luciferase
To demonstrate that mRNA can also be delivered by the membrane permeable constructs as disclosed herein, the delivery of reporter mRNA encoding firefly luciferase was tested on HaCaT cells.
For CPP/mRNA similar approach was used as used for LNP mix+CPP/siRNA. The CPP was added to the ethanolic lipid mixture (group LNP/CPP+mRNA) or added to already formed LNP/mRNA (group LNP/mRNA + CPP). The mRNA was in 1 mM Sodium Citrate pH 6.4, therefore this buffer was attested additionally to HBS pH 7.4, HEPES pH 7.4 and sodium actetate pH 6 and pH 3.
Experimental conditions: 96-well plate format with 30 ng of mRNA per well. Serum containing media. Reporter protein (luciferase) levels were determined from total cell lysate 24 posttransfection.
In the case of mRNA based nucleic acid, the addition of CPP to already formed LNP/mRNA did increase the efficacy significantly more than pre-mixing CPP to ethanolic lipid mixture (see figure 3).
Depending on the mRNA concentration and LNP mixture preparation, the addition of CPPs to the pre-formed LNP/mRNA mix led to 5-50 fold increase in reporter levels compared to LNP/mRNA and thus had clearly superior efficacy in terms of mRNA delivery.
The above experiment was repeated using HeLa cells instead and as shown in figure 4, the effects of treatment in HeLa cells are less pronounced when compared to HaCaT cells used previously (figure 3). HaCaT cells are considered hard to transfect cells, therefore one of the differences may arise from the innate differences between the two cell lines.
Interestingly, in both cases of LNP mixes tested (refer to LNP mix 1 and LNP mix 2 stated above, and LNP/CPP/mRNA), there is an increase of efficacy when CPP is added at a certain charge ratio (CR) to the pre-formed LNP/mRNA particles. CPP is added after the LNP/mRNA has been mixed, incubated, dialysed and dose-matched.
CR refers to charge ratio of charges from the CPP to the negative charges of nucleic acid or to the negative charges of the LNP. For example a value such as CR2 should be understood as a charge ratio of CPP positive charges to negative charges of either LNP, nucleic acid or other cargo of 2:1 .
Example 6: Change of mixing method of LNP and reduced mRNA dose
The same LNP mix 1 and LNP mix 2 were used in this experimental set. In this case similar mixing of LNP was used as referred in Liu 2021 with slight modifications. Briefly, the LNP and mRNA are mixed in an acidic buffer at concentrated conditions, and after formulation, the acidity is neutralized with the addition of buffer at physiologic pH. In the referred paper, PBS was used. As our CPPs are incompatible with PBS, HEPES buffer was used instead. No dialysis was performed. Due to the lack of dialysis, the NA doses were not adjusted, as the input was according to the used doses.
LNP1 = LNP mixture with SM-102 100 mg/ml
LNP2 = LNP mixture with DLin-MC3-DMA 100 mg/ml
LNP1/CPP and LNP2/CPP refer to conditions where the CPP is mixed with the lipid mixture in the ethanolic phase, and then mRNA in an aqueous buffer is added.
For LNP/CPP mix where NA was added after mixing (LNP/CPP), the ethanolic mixture of LNP/CPP (0.05 ul of SM-102 100 mg/ml (working solution concentration), 0.05 ul of 1 ,2-DSPC 25 mg/ml, 0.24 ul of cholesterol 10 mg/ml, 0.61 ul of DMG-PEG(2000) 1 mg/ml, 0.15-0.7 ul CPP in ethanol 4 mM, and extra ethanol if needed to have the same final volume for all tested mixes). The ethanolic mixture or LNP/CPP was mixed with 1 ug of mRNA in an aqueous solution (water and acetate buffer; as we compare the two methods, the mix processing was the same). After incubation, an HEPES buffer was used to dilute the particles to a suitable concentration with 80 ng of mRNA per 10 ul of solution.
LNP1/mRNA + CPP and LNP2/mRNA + CPP refers to conditions where the CPP is added after the LNP/mRNA is already formed, and diluted in HEPES.
For the LNP/mRNA where CPP was added afterward (LNP + CPP), the ethanolic mixture was prepared (consisting of 35 ul of SM-102 100 mg/ml, 32 ul of 1 ,2-DSPC 25 mg/ml, 296 ul of cholesterol 10 mg/ml, 378 ul of DMG-PEG(2000) 1 mg/ml). An oligonucleotide solution was added (620 ul of mRNA adjusted to 1 ml with 50 mM sodium acetate, pH 5.0). After mixing, to the LNPs ethanolic solution of CPP solution was added and after short incubation HEPES was added. The volume adjusted, so after adding the (4 mM working solution diluted accordingly) CPP, there would be 80 ng of mRNA per 10 ul of solution.
Two different GP peptides were used, GP16 (figure 5) and GP (figure 6), and two mRNA dose per 96-well plate well were tested (50 ng and 20 ng, instead of 80 ng or 100 ng).
As can be seen in figures 5 and 6, GP16/LNP formulation with added CPP led to an increase of efficacy when LNP1 was used, but in the case of LNP2, the effects are lacking or efficacy reduced. In the case of GPM, the mixing leads to reduction.
This may be due to the inefficient formation of LNP/mRNA, as the unformulated particles are not dialyzed or dose-matched post-formulation, and the dose is determined as the input mRNA per sample. Nevertheless, at a significantly lower 20 ng mRNA dose, the LNP1/CPP + mRNA leads to similar levels of the reporter as 50 ng of mRNA.
Example 7: Delivery of plasmid expressing firefly luciferase
Transfection experiments were performed in CHO-K1 cells (figure 7) and HeLa cells (figure 8) to test the ability of compositions of membrane-permeable constructs comprising the CPPs GP1 , GP2, GP4, PF14, NF51 , NF71 together with plasmid cargo in figure 7 and compositions of membrane-permeable constructs comprising the CPPs GP11 , GP12, GP13, GPM, GP15, and GP16 together with LNPs and plasmids in figure 8, to transfect cells with plasmids expressing luciferase.
Transfection of CHO-K1 cells
CHO-K1 cells were transfected with membrane-permeable constructs comprising the CPPs GP1 , GP2, GP4, PF14, NF51 , NF71 in a 96 well setup. The CPP/pDNA complexes achieve high expression levels without the addition of LNP mixes. CPP/pDNA complexes were formed at indicated charge rations (CR) with CPP in excess. The CPP was diluted in water prior addition to plasmid in water. Per 96 well plate well 0.1 ug of plasmid dose was used in the 10 ul of complex solution and final volume (complexes and media) of 100 ul per well. 24 h post transfection in serum containing DMEM media, the cells were lysed and reporter (luciferase) detected form the lysate. Negative controls include CHO-K1 cells transfected using plasmid without CPP and an untreated sample. The membrane-permeable construct was prepared in several concentrations based on the charge ratio (CR) of a positive charge excess contributed by the CPP to the negative charge contributed by the plasmid phosphate backbone. The efficacy of the transfection was measured by means of detecting the luminescence signal (RLU) for lysed cells after 24 hours of incubation. As can be seen from figure 7, there appears to be a difference in efficiency of transfection that depends on the charge ratio (CR) between CPP and plasmid of the membrane-permeable construct. Generally, a charge ratio (CR) between 2 and 3 show the highest efficiency of transfection, with CRs above 3 and below 2 yielding a lower luminescence signal. Overall, the highest luminescence signals were obtained with a CPP to plasmid CR of 2:1 , indicating this charge ratio was the most efficient charge ratio among the tested as compared to formulations of plasmid with LNP alone or LNP and CPP together.
Transfection of HeLa cells
At this point, LNP with SM-102 nor DLin-MC3-DMA achieved very high efficacies when plasmid DNA was used as the nucleic acid cargo.
The LNP/CPP/pDNA was prepared as follows.
For LNP/CPP mix where NA was added after mixing, the ethanolic mixture of LNP/CPP (0.05 ul of DLin-MC3-DMA 100 mg/ml (working solution concentration), 0.05 ul of 1 ,2-DSPC 25 mg/ml, 0.24 ul of cholesterol 10 mg/ml, 0.61 ul of DMG-PEG(2000) 1 mg/ml, 0.15-0.7 ul CPP in ethanol 4 mM, and extra ethanol if needed to have the same final volume for all tested mixes). The ethanolic mixture or LNP/CPP was mixed with 1 ug of plasmid in an aqueous solution (water and acetate buffer; as we compare the two methods, the mix processing was the same). After incubation, the particles were dialyzed against HEPES buffer and diluted to a suitable concentration with 80 ng of NA per 10 ul of solution.
For the LNP/NA where CPP was added afterward, the ethanolic mixture was prepared (consisting of 35 ul of DLin-MC3-DMA 100 mg/ml, 32 ul of 1 ,2-DSPC 25 mg/ml, 296 ul of cholesterol 10 mg/ml, 378 ul of DMG-PEG(2000) 1 mg/ml and 259 ul of absolute ethanol). An oligonucleotide solution was added (620 ul of NA adjusted to 3 ml with 50 mM sodium acetate, pH 5.0). After mixing, the LNPs were dialysed against HEPES. Then, the NA concentration was determined and volume adjusted, so after adding the (4 mM working solution diluted accordingly) CPP, there would be 80 ng of NA per 10 ul of solution. Summary
All in all, as demonstrated in the experiments described above, the CPPs tested herein were highly efficient in driving delivery of cargo such as plasmids, mRNA and siRNA when used both on their own and when used together with LNPs as defined herein.
From the experimental results shown herein, it is clear that for the tested CPPs, a CR of 2:1 to 3:1 is considered optimal with highest efficacy and using a CPP in a CR of 2:1 to 3:1 with the CPP in excess forms particles with most efficacy.
There is a tendency to have a better LNP/CPP mix if the proportion of the CPP is below the CR3:1 , with the best performance between CR1 :1 to 2:1.
Similar tendencies can be seen for siRNA, where the molar ratio 15:1 is better than 30:1 if the LNP is involved to the LNP/CPP/siRNA complexes. Whereas for CPP/siRNA the MR30:1 would be preferred.
FIGURES
Figure 1
Figure 1 shows the luminescence signal detected from firefly luciferase expressing cells (U87 cells) treated with various membrane permeable constructs loaded with siRNA as cargo and control samples, where UT is untreated cells and siRNA is siRNA treated cells without membrane permeable construct. The luminescence signal was measured for each sample is normalized onto the luminescence signal measured for untreated cells.
Figure 2
Figure 2 shows the luminescence signal detected from firefly luciferase expressing cells (U87 cells) treated with various membrane permeable constructs loaded with siRNA as cargo and control samples, where UT is untreated cells and siRNA is siRNA treated cells without membrane permeable construct. The membrane permeable constructs comprise the CPPs PF14, NF55 and NF71 respectively in molar ratios (MR) of 15:1 and 30:1 to the concentration of siRNA.
Figure 3
Fig. 3 shows the luminescence signal detected from HaCaT cells transfected with mRNA encoding luciferase using a CPP-mRNA mixture with a charge ratio (CR) of 2:1 , a mixture where mRNA was added to a LNP/CPP preparation, a mixture where a CPP was added to a LNP/mRNA preparation, with controls being an untreated sample, a sample treated with mRNA and a sample treated with LNP without mRNA.
Figure 4
Figure 4 shows the luminescence signal detected from HeLa cells transfected with 80 ng of mRNA encoding luciferase in 10% FBS. The transfection was carried using mixtures of CPP/mRNA, LNP1/mRNA solutions to which CPP was added in various charge ratios (CR), or mixtures of LNP2/mRNA solutions to which a CPP was added in various charge ratios (CR). Controls comprising LNP1 mixture, LNP2 mixture and mRNA were included.
Figure 5
Fig. 5 shows HeLa cells transfected with complexes comprising GP16 and 50 or 20 ng mRNA expressing luciferase in various charge ratios (CR). LNPs were formulated in acidic buffer and neutralised with HEPES after complexion.
Figure 6
Fig. 5 shows HeLa cells transfected with complexes comprising GP14 and 50 or 20 ng mRNA expressing luciferase in various charge ratios (CR). LNPs were formulated in acidic buffer and neutralised with HEPES after complexion.
Figure 7
Fig. 7 shows transfection efficiency as result of a luminescence signal from CHO-K1 cells transfected using mixtures comprising CPP (GP1 , GP2, GP4, PF14, NF51 , and NF71) and cargo plasmid expressing firefly luciferase. Controls include a sample with plasmid (pDNA) without CPP and an untreated sample.
Figure 8
Fig. 8 shows transfection efficiency as result of a luminescence signal from HeLa cells transfected using mixtures comprising LNP, CPP (GP11 , GP12, GP13, GP14, GP15, and GP16) and cargo plasmid expressing firefly luciferase. Controls include an untreated sample, a sample with plasmid (pDNA) without CPP and a sample comprising LNP without mRNA. References
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Claims

1. A membrane-permeable construct for transport of cargo across a lipid membrane and subsequent into cells, the membrane construct comprising a cell penetrating peptide (CPP).
2. The membrane-permeable construct according to claim 1 comprising a lipid nanoparticle (LNP), and a cell penetrating peptide (CPP).
3. The membrane-permeable construct according to any one of claims 1-2, wherein the cell penetrating peptide (CPP) comprises a fatty acid modification.
4. The membrane-permeable construct according to any one of claims 1-3, wherein the cell penetrating peptide (CPP) is lipidated.
5. The membrane permeable construct according to any one of the preceding claims, wherein the cell penetrating peptide (CPP) is stearylated.
6. The membrane-permeable construct according to any one of the preceding claims, wherein the cell penetrating peptide (CPP) is selected as one or more from the list consisting of Transportan (TP), Transportan 10 (TP10), PepFect 14 (PF14), NickFect 51 (NF51), NickFect 55 (NF55), NF554, NF70, NF71 , PF132, PF141 , PF142, PF143, GP1 , GP2, GP3 and GP4.
7. The membrane-permeable construct according to any one of the preceding claims, wherein the membrane-permeable construct further comprises a cargo.
8. The membrane-permeable construct according to any one of the preceding claims, wherein the cargo is selected as one or more of a peptide or protein, a non-peptide pharmaceutical, a nucleic acid, a single stranded or double stranded oligonucleotide, imaging agents, metal chelates, and small molecule drugs.
9. The membrane-permeable construct according to any one of the preceding claims, wherein the membrane-permeable construct consists essentially of cell penetrating peptide and a cargo.
10. The membrane-permeable construct according to any one of the preceding claims comprising CPP to LNP in a charge ratio (CR) of 1.5-3:1 , 2-3:1 , 1 .5:1 , 2:1 or 3:1.
11. The membrane-permeable construct according to any one of the preceding claims comprising CPP to cargo in a charge ratio (CR) of 1.5-3:1 , 2-3:1 , 1.5:1 , 2:1 or 3:1 .
12. A method for producing a membrane-permeable construct comprising the steps providing a cell penetrating peptide (CPP)
13. The method for producing a membrane-permeable construct according to claim 12 comprising the steps providing a lipid nanoparticle (LNP), providing a cell penetrating peptide (CPP), and mixing the lipid nanoparticle (LNP) with the cell penetrating peptide (CPP), wherein the mixing of the lipid nanoparticle (LNP) and the cell penetrating peptide produces the membrane-permeable construct.
14. The method according to claims 12-13, wherein the cell penetrating peptide (CPP) comprises a fatty acid modification.
15. The method according to any one of claims 12-14, wherein the cell penetrating peptide (CPP) is lipidated.
16. The method according to any one of claims 12-15, wherein the cell penetrating peptide (CPP) is stearylated.
17. The method according to any one of claims 12-16, wherein the cell penetrating peptide (CPP) is selected as one or more from the list consisting of PepFect 14 (PF14), NickFect 51 (NF51), NickFect 55 (NF55), NF554, NF70, NF71 , PF132, PF141 , PF142, PF143, GP1 , GP2, GP3 and GP4.
18. A method according to any one of 12-17, wherein the method further comprises the steps providing a cargo, and loading the cargo onto or into the membrane-permeable construct.
19. The method according to claim 18, wherein the loading of the cargo onto or into the membrane-permeable construct is achieved by mixing the membrane-permeable membrane with the cargo.
20. The method according to any of claims 1-19, wherein the cargo is selected as one or more of a peptide or protein, a non-peptide pharmaceutical, a nucleic acid, a single stranded or double stranded oligonucleotide, imaging agents, metal chelates, and small molecule drugs.
21. The method according to any one of claims 12-20, wherein the membrane-permeable construct is produced by mixing CPP and LNP in a charge ratio (CR) of 1.5-3:1 , 2-3:1 , 1.5:1 , 2:1 or 3:1.
22. The method according to any one of claims 12-20, wherein the method for producing a membrane-permeable construct consists essentially of providing a cell penetrating peptide (CPP), and mixing the CPP with a cargo, thereby providing a membrane permeable construct consisting essentially of a cell penetrating peptide and a cargo.
23. The method according to any one of claims 12-20, wherein the membrane-permeable construct is produced by mixing CPP to cargo in a charge ratio (CR) of 1.5-3:1 , 2-3:1 , 1.5:1 , 2:1 or 3:1.
24. A composition comprising the membrane-permeable construct according to any one of claims 1-11 or the membrane-permeable construct as produced by the method as defined in any one of claims 12-23.
25. A pharmaceutical composition comprising the membrane-permeable construct according to any one of claims 1-11 or the membrane-permeable construct as produced by the method as defined in any one of claims 12-23, and a pharmaceutically acceptable carrier.
26. A membrane-permeable construct according to any one of claims 1-11 , the composition according to claim 24 or a pharmaceutical composition according to claim 25 for use as a medicament.
27. A membrane-permeable construct according to any one of claims 1-11 , the composition according to claim 24 or a pharmaceutical composition according to claim 25 for use in a vaccine.
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