WO2023048572A1 - Targeted lipid nanoparticle formulations - Google Patents

Targeted lipid nanoparticle formulations Download PDF

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WO2023048572A1
WO2023048572A1 PCT/NL2022/050538 NL2022050538W WO2023048572A1 WO 2023048572 A1 WO2023048572 A1 WO 2023048572A1 NL 2022050538 W NL2022050538 W NL 2022050538W WO 2023048572 A1 WO2023048572 A1 WO 2023048572A1
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lipid
peg
lnp
ligand
sirna
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PCT/NL2022/050538
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French (fr)
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Olaf Torben HEIDENREICH
Laura Elise SWART
Hasan ISSA
Milad RASOULI
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Prinses Máxima Centrum Voor Kinderoncologie B.V.
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Publication of WO2023048572A1 publication Critical patent/WO2023048572A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
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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
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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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
    • C12N15/87—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
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0368—Animal model for inflammation
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
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    • 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
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
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    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • the present invention is related to the field of targeted drug delivery. More specifically, it discloses a lipid nanoparticle loaded with a therapeutic molecule comprising a ligand capable of binding a receptor on its outer surface.
  • a RUNX1/ETO fusion gene encodes a novel transcription factor that blocks differentiation along the myeloid lineage.
  • Leukemia is generally scored as either lymphocytic or myeloid, and as acute or chronic, whereby lymphocytic leukemias develop from T cells, B cells, or natural killer (NK) cells, and myeloid leukemias develop from granulocytes and monocytes.
  • lymphocytic leukemias develop from T cells, B cells, or natural killer (NK) cells
  • myeloid leukemias develop from granulocytes and monocytes.
  • AML acute myeloid leukemia
  • CLL chronic lymphocytic leukemia
  • Treatment for leukemia often includes chemotherapy and stem cell (bone marrow) transplantation.
  • specific kinase inhibitors such as imatinib, dasatinib, and nilotinib may be used in combination with other chemotherapy drugs.
  • specific inhibitors of Bruton's tyrosine kinase such as ibrutinib
  • inhibitors of the anti- apop totic protein Bcl-2 such as venetoclax
  • phosphoinositide 3-kinase inhibitors such as idelalisib have recently been approved for CLL, amongst other types of leukemias.
  • Immunotherapy including the use of checkpoint inhibitors and chimeric antigen receptor (CAR) T cell therapy has produced encouraging results over the past years in people with certain types of leukemia.
  • the main treatment for most childhood leukemias is chemotherapy.
  • high- dose chemotherapy may be given along with a stem cell transplant.
  • Other treatments might also be used in special circumstances.
  • the long-term and late effects of such treatment on childhood leukemia include cognitive effects, and effects in physical and psychological development. There is thus a need to develop therapies that effectively treat pediatric acute leukemias with reduced side effects.
  • lipid nanoparticle loaded with a therapeutic molecule, said lipid nanoparticle comprising a ligand on its outer surface capable of binding to the Very Late Antigen 4 (VLA-4) receptor, wherein the ligand comprises the structure according to formula (II):
  • a lipid nanoparticle according to the invention is taken up more rapidly by VLA-4 expressing cells, such as hematopoietic cells, compared to a comparable lipid nanoparticle lacking a ligand on its outer surface.
  • VLA-4 expressing cells such as hematopoietic cells
  • the inventors have thereby created a drug delivery approach specifically targeting cells expressing the VLA-4 receptors, thereby achieving increased efficacy compared to a comparable untargeted approach. This provides a promising approach towards treatment of diseases or disorders of cells expressing VLA-4 receptors, e.g. leukemia, in particular AML.
  • Said therapeutic molecule is preferably a small interfering ribonucleic acid (siRNA), preferably siRNA that inhibits functional expression of a RUNX1/ETO fusion gene.
  • siRNA small interfering ribonucleic acid
  • Said siRNA preferably comprises a first RNA strand complementary to an mRNA transcript of the RUNX/ETO gene and, optionally, a second RNA strand complementary to the first RNA strand, wherein said first RNA strand is preferably represented by the sequence as shown in SEQ ID NO:1 and wherein said second RNA strand, when present, is represented by the sequence as shown in SEQ ID NO:2.
  • Said siRNA is preferably a modified siRNA more preferably comprises at least 21 nucleotides and which siRNA comprises one or more of the following modifications:
  • sequence of SEQ ID NO:1 comprises a C F on position 1, a C F on position 2, an U F on position 3, and a C F on position 4, wherein C F and U F represent nucleotides wherein the hydroxyl group on the 2-position of the ribose moiety is substituted for a fluoride;
  • sequence of SEQ ID NO:1 comprises dA on position 17, dG on position 18, dA on position 19, dT on position 20 and dT on position 21, wherein dA dG and dT represent nucleotides wherein the hydroxyl group on the 2-position of the ribose moiety is substituted for a hydrogen;
  • the sequence of SEQ ID NO:1 comprises an Uo Me on position 9, a Co Me on position 10, an Uo Me on position 12, an Co Me on position 14 and a Uo Me on position 15, wherein Co Me and UOME represent nucleotides wherein the hydroxyl group on the 2-position of the ribose moiety is substituted for a methoxy group;
  • sequence of SEQ ID NO:1 comprises a TPS on position 20, and/or wherein the sequence of SEQ ID NO:2 comprises a TPS on position 20, wherein TPS represents a nucleotide wherein the phosphodiester group on the 3-position of the ribose moiety is substituted for a phosphorothioate;
  • sequence of SEQ ID NO: 1 comprises a T on position 20 and a T on position 21 and/or wherein the sequence of SEQ ID NO:2 comprises a T on position 20 and a T on position 21.
  • said lipid nanoparticle comprises at least one cationic or ionizable lipid, at least one helper lipid and preferably at least one poly(ethylene)glycol (PEG)-lipid.
  • PEG poly(ethylene)glycol
  • said at least one cationic or ionizable lipid preferably comprises (6Z,9Z,28Z,31Z)-heptatriacont-6,9,28,31-tetraene-19-yl 4- (dimethylamino)butanoate (Dlin-MC3-DMA), said at least one or more helper lipids is preferably selected from distearoylphosphatidylcholine (DSPC), cholesterol and sphingomyelin and said lipid nanoparticle preferably comprises at least one PEG- lipid selected from l,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol (DMG- PEG)) and a l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [amino(polyethylene glycol)] salt (DSPE-PEG), preferably DMG-PEG 1000-6000 or DSPE-PEG 1000-6000 , more preferably D
  • the lipid nanoparticle according to the invention preferably has a hydrodynamic diameter of about 75 to about 130 nm, preferably about 78 to about 90 nm and/or a polydispersity index of about 0.15 to about 0.25, preferably about 0.18 to about 0.22, as determinable with a Zetasizer Nano (Malvern Instruments, UK).
  • Said lipid nanoparticle further preferably comprises a detectable label, more preferably on its outer surface.
  • the invention further relates to a lipid nanoparticle according to the invention for use in a method of treatment by therapy, preferably for use in a method of acute myeloid leukemia.
  • the invention further pertains to a pharmaceutical composition
  • a pharmaceutical composition comprising a lipid nanoparticle according to the invention and a pharmaceutically acceptable carrier.
  • the invention further provides a method of coupling a ligand to a lipid, comprising
  • lipid functionalized with a strained alkyne moiety preferably a dibenzocyclooctyne (DBCO) moiety
  • the invention further pertains to a method for preparing a lipid nanoparticle according to the invention, comprising
  • Said at least one helper lipid preferably comprises DSPE-PEG, more preferably DSPE-PEG (2000) .
  • Figure 1 A reaction scheme showing the copper-free click reaction between a ligand functionalized with a terminal azide and a lipid functionalized with a strained alkyne moiety to form a ligand coupled to a lipid via a triazole linkage;
  • Figure 2 Cryo-electron microscopy images showing lipid nanoparticles loaded with the therapeutic molecule siRE (right) and loaded with a control siRNA molecule (left).
  • Figure 3 Uptake of a Cy3-labelled lipid nanoparticle loaded with an siRE molecule comprising a ligand according to Formula (II) in Kasumi-1 cells is shown, compared to uptake of a comparable lipid nanoparticle lacking a ligand on its outer surface.
  • Figure 4 A: Graph showing uptake of a Cy3-labelled lipid nanoparticle loaded with siRE RNA and comprising a ligand according to Formula (II) on its outer surface in Kasumi-1 cells, compared to a comparable Cy3-labelled lipid nanoparticle loaded with siMM RNA and comprising a ligand according to Formula (II) on its outer surface, and comparable Cy3-labelled lipid nanoparticles lacking a ligand on their outer surface.
  • Figure 5 Bar graph showing reduction of RUNX1/ETO expression over time in Kasumi-1 and SKNO-1 cells as a result of administration of a lipid nanoparticle loaded with siRE RNA (striped bar) compared to a control lipid nanoparticle loaded with siMM RNA (black bar).
  • Figure 6 A: Bar graph showing reduced expression of RUNX1/ETO fusion gene in t(8;21) AML cells of a lipid nanoparticle loaded with an siRE molecule, comprising DMG-PEG (2000) (white bar) or DSPE-PEG (2000) (striped bar) in the lipid outer layer, compared to a lipid nanoparticle loaded with siMM (control, black bar).
  • B Bar graph showing number of colony formation of cells after administration of a lipid nanoparticle loaded with siRE (striped bar) and loaded with siMM (black bar).
  • Figure 7 A: Graph showing increased expression of Lysosomal Protein Transmembrane 5 (LAPTM5) in (t8;21) AML cells upon administration of lipid nanoparticles loaded with siRE (striped bar) compared to lipid nanoparticles loaded with siMM (black bar).
  • LAPTM5 Lysosomal Protein Transmembrane 5
  • CEBPA CCAAT Enhancer Binding Protein Alpha
  • Figure 8 A: Graph showing decreased expression of the adhesion molecule CD34 in (t8;21) AML cells upon administration of lipid nanoparticles loaded with siRE (striped bar) compared to lipid nanoparticles loaded with siMM (black bar).
  • B Graph showing decreased expression of Angiopoietin-1 (ANGPT1) in (t8;21) AML cells upon administration of lipid nanoparticles loaded with siRE (striped bar) compared to lipid nanoparticles loaded with siMM (black bar).
  • ANGPT1 Angiopoietin-1
  • Cyclin D2 Cyclin D2 (CCND2) in (t8;21) AML cells upon administration of lipid nanoparticles loaded with siRE (striped bars) compared to lipid nanoparticles loaded with siMM (black bars).
  • Figure 9 A: Bar graph showing reduced expression of the RUNX1/ET0 fusion gene over time after administration of a lipid nanoparticle loaded with an siRE molecule and comprising a ligand according to Formula (II) on its outer surface (white bar), compared to expression of the RUNX1/ETO fusion gene upon administration of a comparable lipid nanoparticle loaded with an siMM molecule (black bar) and comparable lipid nanoparticles lacking a ligand on its outer surface (siRE: striped bar; siMM: grey bar).
  • B Bar graph showing reduced expression of the RUNX1/ETO fusion gene over time after sequential administration (indicated with black arrow) of a lipid nanoparticle loaded with an siRE molecule and comprising a ligand according to Formula (II) on its outer surface (white bar), compared to expression of the RUNX1/ETO fusion gene upon administration of a comparable lipid nanoparticle loaded with an siMM molecule (grey bar), and expression of the RUNX1/ETO fusion gene upon administration of comparable lipid nanoparticles loaded with an siRE or siMM molecule and lacking a ligand on its outer surface (siRE: striped bar; siMM: black bar) in absence of MSCs.
  • siRE striped bar
  • siMM black bar
  • C Bar graph showing reduced expression of the RUNX1/ETO fusion gene over time after sequential administration (indicated with black arrow) of a lipid nanoparticle loaded with an siRE molecule and comprising a ligand according to Formula (II) on its outer surface (white bar), compared to expression of the RUNX1/ETO fusion gene upon administration of a comparable lipid nanoparticle loaded with an siMM molecule (grey bar), and expression of the RUNX1/ETO fusion gene upon administration of comparable lipid nanoparticles loaded with an siRE or siMM molecule and lacking a ligand on its outer surface (siRE: striped bar; siMM: black bar) in presence of MSCs.
  • siRE striped bar
  • siMM black bar
  • FIG. 10 Western blot analysis showing two-fold reduction of RUNX1/ETO protein three and six days after addition of a lipid nanoparticle loaded with siRE RNA comprising a ligand according to Formula (II) on its outer surface, both in presence and absence of MSCs after three (upper part) and six days (lower part).
  • T- LNPsiRE targeted LNPsiRE containing LDV ligand.
  • Figure 11 FACS images showing a shift of subpopulations from immature CD34+ cells to more mature CD34- CD 15+ cells indicating strong induction of myeloid differentiation upon administration of a lipid nanoparticle according to the invention, loaded with siRE molecule (lower graphs) compared to a comparable lipid nanoparticle loaded with siMM (control; upper part), both in absence (left) and presence (right) of MSCs.
  • Figure 12 A: Dosing scheme of a lipid nanoparticle according to the invention administered in a mouse model.
  • Figure 13 Graph showing uptake of a Cy3-labelled lipid nanoparticle loaded with siRE RNA and comprising a ligand according to Formula (II) on its outer surface in primary AML cells from three different patients (A B and C), compared to a Cy3-labelled lipid nanoparticle loaded with siMM RNA and comprising a ligand according to Formula (II) on its outer surface, and comparable Cy3-labelled lipid nanoparticles lacking a ligand on its outer surface.
  • Figure 14 A: Graph showing reduced expression of RUNX1/ETO fusion gene in t(8;21) primary cells after sequential administration (indicated with a black arrow) of a lipid nanoparticle loaded with an siRE molecule (white bar) comprising a ligand according to Formula (II) on its outer surface, compared to a lipid nanoparticle loaded with siMM (control, grey bar) comprising a ligand according to Formula (II) on its outer surface.
  • B Western blot analysis showing reduction of RUNX1/ETO protein in primary cells twelve days after sequential addition (6 days after the last dose) of a lipid nanoparticle loaded with siRE RNA comprising a ligand according to Formula (II) on its outer surface.
  • Figure 15 A: t-SNE plot showing a shift of subpopulations from immature CD34+ cells towards CD34- cells as determined by a multiparameter flow analysis upon administration of a lipid nanoparticle according to the invention, loaded with siRE molecule (right) compared to a comparable lipid nanoparticle loaded with siMM (control; left) in patient-derived material.
  • Figure 16 Bar graph showing reduced expression of RUNX1/ETO fusion gene in t(8;21) patient-derived cells of a lipid nanoparticle loaded with an siRE molecule, comprising DMG-PEG (2000) (white bar) or sphingomyelin (20% molar ratio) and cholesterol (20% molar ratio) (white bar, chess blocks) in the lipid outer layer, compared to a lipid nanoparticle loaded with siMM, comprising DMG-PEG (2000) (grey bar) or sphingomyelin (20% molar ratio) and cholesterol (20% molar ratio) (grey bar, black line).
  • Figure 17 A: siRNA targeting the unique breakpoint of the RUNX1/ETO t(8;21) fusion transcript (siRE) and the mismatch control (siMM) generated by swapping two nucleotides in the sequence.
  • B To improve the stability the siRNAs were modified by introducing 2’-deoxy- (2’-H), 2’-fluoro (2’-F) and 2’-methoxy (2’- OMe) ribose modifications and 3’-terminal phosphorothioate (PS) linkages.
  • B To improve the stability the siRNAs were modified by introducing 2’-deoxy- (2’-H), 2’-fluoro (2’-F) and 2’-methoxy (2’- OMe) ribose modifications and 3’-terminal
  • Figure 18 A: Graph showing the cell cycle profile of t(8;21) cells on day 6 upon administration of a lipid nanoparticle according to the invention, loaded with siRE molecule compared to a comparable lipid nanoparticle loaded with siMM (control) in cell lines.
  • the LNPsiRE-mod treated cells accumulate more in the G0/G1 phase and proliferate less as indicated by the decrease in cells in the S- phase.
  • B Bar graph showing the senescent t(8;21) cells on day 6 stained by senescence-associated beta-galactosidase staining upon administration of a lipid nanoparticle according to the invention, loaded with siRE molecule (right) compared to a comparable lipid nanoparticle loaded with siMM (control; left) in cell lines.
  • C Bar graph showing the colony formation units in first (left) and second (right) platings upon administration of a lipid nanoparticle according to the invention, loaded with siRE molecule compared to a comparable lipid nanoparticle loaded with siMM (control) in t(8;21) cell lines.
  • Figure 19 A: Figure (left) showing the distribution of labelled LNPs (LNP/NIR) (top) or control mice treated with free NIR dye in PBS (bottom) as measured by fluorescence. Figure (right) showing the fluorescence of the indicated organs of mice treated with LNP/NIR (top) or control mice (bottom).
  • B Figures (left) showing the bioluminescence signal linked to tumour cells (left), the fluorescence signal linked to LNPs and overlay (overlay of bioluminescence and fluorescence signals) in mice.
  • Figure (right) showing the fluorescence and bioluminescence signal co-localizing in harvested tumours from mice.
  • Figure 20 A: Western blot analysis showing reduction of RUNX1/ETO protein in cells harvested from mice after sequential addition of a lipid nanoparticle loaded with siRE RNA or siMM RNA. Each lane represents one animal. * denotes two tumours from the same animal.
  • B Bar graph showing the senescent t(8;21) cells stained by senescence-associated beta-galactosidase staining upon administration of a lipid nanoparticle according to the invention, loaded with siRE molecule (right) compared to a comparable lipid nanoparticle loaded with siMM (control; left) in cells harvested from mice.
  • C Bar graph showing the colony formation units upon administration of a lipid nanoparticle according to the invention, loaded with siRE molecule (right) compared to a comparable lipid nanoparticle loaded with siMM (control; left) in cells harvested from mice.
  • Figure 21 A: Graph showing the quantification of bioluminescence signal in mice upon administration of a lipid nanoparticle loaded with siRE molecule (round) compared to a comparable lipid nanoparticle loaded with siMM (control; square).
  • B Kaplan-Meier graph showing the survival curves of mice after sequential administration of lipid nanoparticles loaded with siRE molecule compared to lipid nanoparticles loaded with siMM (control).
  • C Graph showing the quantification of bioluminescence signal in mice that were transplanted with cells harvested from mice treated with a lipid nanoparticle loaded with siRE molecule (round) compared to a lipid nanoparticle loaded with siMM (control; square).
  • D Kaplan-Meier graph showing the survival curves of mice after transplantation of cells harvested from mice that were treated sequentially with a lipid nanoparticle loaded with siRE molecule compared to a comparable lipid nanoparticle loaded with siMM (control).
  • E Western blot analysis showing the RUNX1/ETO protein expression in cells harvested from the recipients upon administration of a lipid nanoparticle loaded with siRE molecule compared to a lipid nanoparticle loaded with siMM molecule (control).
  • Figure 22 A: siRNA targeting the unique breakpoint of the MLL/AF4 t(4; 11) fusion transcript.
  • siRNAs were modified by introducing 2’-deoxy- (2’-H), 2’-fluoro (2’-F) and 2’-methoxy (2’-OMe) ribose modifications and 3’-terminal phosphorothioate (PS) linkages.
  • B Bar graph showing reduction of expression of MLL/AF4 and its target gene HOXA7 over time (1 day, black columns; 3 days, grey columns; 6 days, white columns) in SEM cells as a result of administration of a lipid nanoparticle loaded with siMAG or siMAG- M2 RNA compared to a control lipid nanoparticles loaded with siMM or siMM-mod RNA, respectively.
  • C Graph showing proliferation of SEM cells over time after administration of a lipid nanoparticle loaded with siMA6-M2 (diamond), compared to lipid nanoparticles loaded with siMM (control; square), siMM-mod (modified control, triangle down), siMAG (unmodified, triangle up) and a mock sample (circles).
  • lipid nanoparticle refers to a particle comprising a lipid outer layer at least partly encapsulating a lipid-therapeutic molecule complex. Said particle preferably is substantially spherical.
  • the lipid outer layer typically comprises at least an ionizable or charged lipid comprising respectively an ionizable or charged portion and a hydrophobic portion.
  • the ionizable portion is typically positively charged at low pH, i.e. a pH of below 5, and neutral at physiological pH.
  • the charged portion is typically positively charged at physiological pH.
  • the ionizable or cationic lipid further comprises a hydrophobic portion, such as an aliphatic portion of a fatty acid.
  • the charged or ionizable portion is generally projected outwards, or, if present, projected towards a negatively charged therapeutic molecule, whereas the hydrophobic portion is generally projected inwards, or, if present, projected towards a hydrophobic portion of a therapeutic molecule.
  • the ionizable or cationic portion is preferably capable of interacting with a liquid (polar) medium, such as water or, if present, with the negatively charged therapeutic molecule, and the hydrophobic portion is shielded therefrom.
  • the lipid outer layer typically comprises one or more helper lipids, such as cholesterol, a phospholipid or a sphingolipid and may comprise one or more PEG-lipids.
  • the lipid outer layer may further comprise a lipid monolayer, comprising a single layer of lipids, or a lipid bilayer wherein the outer layer is formed of two lipid layers.
  • a lipid monolayer comprising a single layer of lipids
  • a lipid bilayer wherein the outer layer is formed of two lipid layers.
  • the hydrophobic portions of both layers are preferably facing each other.
  • PEG-lipid refers to a lipid comprising at least one poly(ethylene) glycol (PEG) moiety.
  • a polyethylene glycol moiety has a molecular formula of C 2n H 4n+2 O n+1 , wherein n is an integer typically varying from 5 to 100, preferably from 10 to 70, more preferably from 20 to 60, in particular around 40-50.
  • the molecular weight of a PEG moiety may be calculated using the formula 44.05n+ 18.02 g/mol.
  • a PEG molecule is referred to as PEG (average molecular weight) .
  • a PEG (2000) has an average molecular weight of about 1900-2200 Da.
  • a PEG-lipid may comprise one or more PEG moieties per lipid molecule, typically a PEG-lipid comprises between one and five PEG-moieties per lipid molecule, in particular around one PEG-moiety per lipid molecule.
  • therapeutic molecule refers to any molecule that has a pharmaceutical benefit in a subject in need thereof, when provided at a therapeutically effective amount.
  • small interfering ribonucleic acid refers to a short single or double stranded ribonucleic acid molecule that is capable of base pairing with a messenger RNA (mRNA) transcript of a target gene.
  • mRNA messenger RNA
  • mRNA interference a process commonly referred to in the art as RNA interference.
  • translation of the mRNA transcript into a functional protein is inhibited or prevented, thereby effectively inhibiting or preventing functional expression of said target gene.
  • the length of an siRNA molecule typically is between 18 to 27 nucleotides.
  • inhibitor in the context of siRNA refers to the process of reducing the number of mRNA transcripts in a cell compared to the wildtype cell (wherein no siRNA is present), so that a reduced number of proteins will be generated in that cell. Generally, inhibition refers to a reduction of 50 mol% or more of protein or RNA that is produced in a cell, when compared to a wild-type cell wherein no siRNA is present.
  • target gene refers to a section located on a chromosome comprising a protein-coding or an RNA-coding sequence.
  • a target gene in the context of the present invention is involved in the onset or development of a disease or medical condition.
  • fusion gene refers to a combination of two or more genes which are normally located at distinct locations on a chromosome or on different chromosomes, but are joined as a result of a chromosomal aberration.
  • chromosomal aberration refers to an abnormality in one or more chromosomes, typically leading to a structural change or defect, such as translocation of a chromosome fragment.
  • the chromosomal aberration may be genetic, or may be caused by external factors.
  • nucleic acid comprises the requisite information to guide translation of the nucleotide sequence into a specified protein.
  • the information by which a protein is encoded is specified by the use of codons.
  • a nucleic acid encoding a protein may comprise a non-translated sequence, e.g. an intron, interspersed with translated regions of the nucleic acid or may lack such an intervening non-translated sequence.
  • protein or “polypeptide” refers to a chain of amino acids arranged in a specific order determined by the coding sequence in a polynucleotide encoding the polypeptide.
  • RUNX1 refers to a gene on chromosome 21q22 that encodes a transcription factor.
  • the gene is characterized by HUGO Gene Nomenclature Committee accession number 10471; and by Ensembl accession number ENSG00000159216.
  • the encoded protein is characterized by Uniprot accession number Q01196.
  • ETO or “RUNX1 Partner Transcriptional Co- Repressor 1” (RUNX1T1) refers to a gene on chromosome 8q21 that encodes a transcriptional corepressor.
  • the gene is characterized by HUGO Gene Nomenclature Committee accession number 1535; and by Ensembl accession number ENSG00000079102.
  • the encode protein is characterized by Uniprot accession number Q06455.
  • RUNX1/ETO fusion gene refers to a chromosome (8;21)(q21;q22) translocation, which is one of the most frequent karyotypic abnormalities in acute myeloid leukemia.
  • the translocation produces a chimeric gene made up of the 5'-region of the RUNX1 gene fused to the 3'-region of the ETO gene.
  • the chimeric protein is thought to associate with the nuclear corepressor/histone deacetylase complex to block hematopoietic differentiation.
  • RUNX1/ETO polypeptide refers to a polypeptide that is encoded by a RUNX1/ETO fusion gene.
  • the term “functional expression”, in relation to a gene refers to the expression of a gene product, such as a protein product, that is able to completely fulfil its function.
  • a reduction of the functional expression of a gene may be accomplished by reduced expression of the encoded product such as protein product, by expression of a product, such as protein product, with reduced functional activity, or both reduced expression of a product with reduced activity.
  • a reduction of functional expression is preferably accomplished by reduced expression of a gene product.
  • complementary refers to a strand of RNA or DNA that is capable of hybridizing with another strand of RNA or DNA that it is complementary to, thereby forming a duplex structure.
  • Said hybridizing typically occurs through the formation of one or more Watson-Crick base pairs. Not all nucleotides present in complementary RNA or DNA strands are required to form base pairs.
  • the minimal number of base pairs is the number required to form a duplex structure. As is generally known, the formation of a duplex structure depends on the environmental conditions such as the temperature and/or salt concentration. Generally, at least 12 base pairs are required to form a duplex structure, in particular at least 15 base pairs.
  • the maximum number of base pairs formed in complementary strands is the total number of nucleotides present in a strand.
  • RNA or polypeptide refers to the transcription or translation of a nucleic acid into a functional RNA or polypeptide.
  • functional refers to the ability of said RNA or polypeptide to perform its natural function, e.g. as observed in the wildtype.
  • percentage sequence identity refers to the percentage of sequence overlap when comparing a sequence with a reference sequence, i.e. bait sequence. For optimal alignment of the two sequences, the two sequences are preferably compared over the full length of the reference sequence.
  • a polypeptide or polynucleotide is compared to a reference sequence, i.e. a bait sequence
  • the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise an addition or a deletion (i.e. a gap) as compared to the reference sequence (which does not comprise an addition or a deletion).
  • the percentage is calculated by determining the number of positions at which an identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.
  • a nucleotide is meant comprising an unmodified ribose moiety attached to a nucleobase indicated with the letters A (adenine), C (cytosine), G (guanine), thymine (T), or uracil (U).
  • a “T” on a specific position in a sequence of siRNA indicates the presence of a nucleotide comprising a ribose moiety attached to a thymine, also referred to in the art as 5-methyluridine or ribothymidine.
  • a “T” on position 20 of the sequence of SEQ ID NO:1 indicates the presence of a ribothymidine, on position 20 of SEQ ID NO:1.
  • nucleotide comprising a modified ribose moiety attached to any nucleobase (X).
  • T refers to a nucleotide comprising a modified ribose attached to a thymine, wherein the hydroxyl group on the 2- position of the ribose moiety is substituted for a hydrogen
  • T PS refers to a nucleotide attached to a thymine, comprising a modified ribose, wherein the phosphodiester group on the 3-position of the ribose moiety is substituted for a phosphorothioate
  • Co Me refers to a nucleotide comprising a modified ribose attached to a cytosine, wherein the hydroxyl group on the 2-position of the ribose moiety is substituted for a methoxy-group
  • C F refers to a nucleotide comprising a modified ribose attached to a cytosine, wherein the hydroxyl group on the 2-position of the ribose moiety is
  • purifying' refers to at least partly separating a lipid nanoparticle from its environment. “Purifying” may encompass removing a water-miscible organic solvent from an aqueous solution comprising lipid nanoparticles. “Purifying'’ may further encompass removal of impurities and other substances until the solution comprising lipid nanoparticles is substantially pure. “Substantially pure” is used herein to indicate that the substance, e.g.
  • a lipid nanoparticle or a lipid nanoparticle loaded with a therapeutic molecule has a purity of at least 95%, more preferably at least 96%, at least 97%, at least 98%, at least 99% or at least 100% pure, based on the total weight of the substance, e.g. lipid nanoparticle or lipid nanoparticle loaded with a therapeutic molecule, as determined with standard analytical techniques known in the art on the date of filing.
  • the invention relates to a lipid nanoparticle loaded with a therapeutic molecule, which lipid nanoparticle comprises a ligand on its outer surface capable of binding a receptor present on an outer surface of a target cell.
  • a lipid nanoparticle according to the invention advantageously comprises a ligand on its outer surface capable of binding a receptor present on an outer surface of a target cell.
  • a ligand allows the targeting of specific cell types, expressing a receptor on an outer surface of said cell, and thus provides the possibility to target specific cell types (expressing said receptor on an outer surface), whereas other cell types (not expressing, or less abundantly expressing said receptor on an outer surface) are not or less targeted.
  • Such an approach advantageously allows more rapid and more efficient delivery of a therapeutic molecule to a target cell, because a therapeutic molecule may be specifically released in said target cell expressing said receptor on an outer surface. Further, such an approach may result in fewer side effects, because the therapeutic molecule is released to fewer or no cells that do not, or to a lesser extent, express a receptor on its outer surface.
  • any target cell may be suitable in the context of the present invention, but preferably, the target cell is a cell of the hematopoietic lineage.
  • the target cell is a cancer cell, more preferably an acute myeloid leukemia (AML) cell, such as a t(8;21) AML cell, including a Kasumi- 1 cell and a SKNO-1 cell.
  • AML acute myeloid leukemia
  • Said target cell preferably comprises a unique RNA molecule that can be targeted with a negatively charged therapeutic molecule such as an oligonucleotide, in particular short interfering RNA (siRNA), that can specifically hybridize with said unique RNA molecule, thereby inducing RNA interference.
  • a negatively charged therapeutic molecule such as an oligonucleotide, in particular short interfering RNA (siRNA), that can specifically hybridize with said unique RNA molecule, thereby inducing RNA interference.
  • siRNA short interfering RNA
  • a lipid nanoparticle according to the invention preferably comprises one or more, preferably two or more, in particular three or more, or even four or more lipid molecules that are assembled together to form a lipid nanoparticle.
  • said lipid nanoparticle comprises at least one cationic or ionizable lipid.
  • a cationic or ionizable lipid is preferably present in a lipid nanoparticle according to the invention, if a lipid nanoparticle according to the invention is loaded with a negatively charged therapeutic molecule, preferably an oligonucleotide, in particular short interfering RNA (siRNA).
  • a cationic lipid was found to promote encapsulation of said negatively charged therapeutic molecule into said lipid nanoparticle, by forming a lipid-siRNA complex and stabilize a lipid nanoparticle loaded with said negatively charged therapeutic molecule.
  • a cationic lipid is a lipid that carries a net positive charge at physiological pH or lower, e.g. at pH of 7.5 or lower, such as 7.0 or lower.
  • cationic lipids examples include (N-[l-(2,3-Dioleoyloxy)propyl]-N,N,N- trimethylammonium (DOTAP), l,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), dioctadecylamidoglycylspermine (DOGS), 2,3-dioleyloxy-N-[2- (sperminecarboxamido)ethyl]-N,N-dimethyl -1-propanaminium trifluoroacetate (DOSPA), 3 ⁇ - [N-(N',N'-dimethylamino-ethane)carbamoyl] -cholesterol (DC-Chol), N- [3-[2-(l,3-dioleoyloxy)propoxy-carbonyl]propyl]-N,N,N-trimethylammonium iodide (YKS-220), lipofectamine®
  • An ionizable lipid is a lipid that is positively charged at a pH of 6.5 or lower, such as at a pH of 5 or lower, but is typically neutral at physiological pH. Inclusion of an ionizable lipid into a lipid nanoparticle according to the invention typically facilitates loading of lipid nanoparticles with negatively charged therapeutic molecules at acidic pH. Further, inclusion of an ionizable lipid in a lipid nanoparticle according to the invention may restore neutral or even negative surface charge of a lipid nanoparticle at physiological pH, which is associated with increased circulation times and lower aggregation (Zhao et al. 2011. Int J Nanomedicine 3:198).
  • ionizable lipids examples include (6Z,9Z,28Z,31Z)-heptatriacont-6,9,28,31- tetraene- 19-yl 4-(dimethylamino)butanoate (Dlin-MC3-DMA) or 2,2-dilinoleyl-4- dimethylaminoethyl- [ 1, 3] -dioxolane (DLin-KC2-DMA) .
  • a lipid nanoparticle according to the invention comprises at least the ionizable lipid Dlin-MC3-DMA.
  • a lipid nanoparticle according to the invention further preferably comprises one or more helper lipids.
  • helper lipids typically improve one or more physical properties of a lipid nanoparticle, such as, for example the stability of a lipid nanoparticle, in particular the in vivo stability of a lipid nanoparticle, intracellular delivery of a lipid nanoparticle, or release of a therapeutic molecule from a lipid nanoparticle into a target cell.
  • helper lipids include cholesterol or a derivative thereof, ⁇ - sitosterol (Sito), fucosterol (Fuco), campesterol (Camp), stigmastanol (Stig), dioleoylphosphatidylethanolamine (DOPE), phosphatidylcholine, such as distearoylphosphatidylcholine (DSPC), l,2-dipalmitoyl-sn-glycero-3- phosphaoethanolamine, 1,2-dimyristoyl-rac-glycerol and sphingomyelin.
  • derivatives of cholesterol include cholestenol, cholestanone, cholestenone, coprostanol, cholesteryl- 2’-hydroxylethyl ether, cholesteryl-4’- hydroxybutyl ether, and mixtures thereof.
  • a lipid nanoparticle according to the invention comprises one or more, preferably two or more, or all of cholesterol or a derivative thereof, sphingomyelin and DSPC.
  • a lipid nanoparticle according to the invention comprises a PEG- lipid.
  • a lipid nanoparticle comprising a PEG-lipid may improve colloidal stability of a lipid nanoparticle in vitro and may further improve circulation time of the lipid nanoparticle in vivo (Klibanov et al, 1990. FEES Lett. 268:235-7).
  • any PEG-lipid may be used, but preferably a PEG-lipid is used with a molecular weight of PEG in the range of 350 Da to 6000 Da, preferably between 550 Da and 5000 Da, more preferably between about 750 Da and about 4000 Da, even more preferably between about 1000 Da and about 3000 Da, in particular about 2000 Da.
  • Preferred PEG-lipids in a lipid nanoparticle according to the invention is DMG-PEG or DSPE-PEG, more preferably DMG-PEG 1000-6000 or DSPE-PEG 1000-6000 , most preferably DMG-PEG 2000 or DSPE-PEG 2000 .
  • a lipid nanoparticle according to the invention preferably comprises at least the ionizable lipid (6Z,9Z,28Z,31Z)-heptatriacont-6,9,28,31-tetraene-19-yl 4- (dimethylamino)butanoate (Dlin-MC3-DMA), at least one helper lipid selected from the group consisting of distearoylphosphatidylcholine (DSPC), cholesterol and sphingomyelin and at least one PEG-lipid selected from the group consisting of DMG-PEG and DSPE-PEG, preferably DMG-PEG 1000-6000 or DSPE-PEG 1000-6000 , more preferably DMG-PEG (2000) and DSPE-PEG (2000) .
  • DMG-PEG and DSPE-PEG preferably DMG-PEG 1000-6000 or DSPE-PEG 1000-6000 , more preferably DMG-PEG (2000) and DSPE-PEG (2000) .
  • a lipid nanoparticle according to the invention comprises the lipids Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG, preferably DMG-PEG 1000-6000 , more preferably DMG-PEG 2000 .
  • the molar ratio Dlin-MC3- DMA:DSPC:cholesterol:DMG-PEG is between about 1:0.1:0.5:0.02 and about 1:0.4:0.9:0.05, more preferably between about 1:0.2:0.7:0.03 and about 1:0.3:0.8:0.04.
  • a lipid nanoparticle according to the invention comprises the lipids Dlin-MC3-DMA, DSPC, cholesterol and sphingomyelin.
  • An advantage of such a lipid nanoparticle is that the presence of a PEG-lipid is not required in the lipid nanoparticle according to the invention.
  • PEG- lipids may disadvantageously induce an immune response in a subject it is administered to. Therefore, it may be advantageous to provide a lipid nanoparticle that is essentially free of, or comprises a low content of a PEG-lipid, such as DSPE- PEG or DMG-PEG.
  • the ratio Dlin-MC3-DMA:DSPC:cholesterol:spingomyelin is between about l:0.1:0.2:0.2 and about l:0.4:0.5:0.5, more preferably between about l:0.2:0.3:0.3 and about l:0.3:0.4:0.4.
  • a lipid nanoparticle according to the invention comprises the lipids Dlin-MC3-DMA, DSPC, cholesterol, sphingomyelin and a PEG-lipid preferably DMG-PEG or DSPE-PEG, more preferably DSPE- PEG 1000-6000 or DMG-PEG 1000-6000 , even more preferably DSPE-PEG 2000 or DMG- PEG 2000 .
  • lipid nanoparticle In such a lipid nanoparticle, the advantages of the presence of a PEG- lipid, such as a positive effect on the colloidal stability of a lipid nanoparticle in vitro and improvement of circulation time of the lipid nanoparticle in vivo are maintained, whilst the risk of inducing an undesired immune response is markedly reduced.
  • the ratio Dlin-MC3- DMA:DSPC:cholesterol:sphingomyelin:DSPE-PEG is between about 1:0.1:0.2:0.2:0.001 and about 1:0.4:0.5:0.5:0.005, more preferably between about 1:0.2:0.3:0.3:0.002 and about 1:0.3:0.4:0.4:0.003.
  • the therapeutic molecule in a lipid nanoparticle according to the invention may be any molecule having a pharmaceutical benefit when administered in a therapeutically effective amount to a subject in need thereof.
  • the therapeutic molecule may be a small molecule, a natural product, such as Paclitaxel, known under the tradename Taxol®, a biomolecule, such as an oligonucleotide, for example a small interfering ribonucleic acid (siRNA), an antisense oligonucleotide (ON), messenger RNA (mRNA), or microRNA (miRNA) or a polypeptide, such as an antibody, a conjugate, or a combination thereof.
  • a small interfering ribonucleic acid siRNA
  • mRNA messenger RNA
  • miRNA microRNA
  • said therapeutic molecule is a negatively charged molecule, more preferably an oligonucleotide, in particular an siRNA molecule.
  • Said siRNA molecule may be single stranded or double stranded RNA preferably double stranded RNA.
  • Said siRNA molecule preferably comprises a first RNA strand which is complementary to an mRNA transcript of a target gene, or a portion of a target gene.
  • said siRNA molecule comprises a second RNA strand which is complementary to said first RNA strand.
  • Said first RNA strand may efficiently mediate RNA interference, by hybridizing to an mRNA transcript of a target gene, or a part thereof. Upon hybridizing, said siRNA molecule may direct an enzyme complex to degrade said mRNA transcript, thereby preventing translation of said mRNA transcript into a polypeptide and thus effectively inhibiting functional expression of said target gene.
  • Said target gene preferably expresses an unique RNA molecule that can be targeted with a negatively charged therapeutic molecule such as an oligonucleotide, in particular short interfering RNA (siRNA), that can specifically hybridize with said unique RNA molecule, thereby inducing RNA interference.
  • a negatively charged therapeutic molecule such as an oligonucleotide, in particular short interfering RNA (siRNA), that can specifically hybridize with said unique RNA molecule, thereby inducing RNA interference.
  • Said unique RNA molecule may be the resultant of an alteration or rearrangement of a gene, for example a deletion or a duplication of a part of the RNA transcript of said gene, or the resultant of translocation resulting in a gene fusion by which part of a first gene becomes connected to a part of a second gene.
  • the sequences of the gene at around the deletion, duplication or gene fusion break points provide an unique RNA molecule that can be targeted with a negatively charged therapeutic
  • RNA molecules are provided by gene alterations including mutations in several epigenetic regulators such as ASXL1, DNMT3A EZH2, IDH1/2 and TET2.2, 3 DNMT3A IDH1/2 and TET2 (Brecqueville et al., 2011. Blood Cancer J 1, e33), IKZF1 (Stanulla et al., 2020. Blood 135: 252-260) and UBTF-TD (Umeda et al., 2022. Blood Cancer Discovery 2: 193-207).
  • epigenetic regulators such as ASXL1, DNMT3A EZH2, IDH1/2 and TET2.2, 3 DNMT3A IDH1/2 and TET2 (Brecqueville et al., 2011. Blood Cancer J 1, e33), IKZF1 (Stanulla et al., 2020. Blood 135: 252-260) and UBTF-TD (Umeda et al., 2022. Blood Cancer Discovery 2: 193-207).
  • RNA molecules are provided by the BCR/ABL fusion gene product, the MLL/AF4 fusion gene product, the RUNX1/ETO fusion gene product, the CTLA4/CD28 fusion gene product, the NRIP1/MIR99AHG fusion gene product, the LATS2/ZMYM2 fusion gene product, the ATP11A/ING1 fusion gene product, the MBP/SLC66A2 fusion gene product, or the PRDM16/SKI fusion gene product. See, for example, Chen et al., 2021. Blood Cancer J 11: 112 https://doi.org/10.1038/s41408-021-00504-5.
  • siRNA hybridizes to an mRNA transcript of the RUNX1/ETO gene, thereby inhibiting functional expression of the RUNX1/ETO fusion gene.
  • a RUNX1/ETO fusion gene By inhibiting function expression of a RUNX1/ETO fusion gene, translation of said fusion gene into the RUNX1/ETO protein is inhibited and/or silenced, which protein is believed to be involved in inhibition of hematopoietic differentiation.
  • inhibiting and/or silencing of functional expression of the RUNX1/ETO fusion gene may enhance hematopoietic differentiation.
  • inhibition or silencing of functional expression of the RUNX1/ETO fusion gene provides a fruitful approach towards treating leukemia, preferably amyloid myeloid leukemia (Agrawal et al., 2020. Leukemia 34: 630-634).
  • functional expression of the RUNX1/ETO gene is inhibited by at least 25%, more preferably at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, most preferably about 100%.
  • functional expression of the RUNX1/ETO gene is silenced, by inhibiting functional expression of the RUNX1/ETO gene such that its function is essentially lost.
  • functional expression of the RUNX1/ETO gene is silenced, by inhibiting functional expression of the RUNX1/ETO gene by more than 70%, preferably more than 80%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, in particular 100%.
  • said siRNA comprises a first RNA strand complementary to an mRNA transcript of the RUNX1/ETO fusion gene, and optionally a second RNA strand complementary to the first RNA strand.
  • said siRNA molecule may be any siRNA molecule that is capable of hybridizing with an mRNA transcript from the RUNX1/ETO fusion gene.
  • an siRNA molecule is capable of hybridizing to an mRNA transcript of an RUNX1/ETO fusion gene when at least 50% of the nucleotides, preferably at least 75% of the nucleotides, more preferably at least 85% of the nucleotides are capable of forming a base pair.
  • siRNA molecule wherein said first RNA strand is represented by the sequence 5’- CCUCGAAAUCGUACUGAGAUU -3’ (SEQ ID NO:1).
  • said second RNA strand is preferably represented by the sequence 5’- UCUCAGUACGAUUUCGAGGUU-3’ (SEQ ID NO:2).
  • lipid nanoparticle according to the invention comprising an siRNA molecule represented by SEQ ID NO:1 was effective in inhibiting functional expression of a RUNX1/ETO fusion gene when administered to (t8;21) AML cells.
  • lipid nanoparticle comprising an siRNA molecule represented by SEQ ID NO:1
  • LAPTM5 Lysosomal Protein Transmembrane 5
  • CEBPA CCAAT Enhancer Binding Protein Alpha
  • ANGPT1 Angiopoietin 1
  • CCND2 Cyclin D2
  • the invention also preferably relates to a lipid nanoparticle according to the invention, wherein the therapeutic molecule is an siRNA molecule comprising a first strand represented by the sequence as shown in SEQ ID NO:1, or a functional derivative having at least 70% sequence identity thereto, preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity thereto.
  • the therapeutic molecule is an siRNA molecule comprising a first strand represented by the sequence as shown in SEQ ID NO:1, or a functional derivative having at least 70% sequence identity thereto, preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity thereto.
  • said siRNA molecule comprises a second strand complementary to said first strand represented by the sequence as shown in SEQ ID NO:2 or a functional derivative having at least 70% sequence identity thereto, preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity thereto.
  • the siRNA molecule preferably comprises between about 18 to about 27 nucleotides, more preferably between 19 and 25 nucleotides, between 20 and 23 nucleotides, in particular 21 nucleotides.
  • the siRNA molecule comprises at least 18 nucleotides, more preferably at least 19 nucleotides, in particular at least 21 nucleotides.
  • the siRNA molecule comprises at most 27 nucleotides, more preferably at most 25 nucleotides, in particular at most 24 nucleotides.
  • Said siRNA molecule may be modified to increase thermal stability of the siRNA molecule, to increase resistance of the siRNA molecule against nuclease degradation, and/or to increase the resistance of the siRNA molecule towards 2’- OH-dependent RNases including the RNase A family.
  • Said modification may include one or more phosphorothioate (PS) bonds, substitution of one or more hydroxyl groups with one or more fluorides, substitution of one or more hydroxyl groups with one or more alkoxy groups including methoxy groups, substitution of one or more hydroxyl groups with hydrogen atoms and substitution of one or more uracil groups with one or more thymine groups.
  • PS phosphorothioate
  • said siRNA is modified, more preferably comprises one or more, more preferably two or more, even more preferably three or more, even more preferably four or more, in particular all five of a modification selected from the group consisting of a substitution of a hydroxyl group, preferably a 2-OH group of a ribose moiety of said siRNA with a methoxy group, a substitution of a hydroxyl group, preferably a 2-OH group of a ribose moiety of said siRNA with a fluoride or a substitution of a hydroxyl group, preferably a 2-OH group of a ribose moiety of said siRNA with a hydrogen, a substitution of a phosphodiester group, preferably a phosphodiester group on a 3-position of a ribose moiety of said siRNA, for a phosphorothioate group and a substitution of a uracil group with a thymine group.
  • a preferred siRNA molecule in a lipid nanoparticle according to the invention preferably comprises at least 21 nucleotides, wherein the siRNA molecule comprises one or more, preferably two or more, more preferably three or more, even more preferably four or more, in particular all five of the following modifications:
  • the sequence of SEQ ID NO:1 comprises a C F on position 1, aC F on position 2, anUr on position 3 and a C F on position 4, wherein C F and U F represent nucleotides wherein the hydroxyl group on the 2-position of the ribose moiety is substituted for a fluoride;
  • the sequence of SEQ ID NO:1 comprises dA on position 17, dG on position 18, dA on position 19, dT on position 20 and dT on position 21, wherein dA dG and dT represent nucleotides wherein the hydroxyl group on the 2-position of the ribose moiety is substituted for a hydrogen;
  • the sequence of SEQ ID NO:1 comprises an Uo Me on position 9, a Co Me on position 10, an Uo Me on position 12, an Co Me on position 14 and a Uo Me on position 15, wherein Co Me and UOME represent nucleotides wherein the hydroxyl group on the 2-position of the ribose moiety is substituted for a methoxy- group;
  • sequence of SEQ ID NO:1 comprises a TPS on position 20, and/or wherein the sequence of SEQ ID NO:2 comprises a TPS on position 20, wherein TPS represents a nucleotide wherein the phosphodiester group on the 3-position of the ribose moiety is substituted for a phosphorothioate.
  • sequence of SEQ ID NO: 1 comprises a T on position 20 and a T on position 21 and/or wherein the sequence of SEQ ID NO:2 comprises a T on position 20 and a T on position 21.
  • sequence of SEQ ID NO:1 comprises a dTps on position 20 and a dT on position 21 and/or the sequence of SEQ ID NO:2 comprises a dTps on position 20 and a dT on position 21.
  • siRNA molecule comprising modification a) renders the siRNA molecule more thermally stable.
  • modifications a) and b) independently or in combination increase the resistance of the siRNA molecule towards 2’-OH- dependent RNases including the RNase A family (Sipa et al., 2007. RNA 13: 1301- 16; Khvorova et al., 2003. Cell 115: 209-16).
  • modification c) increases the resistance of said siRNA molecule against nuclease degradation (Selvam et al., 2017. Chem Biol Drug Des 90: 665-678).
  • modification d) is envisaged to protect the siRNA molecule against exonuclease degradation (Stein, 1996. Chem Biol 3: 319- 23; Taylor et al., 1985. Nucleic Acids Res 13: 8765-85; Heidenreich et al., 1994. J Biol Chem 269: 2131-8).
  • the invention further relates to a lipid nanoparticle loaded with modified siRNA wherein said modified siRNA comprises a first RNA strand that is complementary to an mRNA transcript of the RUNX1/ETO gene and, optionally, a second RNA strand complementary to the first RNA strand, wherein said first RNA strand is preferably represented by the sequence as shown in SEQ ID NO:3 and wherein said second RNA strand, when present, is represented by the sequence as shown in SEQ ID NO:4.
  • a modified siRNA molecule in a lipid nanoparticle according to the invention comprises about 5% to about 100% of modified nucleotides, more preferably between 20% and 90%, between 30% and 85%, between 40% and 80%, between 50% and 75%, in particular between 60% and 75% of the nucleotides are modified nucleotides compared to a reference sequence, preferably compared to a sequence according to SEQ ID NO:1 and/or SEQ ID NO:2.
  • said modified nucleotides are nucleotides comprising a modified ribose moiety.
  • a modified siRNA molecule in a lipid nanoparticle according to the invention preferably about 15% to about 35% of the nucleotides are modified nucleotides, wherein a modified nucleotide comprises a modified ribose moiety, wherein the hydroxyl group on the 2-position of the ribose moiety is substituted for a fluoride. More preferably, about 20% to about 30% of the nucleotides are modified nucleotides, wherein the modified nucleotide comprises a modified ribose moiety, wherein the hydroxyl group on the 2-position of the ribose moiety is substituted for a fluoride.
  • a modified siRNA molecule in a lipid nanoparticle according to the invention preferably about 5% to about 30% of the nucleotides are modified nucleotides comprising a modified ribose moiety, wherein the hydroxyl group on the 2-position of the ribose moiety is substituted for a hydrogen. More preferably, about 10 to about 25% of the nucleotides are modified nucleotides, wherein the modified nucleotide comprises a modified ribose moiety wherein the hydroxyl group on the 2-position of the ribose moiety is substituted for a hydrogen.
  • a modified siRNA molecule in a lipid nanoparticle according to the invention preferably about 10% to about 30% of the nucleotides are modified nucleotides, wherein the modified nucleotide comprises a modified ribose moiety, wherein the hydroxyl group on the 2-position of the ribose moiety is substituted for a methoxy- group. More preferably, about 15% to about 25% of the nucleotides are modified nucleotides, wherein the modified nucleotide comprises a modified ribose moiety wherein the hydroxyl group on the 2-position of the ribose moiety is substituted for a methoxy- group.
  • a modified siRNA molecule in a lipid nanoparticle according to the invention about 3% to about 8% of the nucleotides are modified nucleotides, wherein the modified nucleotide comprises a modified ribose moiety, wherein the phosphodiester group on the 3-position of the ribose moiety is substituted for a phosphorothioate. More preferably, wherein about 4% to about 6% of the nucleotides are modified nucleotides, wherein the modified nucleotide comprises a modified ribose moiety wherein the phosphodiester group on the 3-position of the ribose moiety is substituted for a phosphorothioate.
  • a modified siRNA molecule in a lipid nanoparticle according to the invention about 4% to about 11% of the nucleotides are modified nucleotides, wherein said modified nucleotide is a nucleotide comprising a ribose moiety attached to a thymine nucleobase.
  • the molar ratio between lipid and therapeutic molecule is preferably between about 1:500 and about 1:150, more preferably between about 1:400 and about 1:250, in particular around 1:330.
  • the molar ratio between cationic or ionizable lipids and therapeutic molecule is preferably between 1:250 and 1: 75, more preferably between about 1:200 and about 1:130, in particular around 1:160.
  • the ratio between positively charged groups, preferably amine groups (typically provided by a cationic or ionizable lipid) and negatively charged groups, preferably phosphate groups (typically provided by a therapeutic molecule) is preferably between 6:1 and 2:1, more preferably between 5:1 and 3:1, in particular about 4:1.
  • the receptor present on an outer surface may be any suitable receptor that is present on a target cell.
  • the receptor is abundantly expressed on an outer surface of a target cell and/or undergoes endosomal recycling thus minimizing exposure of bound lipid nanoparticles to lysosomal degradation.
  • the receptor is preferably specifically expressed on an outer surface of a target cell, i.e. less abundantly or not expressed on a cell that are not a target cell.
  • concentration of the receptor on the outer surface of the target cell is twofold higher, more preferably fivefold higher, even more preferably tenfold higher, in particular one hundredfold higher compared to the concentration of the receptor on another cell that is not a target cell.
  • the receptor is preferably twice as abundant on a target cell compared to another non-target cell, more preferably five times as abimdant, ten times as abundant, in particular one hundred times as abundant on a target cell compared to another non-target cell.
  • Suitable receptors include integrin receptors, such as the Very Late Antigens VLA-4, VLA-5, VLA-6, LFA-1, CR3, and the transferrin receptor.
  • the receptor is the VLA-4 receptor or a VLA-5 receptor, more preferably a VLA-4 receptor.
  • the invention relates to a lipid nanoparticle loaded with a therapeutic molecule, which lipid nanoparticle comprises a ligand on its outer surface capable of binding to the VLA-4 receptor.
  • the VLA-4 receptor or integrin a4Bl, is an integrin dimer composed of CD49d (a4) and CD29 (Bl). It is expressed on leukocytes, such as lymphocytes, eosinophils, monocytes, macrophages, natural killer cells, basophils and mast cells and mediates homing, trafficking, differentiation, activation, and survival of VLA-4 expressing cells.
  • leukocytes such as lymphocytes, eosinophils, monocytes, macrophages, natural killer cells, basophils and mast cells and mediates homing, trafficking, differentiation, activation, and survival of VLA-4 expressing cells.
  • a nanoparticle according to the invention comprising a ligand on its outer surface capable of binding to the VLA-4 receptor is taken up by leukocytes more rapidly compared to a comparable lipid nanoparticle that lacks a ligand capable of binding the VLA-4 receptor.
  • a lipid nanoparticle according to the invention may release a therapeutic molecule, having a therapeutic effect in said target cell.
  • a lipid nanoparticle according to the invention is particularly suitable for use in a method of treatment of diseases of leukocytes, including leukemia, such as amyloid myeloid leukemia.
  • any ligand capable of binding the VLA-4 receptor is suitable to be present on the outer surface of a lipid nanoparticle according to the invention.
  • natural ligands of VLA-4 include vascular cell adhesion molecule 1 (VCAM-1), mucosal vascular addressin cell adhesion molecule- 1 (MAdCAM-1), fibronectin and junctional adhesion molecule-B (JAM-B) (Imhof, 2004. Nat Rev Immunol 4: 432-444).
  • the ligand comprises a structure according to Formula (I).
  • R 1 is preferably selected from a functional group selected from R 1a to R 1e , wherein in R 1c , the chiral carbon atom is in either in (R) or (S) configuration, preferably in (S) configuration; wherein in R 1d , the chiral carbon atoms are independently in either (R) or (S) configuration, preferably both chiral carbon atoms are in (S) configuration;
  • X is preferably selected from a functional group selected from X 1a and a peptide
  • the peptide is a dipeptide, a tripeptide or a tetrapeptide, more preferably a tripeptide having the sequence leucine-asparagine-valine (Leu-Asp- Val or LDV), in particular L-Leu-L-Asp-L-Val;
  • W 1 is an aliphatic group, preferably a benzyl, cyclohexyl, cyclobutyl, cyclopropyl, methyl cyclopropyl, methyl cyclobutyl, methyl cyclopentyl, methyl cyclohexyl, furan, methyl furan, pyrrole, methyl pyrrole, thiophene, methyl thiophene, methyl, ethyl, propyl, butyl or isopropyl group, more preferably wherein W 1 is a cyclopropyl group or a benzyl group; wherein W 2 is a hydrogen
  • lipid nanoparticle comprising a ligand comprising a structure according to formula (I)
  • enhanced uptake said lipid nanoparticles in (8;21) AML cells was obtained compared to a comparable lipid nanoparticle lacking a ligand comprising a structure according to formula (I).
  • a lipid nanoparticle according to the invention performs superiorly compared to comparable lipid nanoparticles in terms of uptake by VLA-4 expressing cells, such as (t8;21) AML cells and in terms of reducing functional expression of a target gene, such as RUNX1/ETO.
  • lipid nanoparticles comprising a ligand comprising a structure according to formula (I) caused a massive shift of subpopulations from immature CD34+ cells to more mature CD34- CD15+ cells indicating strong induction of myeloid differentiation by a lipid nanoparticle according to the invention.
  • a comparable lipid nanoparticle lacking a ligand on its outer surface did not significantly affect myeloid differentiation.
  • the invention preferably relates to a lipid nanoparticle loaded with a therapeutic molecule, which lipid nanoparticle comprises a ligand comprising the structure according to formula (I), wherein R 1 is X and wherein X is a peptide having the sequence LDV on its outer surface.
  • Said ligand is represented by formula (II):
  • the ligand present on the outer surface of a lipid nanoparticle according to the invention may comprise any of the structures shown in Table 1 of Baiula et al., 2019. Front Chem 7: 489.
  • the ligand present on an outer surface of the nanoparticle according to the invention has a KD value for the VLA-4 receptor between 1 pM and 100 nM, preferably between 2 pM and 20 nM as determinable with a suitable binding assay, for example, Jurkat cells in the presence of 2 mM MnCl 2 at room temperature.
  • a low KD value advantageously indicates strong binding to the VLA-4 receptor, and thus indicates rapid and efficient uptake of a lipid nanoparticle according to the invention by a target cell expressing the VLA-4 receptor on an outer surface.
  • the ligand is preferably coupled to a lipid molecule via a suitable linker to enable incorporation of the ligand into the lipid nanoparticle, such that the ligand is presented on the outer surface of the lipid nanoparticle.
  • said linker comprises a tetrazole, more preferably the linker comprises a structure as represented by Formula (III) Formula (III), wherein R 2 is an aliphatic group, preferably an -NHC(CH 2 ) n COOH, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, in particular wherein n is 4, such that R 2 is - NHC(CH 2 ) 4 COOH; wherein R 3 is CO(CH 2 ) m CONH, wherein m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, in particular wherein m is 2, such that R 3 is CO(CH 2 ) 2 CONH.
  • the linker is connected to said lipid and said ligand via a suitable linkage, preferably via a covalent bond, more preferably a peptide bond.
  • said lipid attached via said linker to said ligand is selected from the group consisting of DSPE-PEG, DMG-PEG and Cholesterol- PEG, preferably DSPE-PEG 1000-6000 , DMG-PEG 1000-6000 and Cholesterol-PEG 1000-6000 , more preferably DSPE-PEG (2000) , DMG-PEG (2000) and Cholesterol-PEG (2000) .
  • a lipid nanoparticle according to the invention preferably has a hydrodynamic diameter of about 75 to about 180 nm, preferably about 78 to about 120 nm, more preferably between about 79 nm and about 90 nm as determinable with a Zetasizer Nano (Malvern Instruments, UK).
  • the hydrodynamic diameter of a lipid nanoparticle according to the invention is at most 100% larger compared to a comparable lipid nanoparticle lacking a ligand on its outer surface, more preferably at most 80% larger, at most 60% larger, at most 40% larger, at most 35% larger, at most 30% larger, in particular at most 25% larger, such as at most 24% larger, or at most 23% larger compared to a comparable lipid nanoparticle lacking a ligand on its outer surface.
  • a lipid nanoparticle according to the invention is preferably between 23% and 100% larger compared to a comparable lipid nanoparticle lacking a ligand on its outer surface, more preferably between 24% and 80%, between 25% and 60%, between 30% and 50%, such as between 35% and 40% larger compared to a comparable lipid nanoparticle lacking a ligand on its outer surface.
  • a lipid nanoparticle according to the invention is at most 100%, such as at most 23% larger than a comparable nanoparticle lacking a ligand on its outer surface, because a smaller lipid nanoparticle has a longer circulation time in the body, typically combined with superior tissue penetration properties and is therefore more likely to effectively target the target cell.
  • a lipid nanoparticle according to the invention preferably has a polydispersity index of about 0.15 to about 0.25, preferably about 0.18 to about 0.22 as determinable with a Zetasizer Nano (Malvern Instruments, UK).
  • polydispersity index of a lipid nanoparticle according to the invention is at most 3 fold larger compared to a comparable lipid nanoparticle lacking a ligand on its outer surface, more preferably at most 2.8 fold, at most 2.5 fold larger, at most 2.0 fold larger, at most 1.9 fold larger, in particular at most 1.8 fold larger compared to a comparable lipid nanoparticle lacking a ligand on its outer surface.
  • a lipid nanoparticle according to the invention is preferably between 1.8 and 3.0 fold larger compared to a comparable lipid nanoparticle lacking a ligand on its outer surface, more preferably between 1.9 and 2.5 fold larger.
  • a lipid nanoparticle according to the invention preferably comprises a detectable label, more preferably a detectable label on its outer surface.
  • any detectable label suitable for in vivo use may be present in a lipid nanoparticle according to the invention, such as a fluorescent label, a radioactive label, a magnetic label, or a combination thereof.
  • the detectable label is a fluorescent label.
  • fluorescent labels include Cy3, Cy5, Cy7, Cy9, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 561, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Bodipy Fl, coumarin, fluorescein, Oregon green, pacific blue, pacific green, pacific orange, PE-cyanine7, PerCP-Cyanine5.5, tetramethyl rhodamine and Texas red.
  • the detectable label is a fluorescent label having an excitation wavelength in the range of 488 and 554 nm.
  • a fluorescent label is typically excitable with a 532 nm laser line.
  • the detectable label is a fluorescent label having an emission wavelength in the range of between 560 and 568 nm.
  • a fluorescent label is typically well detectable in the context of the present invention.
  • the invention further relates to a composition comprising a lipid nanoparticle according to the invention and a pharmaceutically acceptable carrier.
  • suitable pharmaceutically acceptable carriers include liquid carriers such as water and aqueous buffer solutions, such as physiological saline, phosphate-buffered saline (PBS) or phosphate buffer and solid carriers such as lactose, calcium chloride, pectin and dextrin.
  • liquid carriers such as water and aqueous buffer solutions, such as physiological saline, phosphate-buffered saline (PBS) or phosphate buffer and solid carriers such as lactose, calcium chloride, pectin and dextrin.
  • PBS phosphate-buffered saline
  • solid carriers such as lactose, calcium chloride, pectin and dextrin.
  • lactose calcium chloride
  • pectin and dextrin phosphate buffer and solid carriers
  • said pharmaceutically acceptable carrier is a liquid carrier, preferably buffered water, more preferably PBS.
  • composition may optionally comprise one or more pharmaceutically acceptable excipients, such as salts, wetting agents, flavoring agents, texturing agents, and stabilizers.
  • pharmaceutically acceptable excipients such as salts, wetting agents, flavoring agents, texturing agents, and stabilizers.
  • the invention further relates to a lipid nanoparticle or composition according to the invention for use in a method of treatment by therapy, preferably for use in a method of treatment of cancer.
  • a lipid nanoparticle or composition according to the invention is for use in a method of treating lung cancer, colon cancer, rectal cancer, anal cancer, bile cancer, small intestine cancer, gastric cancer, gallbladder cancer, liver cancer, pancreatic cancer, breast cancer, ovarian cancer, endometrium cancer, cervical cancer, prostate cancer, renal cancer, glioblastoma, skin cancer, bone cancer, blood cancer, cancer of the central nervous system, head- and neck cancer and lymphoma.
  • a lipid nanoparticle or composition according to the invention is for use in a method of treatment of leukemia, more preferably acute myeloid leukemia.
  • the invention preferably relates to a lipid nanoparticle or composition according to the invention for use in a method of treatment of leukemia, preferably acute myeloid leukemia, in a human, wherein the human has an age of 18 years or lower, preferably 15 years or lower, more preferably 12 years or lower, 10 years or lower, 8 years or lower, or 6 years or lower.
  • said human is a human having an age of at least 6 months, more preferably at least 1 year, at least 2 years, at least 3 years, at least 4 years, in particular at least 5 years of age.
  • the human is preferably between 6 months and 18 years of age, such as between 1 and 15 years of age, between 2 and 12 years of age, between 3 and 10 years of age, such as between 4 and 8 years of age.
  • the invention further pertains to a method for preparing a lipid nanoparticle according to the invention, comprising
  • Mixing of said at least one cationic or ionizable lipid and said at least one helper lipid may be achieved using any suitable method known in the art, e.g. by pipetting or vortexing said lipids in a suitable holder, or by using a microfluidic mixer to obtain said lipid mixture.
  • Said lipid mixture may optionally comprise in addition to said at least one cationic or ionizable lipid and said at least one helper lipid, one or more PEG-lipids.
  • Said one or more cationic or ionizable lipid, one or more helper lipids and optionally said one or more PEG-lipids are as defined herein above.
  • said one or more cationic lipids comprises at least Dlin-MC3-DMA.
  • said one or more helper lipids comprises at least DSPC and cholesterol and optionally sphingomyelin.
  • a lipid nanoparticle according to the invention comprises one or more PEG-lipids, preferably DSPE-PEG 1000-6000 or DMG-PEG 1000- 6000 , more preferably DSPE-PEG (2000) or DMG-PEG (2000) .
  • any water-miscible polar organic solvent is suitable for use in the method according to the invention.
  • examples include methanol, ethanol, propanol, isopropanol, butanol, isobutanol, acetonitrile, 1,4-dioxane, chloroform, ethyl acetate, acetone, dimethylformamide (DMF), dimethylsulfoxide (DMSO), tetrahydrofuran and mixtures thereof.
  • the polar organic solvent is an alcohol, more preferably ethanol.
  • the ratio between the one or more cationic lipids, the one or more helper lipids and the optionally one or more PEG-lipids is preferably between l:0.5:0 and 1:1.4:0.05, more preferably said ratio is between 1:0.8:0.01 and 1:1.2:0.03, in particular around 1:1:0.03 or 1:1:0.04.
  • said lipid mixture comprises the lipids Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG, preferably DMG-PEG 1000-6000 , more preferably DMG- PEG 2000 .
  • the ratio Dlin-MC3-DMA:DSPC:cholesterol:DMG-PEG is between about 1:0.1:0.5:0.02 and about 1:0.4:0.9:0.05, more preferably between about 1:0.2:0.7:0.03 and about 1:0.3:0.8:0.04.
  • said lipid mixture comprises the lipids Dlin-MC3-DMA, DSPC, cholesterol and sphingomyelin.
  • the ratio Dlin-MC3-DMA:DSPC:cholesterol:spingomyelin is between about l:0.1:0.2:0.2 and about l:0.4:0.5:0.5, more preferably between about l:0.2:0.3:0.3 and about l:0.3:0.4:0.4.
  • said lipid mixture comprises the lipids Dlin-MC3-DMA, DSPC, cholesterol, sphingomyelin and a PEG-lipid, preferably DSPE-PEG or DMG-PEG, more preferably DSPE 1000-6000 or DMG-PEG 1000-6000 , more preferably DMG-PEG 2000 or DSPE-PEG 2000 .
  • the lipid concentration in the lipid mixture is preferably between 2.5 and 25 mM, more preferably between 10 and 25 mM, in particular about 25 mM. Further, the concentration of cationic or ionizable lipids in the lipid mixture is preferably between about 1.25 mM and 12.5 mM, more preferably between 5 mM and 12.5 mM, in particular about 12.5 mM.
  • Said aqueous solution comprising said therapeutic molecule has a pH between about 2 and about 6, preferably between 3 and 5, more preferably around 4.
  • Said pH may be determined using any suitable method known in the art, for example using a pH meter.
  • a therapeutic molecule preferably a charged therapeutic molecule, is typically well soluble in an aqueous medium.
  • said aqueous solution is an aqueous buffer solution, such as an acetate buffer or a citric acid buffer, in particular an acetate buffer.
  • the molar strength of said aqueous buffer solution is between 5 and 50 mM, more preferably between 10 and 40 mM, in particular around 25 mM. Most preferably, said aqueous solution is a 25 mM acetate buffer.
  • Said therapeutic molecule is as defined herein above, but is preferably an oligonucleotide, more preferably an siRNA molecule.
  • the concentration of therapeutic molecule in the aqueous solution is preferably between 5 and 50 ⁇ M, more preferably between 20 and 40 ⁇ M, in particular about 26 ⁇ M.
  • lipid mixture and said aqueous solution comprising said therapeutic molecule is preferably achieved using a microfluidic mixer, for example using a NanoAssemblr benchtop (Precision nanosystems).
  • both lipid mixture and aqueous solution are preferably loaded in separate syringes and pumped through a microfluidic mixture to obtain a mixture comprising lipid nanoparticles loaded with said therapeutic molecule.
  • the total flow rate in the microfluidic mixer is between 1 ml/min and 12 ml/min, more preferably between 2 ml/min and 6 ml/min, in particular around 4 ml/min.
  • the ratio between the volume of the aqueous mixture comprising a therapeutic molecule and the lipid mixture is between 1:1 and 5:1, more preferably between 2:1 and 4:1, in particular around 3:1.
  • the molar ratio between therapeutic molecule and lipid in the mixture comprising lipid nanoparticles loaded with a therapeutic molecule is preferably between about 1:500 and about 1:150, more preferably between about 1:400 and about 1:250, in particular around 1:330.
  • the molar ratio between therapeutic molecule and cationic lipid or ionizable lipid in the mixture comprising lipid nanoparticles loaded with a therapeutic molecule is preferably between 1:750 and 1:250, more preferably between about 1:200 and about 1:130, in particular around 1:160.
  • the efficiency of encapsulating a therapeutic molecule in a lipid nanoparticle is relatively high.
  • the encapsulation efficiency is at least 70%, more preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, most preferably 100%.
  • the obtained lipid nanoparticles loaded with a therapeutic molecule may be contacted directly with a ligand coupled to a PEG lipid, or may optionally be at least partly purified prior to said contacting.
  • Any method of purification that is capable of exchanging the organic solvent used to prepare said lipid mixture and the aqueous buffer used to prepare said aqueous solution comprising a therapeutic molecule may be used herein, such as dialysis or centrifugation over a suitable membrane.
  • the mixture comprising lipid nanoparticles loaded with said therapeutic molecule is subjected to one or more dialysis steps. It has been found that when said lipid nanoparticles loaded with a therapeutic molecule have been at least partly purified prior to contacting with a ligand coupled to a lipid, a nanoparticle according to the invention exhibits superior physicochemical properties, in particular superior hydrodynamic diameter and superior polydispersity index, compared to a lipid nanoparticle that has been obtained without at least partly purification.
  • said mixture comprising lipid nanoparticles loaded with said therapeutic molecule is dialyzed against an aqueous buffer having a pH of between 6 and 8, preferably around 7.
  • buffers examples include 2,2-Bis(hydroxymethyl)-2,2’,2”- nitrilotriethanol (Bis-Tris), N-(2-Acetamido)iminodiacetic acid, (ADA), N-(2- Acetamido)-2-aminoethanesulfonic acid (ACES), Piperazine-N,N'-bis(2- ethanesulfonic acid) (PIPES), 3-Morpholino-2-hydroxypropanesulfonic acid (MOPS), 1,3-Bis(tris(hydroxymethyl)methylamino)propane (Bis-Tris propane), N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 2- ⁇ [l,3-Dihydroxy- (hydroxymethyl)propan-2-yl]amino ⁇ ethane-l-sulfonic acid (TES), 4-(2- Hydroxyethyl)-l-piperazineethanesulfonic acid
  • said dialysis takes place at a temperature between 0 °C and 20 C, preferably between 2 °C and 15 °C, in particular between 4 °C and 6 °C.
  • Said dialysis preferably takes place for at least 1 h, more preferably at least 2 h, at least 4 h, at least 8 h, in particular at least 12 h, or at least 16 h.
  • the dialysis is rim over night, e.g. between 12 h and 16 h.
  • said aqueous buffer is replaced at least 1 time with fresh aqueous buffer, more preferably at least 2 times, most preferably at least 3 times.
  • the dialysis membrane preferably has a molecular weight cut off of at most 30 kDa, preferably at most 20 kDa, in particular at most 10 kDa.
  • Said mixture of lipid nanoparticles loaded with said therapeutic molecule is contacted with a ligand coupled to a lipid via a suitable linker.
  • said ligand comprises a structure according to formula (II).
  • said lipid is DSPE-PEG, more preferably DSPE-PEG 1000-6000 , even more preferably DSPE- PEG (2000) .
  • Said contacting may be achieved using any suitable method known in the art, such as by pipetting, stirring or mixing of the mixture comprising lipid nanoparticles loaded with a therapeutic molecule and the ligand coupled to the lipid.
  • the molar ratio between said mixture comprising the lipid nanoparticles loaded with the therapeutic molecule and said ligand coupled to said lipid is between 10000:1 and 500:1, more preferably between 5000:1 and 1000:1, in particular around 3000:1.
  • the molar fraction of ligand in a lipid nanoparticle according to the invention is between 0.01% and 0.2%, preferably between about 0.02% and about 0.1%, in particular around 0.03%.
  • the concentration of lipid nanoparticles loaded with the therapeutic molecule is between 5 and 40 mM, more preferably between 10 and 25 mM.
  • the concentration of ligand coupled to lipid is between about 5 and about 40 ⁇ M, more preferably between about 10 and about 25 ⁇ M.
  • the mixture comprising the lipid nanoparticles loaded with said therapeutic molecule and said ligand coupled to said lipid were contacted for at least 15 minutes, preferably at least 30 minutes, at least 1 h, in particular at least 2 h, to obtain a lipid nanoparticle according to the invention.
  • said mixture comprising the lipid nanoparticles loaded with said therapeutic molecule and said ligand coupled to said lipid were preferably contacted at a temperature of between about 4 °C and about 80 °C, more preferably between about 10 °C and about 70 °C, between about 20 °C and about 60 °C, in particular between about 40 °C and about 50 °C, such as about 45 °C.
  • Said ligand is preferably coupled to said lipid via a linker comprising a triazole moiety to obtain a ligand coupled to a lipid via a triazole linkage. It was found that this molecule may be efficiently obtained using a copper-free click reaction protocol.
  • the invention further pertains to a method of coupling a ligand to a lipid, comprising
  • lipid functionalized with a strained alkyne moiety preferably a dibenzocyclooctyne (DBCO) moiety
  • said method does not require the use of Cu 2+ species, which is associated with copper-related toxicity when administered to animals, such as humans.
  • said ligand functionalized with a terminal azide moiety is a molecule comprising the structure of formula (IV).
  • Said ligand according to formula (I) functionalized with a terminal azide, preferably said molecule comprising the structure of formula (IV) may be prepared via a chemical synthesis protocol.
  • a corresponding amine (lysine, preferably L-lysine) may be used as starting material.
  • Said amine may be functionalized with a suitable leaving group, such as a sulfonamide group and subsequently substituting said sulfonamide with an azide group to obtain a ligand according to formula (I), preferably a ligand according to formula (II), functionalized with a terminal azide moiety.
  • Said molecule according to formula (IV) may also be obtained from a commercial source, for example from EMC Microcollections (Tubingen, Germany).
  • Said lipid functionalized with a strained alkyne molecule is preferably a molecule according to formula (V).
  • Said lipid functionalized with a strained alkyne molecule preferably said molecule according to formula (V) may be obtained via a chemical synthesis protocol.
  • Said molecule according to formula (V) may also be obtained from a commercial source, for example from Avanti Polar Lipids.
  • Said ligand functionalized with said azide and said lipid functionalized with said strained alkyne molecule are contacted using any suitable method known in the art, preferably by mixing, e.g. by pipetting, stirring or vortexing said functionalized ligand and said functionalized lipid.
  • said ligand functionalized with an azide and said lipid functionalized with a strained alkyne are dissolved in a suitable liquid medium to obtain a solution of said ligand functionalized with an azide and said lipid functionalized with a strained alkyne in a suitable liquid medium.
  • said suitable liquid medium comprises DMSO, an alcohol, preferably a lower alcohol such as methanol, ethanol and propanol, H 2 O, PBS or combinations thereof, more preferably PBS.
  • the ratio between said ligand functionalized with azide and said lipid functionalized with alkyne is preferably between 1:1 and 1:5, more preferably between 1:2 and 1:4, in particular about 1:3.
  • a ratio promotes full conversion of said azide into the corresponding triazole.
  • the functionalized ligand and the functionalized lipid were contacted for an amount of time sufficient to convert at least 90% of the functionalized azide into the corresponding triazole.
  • said functionalized ligand and said functionalized lipid were contacted for at least 1 h, preferably at least 2 h, at least 4 h, at least 8 h, at least 12 h, at least 16 h, at least 24 h. Conversion of the reaction may be monitored by following conversion of a fluorescently labelled mock substrate on a 1% agarose gel.
  • the functionalized ligand the functionalized lipid were preferably contacted at a temperature of between 0 °C and 50 °C, more preferably between 4 °C and 37 °C, between 10 °C and 20 °C, in particular between 20 °C and 25 °C.
  • Said ligand coupled to said lipid is optionally isolated from the reaction mixture. Any method suitable for isolation known in the art may be used, for example size exclusion chromatography or column chromatography.
  • the invention further pertains to a method of treating a subject suffering from cancer, preferably leukemia, more preferably acute myeloid leukemia, comprising administering a therapeutically effective amount of a lipid nanoparticle or pharmaceutical composition according to the invention to a subject in need thereof, preferably a human.
  • Said human preferably is a human having an age of 18 years or lower, preferably 15 years or lower, more preferably 12 years or lower, 10 years or lower, 8 years or lower, or 6 years or lower.
  • said human is a human having an age of at least 6 months, more preferably at least 1 year, at least 2 years, at least 3 years, at least 4 years, in particular at least 5 years of age.
  • the human is preferably between 6 months and 18 years of age, such as between 1 and 15 years of age, between 2 and 12 years of age, between 3 and 10 years of age, such as between 4 and 8 years of age.
  • said lipid nanoparticle or composition is as described herein above.
  • said lipid nanoparticle comprises a ligand according to formula (II).
  • said lipid nanoparticle is preferably loaded with an oligonucleotide, more preferably an siRNA molecule, most preferably an siRNA molecule comprising a first strand as represented by SEQ ID NO:1.
  • Said lipid nanoparticle or composition according to the invention may be administered via any suitable route of administration known in the art.
  • routes of administration include injection, oral administration, inhalation, transdermal application or rectal administration.
  • said lipid nanoparticle according to the invention is administered via injection.
  • said lipid nanoparticle or said composition according to the invention may be administered parenterally, such as intraarticularly, intravenously, intraperitoneally, subcutaneously or intramuscularly.
  • said lipid nanoparticle or composition is administered intramuscularly.
  • the invention further relates to a use of a lipid nanoparticle according to the invention in the preparation of a medicament for the treatment of cancer, preferably leukemia, more preferably acute myeloid leukemia.
  • Example 1 siRNA linid nanon articles (LNP) components and preparation Preparation of lipid mixtures
  • a second lipid mixture was prepared by replacing DMG-PEG (2000) and part of the cholesterol by sphingomyelin, a major component of viral envelopes, mammalian membranes and exosomes.
  • the lipid composition of the second lipid mixture had a molar ratio of 50:10:20:20 for Dlin-MC3-DMA:DSPC:cholesterol:sphingomyelin, respectively.
  • siRNA siRNA
  • concentration of siRNA (csiRNA) in the stock solution was calculated according to formula (I): wherein C DLin is the concentration of Dlin-MC3-DMA in the lipid mixture, equaling the concentration of positively charged amines, N/P is the ratio of positively chargeable amine groups to negatively charged nucleic acid phosphate groups (here 4, which was found to be optimal for siRNA LNP formulations), np defines the number of phosphate groups in the siRNA (here 40), V SiRNA and V lipid are the volumes of stock solutions of siRNA (in 25 mM acetate buffer) and lipid mixture (in ethanol), respectively, with a V lipid /V siRNA ratio of 1:3.
  • cDlin is 12.5 mM and C siRNA is 26 ⁇ M.
  • the LNPs were prepared using a NanoAssemblr benchtop (Precision Nanosystems). Syringes were loaded with stock solutions comprising a lipid mixture and siRNA at a V lipid /V siRNA ratio of 1:3, and pumped through a microfluidic mixer at a total flow rate of 4 ml/min to obtain a solution comprising LNPs loaded with siRNA (siRNA LNPs).
  • siRNA LNP solutions were dialysed against phosphate-buffered saline (PBS) overnight at 4 °C, using the Gamma- Irradiated Slide- A-Lyzers with lOkDa molecular weight cut-off (ThermoFisher Scientific). PBS was replaced after 1 and 2 hours, to obtain a solution of siRNA loaded LNPs in PBS buffer.
  • PBS phosphate-buffered saline
  • VLA-4 Very Late Antigen 4
  • ITGA4 Integrin Subunit Alpha 4
  • IGB1 Integrin Subunit Beta 1
  • siRNA LNPs were decorated with the fluorophore Cy3 to monitor cell association and uptake.
  • DBCO restrained alkyne dibenzocyclooctyne
  • the ligand or fluorophore was functionalized with an azide moiety.
  • the solvent was dependent on the hydrophobicity of the azide moiety and was most preferably H 2 O or DMSO.
  • a solution of 1 molar eq. of ligand functionalized with an azide moiety dissolved in DMSO:H 2 O in a ratio of 3:2 was mixed with a solution of 3 molar eq.
  • siRNA LNPs were analysed.
  • the siRNA LNPs with and without post-inserted ligands were analysed in terms of size (hydrodynamic diameter), polydispersity index (PDI) using a Zetasizer Nano (Malvern Instruments, UK) and encapsulation efficiency. The results are shown in table 1.
  • LNP siRE LNP loaded with siRNA comprising a first strand as represented by SEQ ID NO:3 and a second strand complementary to the first strand represented by SEQ ID NO:4
  • LNP siMM Control siRNA comprising a first strand as represented by SEQ ID NO:5 and a second strand complementary to the first strand represented by SEQ ID NO:6.
  • Ligand ligand according to formula (II).
  • the hydrodynamic diameter and PDI were increased by 100% compared to siRNA LNP free of ligand (entry 1), while post-insertion of the ligand prior to dialysis (entry 3) or post-insertion of DBCO-DSPE-PEG (2000) (entry 2) in the LNP caused a substantial increase in the hydrodynamic diameter by more than 140% (table 2; comparison entry 1 with entries 2 and 3).
  • the hydrodynamic diameter of the decorated ligand could be further reduced by post-insertion of DBCO-DSPE-PEG (2000) -ligand at 0.3% molar ratio into siRNA LNP containing 1.2% DMG-PEG (2000) .
  • the hereby obtained ligand-decorated siRNA LNP had a size of 79 nm, which corresponds to an increase of only 23% compared to non- decorated siRNA LNP.
  • the ligand-decorated siRNA LNP showed a PDI of 0.19, which corresponds to an increase in PDI of approximately 78% compared to non-decorated siRNA LNP (Table 2, entries 5 and 8).
  • siRNA LNP The non-decorated siRNA LNP were further analyzed in terms of stability over time (table 3). It was found that the size and PDI of siRNA LNP did not increase significantly when stored at 4 °C during an observation period of up to two months (table 3).
  • LNP siRE LNP loaded with siRNA comprising a first strand as represented by SEQ ID NO:3 and a second strand complementary to the first strand represented by SEQ ID NO:4
  • LNP siMM control siRNA comprising a first strand as represented by SEQ ID NO:5 and a second strand complementary to the first strand represented by SEQ ID NO:6.
  • Example 4 Uptake of fluorescent siRNA LNP in t(8:21) AML cell lines and patient cells
  • the following example shows the uptake of Cy3-labelled siRNA LNP in t(8;21) Kasumi-1 cells.
  • Cy3-labelled DSPE-PEG (2000) was post-inserted together with or without a ligand coupled to a lipid according to Formula (VI) into siRE LNP and siMM LNP.
  • FIG 4a the time course of uptake for both “targeted” and “non- targeted” siRE LNP and siMM LNP formulations is shown.
  • Figures 3 and 4 both demonstrate that “targeted” siRNA LNPs showed higher and faster uptake compared to the “non-targeted” counterparts independent of the cargo siRNA.
  • This example shows the efficacy of “non-targeted” siRNA LNPs to knockdown RUNX1/ETO in the t(8;21) positive cell lines Kasumi-1 and SKNO-1.
  • the cells were diluted in 2.5 ml semi-solid medium (RPMI1640 with 10% FCS and 5% methylcellulose) and 0.5 ml of this solution was transferred to a 24- well plate, giving a concentration of about 3125 cells per well.
  • semi-solid medium RPMI1640 with 10% FCS and 5% methylcellulose
  • siRE LNPs affected the expression of established RUNX1/ETO genes.
  • LAPTM5 Lysosomal Protein Transmembrane 5
  • CEBPA Cyclin D2
  • ANGPT1 Angiopoietin 1
  • CCND2 Cyclin D2
  • Example 6 siRNA LNP efficacy in patient-derived t(8;21) AML cells
  • This example demonstrates superior RUNX1/ETO knockdown by the “targeted” siRNA LNPs in patient-derived AML cells compared to the non-decorated siRNA LNP.
  • siRE LNP siRE LNP
  • targeted and “non-targeted” siMM LNPs
  • siRE LNPs did not cause any substantial changes in cell differentiation when compared to untreated cells.
  • only “targeted” siRE LNPs affected the expression of established RUNX1/ETO genes.
  • Example 7 Tolerability of siRE LNPs decorated with an LDV ligand in BALB/cJRag2 tm1 1Flv 112rg tm1 1Flv /J mice
  • mice subjected to treatment with “targeted” LNP loaded with siRE were treated with “targeted” LNP loaded with siRE, compared to control.
  • siRNA LNPs can be safely applied in mice at the maximal dose tested without compromising animal welfare and overall health.
  • Example 8 Improved uptake of LNPs in AML primary cells
  • Example 9 siRNA LNP efficacy in t(8:21) primary cells
  • Example 10 LNPs without DMG-PEG are still functionally active in patient- derived material
  • Example 11 Modified siRE has high efficacy in t(8:21) cell lines
  • Figure 17A shows the siRNAs that were designed specifically targeting the unique breakpoint of the RUNX1/ETO t(8;21) fusion transcript (siRE) and the mismatch control (siMM) generated by swapping two nucleotides in the sequence.
  • siRNAs were modified by site-specifically introducing 2’-deoxy- (2’- H), 2’-fluoro (2’-F) and 2’-methoxy (2’-OMe) ribose modifications and 3’-terminal phosphorothioate (PS) backbones (Figure 17B).
  • Example 12 Lipid composition reaches the bone-marrow and tumour-site in vivo
  • the cells that were harvested from the secondary recipients were not resistant to LNP siRE treatment as shown by Western Blot analysis were the RUNX1/ETO protein expression decreased in the cells that were treated with the siRE LNP, even the cells that were harvested from the secondary recipients treated with the siRE LNPs ( Figure 2 IE). This indicates that the cells remained dependent on RUNX1/ETO and are still susceptible to repeated LNPs treatment.
  • Example 13 siRNA LNP efficacy in a t(4:11) ALL cell line
  • This example shows the efficacy of “non-targeted” siRNA LNPs to knockdown fusions of mixed lineage leukemia gene 1 (MLL; Lysine Methyltransferase 2A; KMT2A) with ALF Transcription Elongation Factor 1 (AF4 or AFF1) in the t(4; 11) positive cell line SEM (Greil et al., 1994. Br J Haematol 86: 275-283).
  • MLL mixed lineage leukemia gene 1
  • KMT2A Lysine Methyltransferase 2A
  • AF4 or AFF1 ALF Transcription Elongation Factor 1
  • FIG 22AB the reduction of expression of MLL/AF4 and of its target gene Homeobox A7 (H0XA7) over time in SEM cells is shown.
  • Expression of the MLL/AF4 transcript is reduced approximately two-fold after a single administration of siMA6 LNP and three-fold by siMAl-Ml LNPs when compared to the control siMM and siMM-mod LNPs, respectively, while expression of H0XA7 is reduced two-fold.
  • siMA6-Ml LNPs Two sequential applications of siMA6-Ml LNPs at day 1 and day 3 blocked proliferation of SEM stronger than control LNP formulations ( Figure 22C).
  • the effect of the knockdown was also investigated in clonogenicity assays. Therefore SEM cells were seeded in semi-solid medium (RPMI1640 with 10% FCS and 5% methylcellulose) at a cell concentration of 2000 cells/ml. After 14 days pictures were taken using the Leica DMi8 and the colonies, defined as > 25 cells present, were counted (both manually as computationally).
  • siMA6-Ml LNPs reduced clonogenic growth more than 7 fold when compared to siMM-mod LNPs ( Figure 22D).

Abstract

The invention relates to a lipid nanoparticle loaded with a therapeutic molecule, which lipid nanoparticle comprises a ligand on its outer surface capable of binding to the Very Late Antigen 4 (VLA-4) receptor, wherein the ligand comprises the structure according to general Formula (II):

Description

Title: Targeted lipid nanoparticle formulations
FIELD
The present invention is related to the field of targeted drug delivery. More specifically, it discloses a lipid nanoparticle loaded with a therapeutic molecule comprising a ligand capable of binding a receptor on its outer surface.
INTRODUCTION
More than 50% of all pediatric acute leukemias are characterized by chromosomal rearrangements and therewith linked expression of novel fusion genes that do not exist in healthy tissues. In general, these fusion genes initiate leukemia development and are also indispensable for maintaining the leukemic phenotype. For instance, a RUNX1/ETO fusion gene encodes a novel transcription factor that blocks differentiation along the myeloid lineage.
Leukemia is generally scored as either lymphocytic or myeloid, and as acute or chronic, whereby lymphocytic leukemias develop from T cells, B cells, or natural killer (NK) cells, and myeloid leukemias develop from granulocytes and monocytes. In acute leukemias, the malignant cells are often immature and are termed blasts, while chronic leukemias develop in more-mature cells. Most common types of leukemias are acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL).
Treatment for leukemia often includes chemotherapy and stem cell (bone marrow) transplantation. For some specific fusion genes, for example for Philadelphia-positive Acute Lymphocytic Leukemia (ALL), specific kinase inhibitors such as imatinib, dasatinib, and nilotinib may be used in combination with other chemotherapy drugs. Similarly, specific inhibitors of Bruton's tyrosine kinase such as ibrutinib, inhibitors of the anti- apop totic protein Bcl-2 such as venetoclax, and phosphoinositide 3-kinase inhibitors such as idelalisib have recently been approved for CLL, amongst other types of leukemias.
Immunotherapy, including the use of checkpoint inhibitors and chimeric antigen receptor (CAR) T cell therapy has produced encouraging results over the past years in people with certain types of leukemia. The main treatment for most childhood leukemias is chemotherapy. For some children with higher risk leukemias, high- dose chemotherapy may be given along with a stem cell transplant. Other treatments might also be used in special circumstances. The long-term and late effects of such treatment on childhood leukemia include cognitive effects, and effects in physical and psychological development. There is thus a need to develop therapies that effectively treat pediatric acute leukemias with reduced side effects.
BRIEF DESCRIPTION OF THE INVENTION
Provided herein is a lipid nanoparticle loaded with a therapeutic molecule, said lipid nanoparticle comprising a ligand on its outer surface capable of binding to the Very Late Antigen 4 (VLA-4) receptor, wherein the ligand comprises the structure according to formula (II):
Figure imgf000004_0001
By targeting the VLA-4 receptor, a lipid nanoparticle according to the invention, is taken up more rapidly by VLA-4 expressing cells, such as hematopoietic cells, compared to a comparable lipid nanoparticle lacking a ligand on its outer surface. The inventors have thereby created a drug delivery approach specifically targeting cells expressing the VLA-4 receptors, thereby achieving increased efficacy compared to a comparable untargeted approach. This provides a promising approach towards treatment of diseases or disorders of cells expressing VLA-4 receptors, e.g. leukemia, in particular AML.
Said therapeutic molecule is preferably a small interfering ribonucleic acid (siRNA), preferably siRNA that inhibits functional expression of a RUNX1/ETO fusion gene.
Said siRNA preferably comprises a first RNA strand complementary to an mRNA transcript of the RUNX/ETO gene and, optionally, a second RNA strand complementary to the first RNA strand, wherein said first RNA strand is preferably represented by the sequence as shown in SEQ ID NO:1 and wherein said second RNA strand, when present, is represented by the sequence as shown in SEQ ID NO:2.
Said siRNA is preferably a modified siRNA more preferably comprises at least 21 nucleotides and which siRNA comprises one or more of the following modifications:
(a) the sequence of SEQ ID NO:1 comprises a CFon position 1, a CFon position 2, an UFon position 3, and a CF on position 4, wherein CF and UF represent nucleotides wherein the hydroxyl group on the 2-position of the ribose moiety is substituted for a fluoride;
(b) the sequence of SEQ ID NO:1 comprises dA on position 17, dG on position 18, dA on position 19, dT on position 20 and dT on position 21, wherein dA dG and dT represent nucleotides wherein the hydroxyl group on the 2-position of the ribose moiety is substituted for a hydrogen;
(c) the sequence of SEQ ID NO:1 comprises an UoMe on position 9, a CoMe on position 10, an UoMe on position 12, an CoMe on position 14 and a UoMe on position 15, wherein CoMe and UOME represent nucleotides wherein the hydroxyl group on the 2-position of the ribose moiety is substituted for a methoxy group;
(d) the sequence of SEQ ID NO:1 comprises a TPS on position 20, and/or wherein the sequence of SEQ ID NO:2 comprises a TPS on position 20, wherein TPS represents a nucleotide wherein the phosphodiester group on the 3-position of the ribose moiety is substituted for a phosphorothioate;
(e) the sequence of SEQ ID NO: 1 comprises a T on position 20 and a T on position 21 and/or wherein the sequence of SEQ ID NO:2 comprises a T on position 20 and a T on position 21.
Preferably, said lipid nanoparticle comprises at least one cationic or ionizable lipid, at least one helper lipid and preferably at least one poly(ethylene)glycol (PEG)-lipid.
Herein, said at least one cationic or ionizable lipid preferably comprises (6Z,9Z,28Z,31Z)-heptatriacont-6,9,28,31-tetraene-19-yl 4- (dimethylamino)butanoate (Dlin-MC3-DMA), said at least one or more helper lipids is preferably selected from distearoylphosphatidylcholine (DSPC), cholesterol and sphingomyelin and said lipid nanoparticle preferably comprises at least one PEG- lipid selected from l,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol (DMG- PEG)) and a l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [amino(polyethylene glycol)] salt (DSPE-PEG), preferably DMG-PEG1000-6000 or DSPE-PEG1000-6000, more preferably DMG-PEG2000 or DSPE-PEG2000.
The lipid nanoparticle according to the invention, preferably has a hydrodynamic diameter of about 75 to about 130 nm, preferably about 78 to about 90 nm and/or a polydispersity index of about 0.15 to about 0.25, preferably about 0.18 to about 0.22, as determinable with a Zetasizer Nano (Malvern Instruments, UK).
Said lipid nanoparticle further preferably comprises a detectable label, more preferably on its outer surface.
The invention further relates to a lipid nanoparticle according to the invention for use in a method of treatment by therapy, preferably for use in a method of acute myeloid leukemia.
The invention further pertains to a pharmaceutical composition comprising a lipid nanoparticle according to the invention and a pharmaceutically acceptable carrier.
The invention further provides a method of coupling a ligand to a lipid, comprising
-providing a ligand according to general Formula (II) functionalized with a terminal azide moiety;
-providing a lipid functionalized with a strained alkyne moiety, preferably a dibenzocyclooctyne (DBCO) moiety;
-contacting the functionalized ligand with the functionalized lipid, to obtain a ligand coupled to a lipid via a triazole linkage; and
-optionally isolating the ligand coupled to the lipid.
The invention further pertains to a method for preparing a lipid nanoparticle according to the invention, comprising
-mixing at least one cationic or ionizable lipid and at least one helper lipid in a polar organic solvent to obtain a lipid mixture;
-mixing the lipid mixture with an aqueous solution comprising a therapeutic molecule at a pH between about 2 and about 6, preferably about 4, to form a mixture comprising lipid nanoparticles loaded with a therapeutic molecule;
-optionally at least partially purifying, preferably dialyzing, the mixture comprising lipid nanoparticles loaded with said therapeutic molecule; and -contacting the mixture comprising the lipid nanoparticles loaded with the therapeutic molecule or the at least partially purified, preferably dialyzed, mixture comprising lipid nanoparticles loaded with the therapeutic molecule with a ligand coupled to a lipid as described herein to obtain a lipid nanoparticle according to the invention.
Said at least one helper lipid preferably comprises DSPE-PEG, more preferably DSPE-PEG(2000).
FIGURE LEGENDS
Figure 1: A: A reaction scheme showing the copper-free click reaction between a ligand functionalized with a terminal azide and a lipid functionalized with a strained alkyne moiety to form a ligand coupled to a lipid via a triazole linkage;
B: 1% agarose gel showing conversion of a Cy3-labelled azide and DBCO- DSPE-PEG(2000) at different temperatures.
C: Reaction scheme showing the incorporation of a ligand coupled to a lipid into a lipid nanoparticle loaded with a therapeutic molecule.
Figure 2: Cryo-electron microscopy images showing lipid nanoparticles loaded with the therapeutic molecule siRE (right) and loaded with a control siRNA molecule (left).
Figure 3: Uptake of a Cy3-labelled lipid nanoparticle loaded with an siRE molecule comprising a ligand according to Formula (II) in Kasumi-1 cells is shown, compared to uptake of a comparable lipid nanoparticle lacking a ligand on its outer surface.
Figure 4: A: Graph showing uptake of a Cy3-labelled lipid nanoparticle loaded with siRE RNA and comprising a ligand according to Formula (II) on its outer surface in Kasumi-1 cells, compared to a comparable Cy3-labelled lipid nanoparticle loaded with siMM RNA and comprising a ligand according to Formula (II) on its outer surface, and comparable Cy3-labelled lipid nanoparticles lacking a ligand on their outer surface.
B: Graph showing uptake of a Cy3-labelled lipid nanoparticle loaded with siRE RNA and comprising a ligand according to Formula (II) on its outer surface in patient-derived AML cells in presence of mesenchymal stem cells (MSCs), compared to a comparable Cy3-labelled lipid nanoparticle loaded with siMM RNA and comprising a ligand according to Figure (II) on its outer surface, and comparable Cy3-labelled lipid nanoparticles lacking a ligand on its outer surface.
C: Graph showing uptake of a Cy3-labelled lipid nanoparticle loaded with siRE RNA and comprising a ligand according to Formula (II) on its outer surface in patient-derived AML cells in absence of MSCs, compared to a comparable Cy3- labelled lipid nanoparticle loaded with siMM RNA and comprising a ligand according to Formula (II) on its outer surface, and comparable Cy3-labelled lipid nanoparticles lacking a ligand on its outer surface.
Figure 5: Bar graph showing reduction of RUNX1/ETO expression over time in Kasumi-1 and SKNO-1 cells as a result of administration of a lipid nanoparticle loaded with siRE RNA (striped bar) compared to a control lipid nanoparticle loaded with siMM RNA (black bar).
Figure 6: A: Bar graph showing reduced expression of RUNX1/ETO fusion gene in t(8;21) AML cells of a lipid nanoparticle loaded with an siRE molecule, comprising DMG-PEG(2000) (white bar) or DSPE-PEG(2000) (striped bar) in the lipid outer layer, compared to a lipid nanoparticle loaded with siMM (control, black bar).
B: Bar graph showing number of colony formation of cells after administration of a lipid nanoparticle loaded with siRE (striped bar) and loaded with siMM (black bar).
C: Graph showing proliferation of cells over time after administration of a lipid nanoparticle loaded with siRE (square), compared to a lipid nanoparticle loaded with siMM (control; triangle) and a mock sample (circles).
Figure 7: A: Graph showing increased expression of Lysosomal Protein Transmembrane 5 (LAPTM5) in (t8;21) AML cells upon administration of lipid nanoparticles loaded with siRE (striped bar) compared to lipid nanoparticles loaded with siMM (black bar).
B: Graph showing increased expression of CCAAT Enhancer Binding Protein Alpha (CEBPA) in (t8;21) AML cells upon administration of lipid nanoparticles loaded with siRE (striped bar) compared to lipid nanoparticles loaded with siMM (black bar).
Figure 8: A: Graph showing decreased expression of the adhesion molecule CD34 in (t8;21) AML cells upon administration of lipid nanoparticles loaded with siRE (striped bar) compared to lipid nanoparticles loaded with siMM (black bar). B: Graph showing decreased expression of Angiopoietin-1 (ANGPT1) in (t8;21) AML cells upon administration of lipid nanoparticles loaded with siRE (striped bar) compared to lipid nanoparticles loaded with siMM (black bar).
C: Graph showing decreased expression of Cyclin D2 (CCND2) in (t8;21) AML cells upon administration of lipid nanoparticles loaded with siRE (striped bars) compared to lipid nanoparticles loaded with siMM (black bars).
Figure 9: A: Bar graph showing reduced expression of the RUNX1/ET0 fusion gene over time after administration of a lipid nanoparticle loaded with an siRE molecule and comprising a ligand according to Formula (II) on its outer surface (white bar), compared to expression of the RUNX1/ETO fusion gene upon administration of a comparable lipid nanoparticle loaded with an siMM molecule (black bar) and comparable lipid nanoparticles lacking a ligand on its outer surface (siRE: striped bar; siMM: grey bar).
B: Bar graph showing reduced expression of the RUNX1/ETO fusion gene over time after sequential administration (indicated with black arrow) of a lipid nanoparticle loaded with an siRE molecule and comprising a ligand according to Formula (II) on its outer surface (white bar), compared to expression of the RUNX1/ETO fusion gene upon administration of a comparable lipid nanoparticle loaded with an siMM molecule (grey bar), and expression of the RUNX1/ETO fusion gene upon administration of comparable lipid nanoparticles loaded with an siRE or siMM molecule and lacking a ligand on its outer surface (siRE: striped bar; siMM: black bar) in absence of MSCs.
C: Bar graph showing reduced expression of the RUNX1/ETO fusion gene over time after sequential administration (indicated with black arrow) of a lipid nanoparticle loaded with an siRE molecule and comprising a ligand according to Formula (II) on its outer surface (white bar), compared to expression of the RUNX1/ETO fusion gene upon administration of a comparable lipid nanoparticle loaded with an siMM molecule (grey bar), and expression of the RUNX1/ETO fusion gene upon administration of comparable lipid nanoparticles loaded with an siRE or siMM molecule and lacking a ligand on its outer surface (siRE: striped bar; siMM: black bar) in presence of MSCs.
Figure 10: Western blot analysis showing two-fold reduction of RUNX1/ETO protein three and six days after addition of a lipid nanoparticle loaded with siRE RNA comprising a ligand according to Formula (II) on its outer surface, both in presence and absence of MSCs after three (upper part) and six days (lower part). T- LNPsiRE: targeted LNPsiRE containing LDV ligand.
Figure 11: FACS images showing a shift of subpopulations from immature CD34+ cells to more mature CD34- CD 15+ cells indicating strong induction of myeloid differentiation upon administration of a lipid nanoparticle according to the invention, loaded with siRE molecule (lower graphs) compared to a comparable lipid nanoparticle loaded with siMM (control; upper part), both in absence (left) and presence (right) of MSCs.
Figure 12: A: Dosing scheme of a lipid nanoparticle according to the invention administered in a mouse model.
B: Graph showing relationship between weight as a result of treatment with a lipid nanoparticle according to the invention.
C: Picture of spleen of mice treated with a lipid nanoparticle loaded with siRE RNA according to the invention (HB279; HB280; HB281) compared to control (HB282 and HB283).
Figure 13: Graph showing uptake of a Cy3-labelled lipid nanoparticle loaded with siRE RNA and comprising a ligand according to Formula (II) on its outer surface in primary AML cells from three different patients (A B and C), compared to a Cy3-labelled lipid nanoparticle loaded with siMM RNA and comprising a ligand according to Formula (II) on its outer surface, and comparable Cy3-labelled lipid nanoparticles lacking a ligand on its outer surface.
Figure 14: A: Graph showing reduced expression of RUNX1/ETO fusion gene in t(8;21) primary cells after sequential administration (indicated with a black arrow) of a lipid nanoparticle loaded with an siRE molecule (white bar) comprising a ligand according to Formula (II) on its outer surface, compared to a lipid nanoparticle loaded with siMM (control, grey bar) comprising a ligand according to Formula (II) on its outer surface. B: Western blot analysis showing reduction of RUNX1/ETO protein in primary cells twelve days after sequential addition (6 days after the last dose) of a lipid nanoparticle loaded with siRE RNA comprising a ligand according to Formula (II) on its outer surface.
Figure 15: A: t-SNE plot showing a shift of subpopulations from immature CD34+ cells towards CD34- cells as determined by a multiparameter flow analysis upon administration of a lipid nanoparticle according to the invention, loaded with siRE molecule (right) compared to a comparable lipid nanoparticle loaded with siMM (control; left) in patient-derived material. B: UMAP showing a shift of subpopulations from immature CD34+ cells towards CD34- cells as determined by single cell RNAseq. This confirms the loss of CD34 as determined by the antibody panel (Figure 15A) and indicates induction of myeloid differentiation upon administration of a lipid nanoparticle according to the invention, loaded with siRE molecule (right) compared to a comparable lipid nanoparticle loaded with siMM (control; left) in patient-derived material. C: t-SNE plot showing a shift of RUNX1/ETO positive cells towards RUNX1/ETO negative cells as determined by a multiparameter flow analysis upon administration of a lipid nanoparticle according to the invention, loaded with siRE molecule (right) compared to a comparable lipid nanoparticle loaded with siMM (control; left) in patient-derived material. D: Immunohistochemistry of primary t(8;21) cells show the loss of the ETO protein upon administration of a lipid nanoparticle according to the invention, loaded with siRE molecule (middle) compared to a comparable lipid nanoparticle loaded with siMM (control; bottom) and untreated primary cells (control; top). This indicates the loss of the leukaemic fusion protein.
Figure 16: Bar graph showing reduced expression of RUNX1/ETO fusion gene in t(8;21) patient-derived cells of a lipid nanoparticle loaded with an siRE molecule, comprising DMG-PEG(2000) (white bar) or sphingomyelin (20% molar ratio) and cholesterol (20% molar ratio) (white bar, chess blocks) in the lipid outer layer, compared to a lipid nanoparticle loaded with siMM, comprising DMG-PEG(2000) (grey bar) or sphingomyelin (20% molar ratio) and cholesterol (20% molar ratio) (grey bar, black line).
Figure 17: A: siRNA targeting the unique breakpoint of the RUNX1/ETO t(8;21) fusion transcript (siRE) and the mismatch control (siMM) generated by swapping two nucleotides in the sequence. B: To improve the stability the siRNAs were modified by introducing 2’-deoxy- (2’-H), 2’-fluoro (2’-F) and 2’-methoxy (2’- OMe) ribose modifications and 3’-terminal phosphorothioate (PS) linkages.
Figure 18: A: Graph showing the cell cycle profile of t(8;21) cells on day 6 upon administration of a lipid nanoparticle according to the invention, loaded with siRE molecule compared to a comparable lipid nanoparticle loaded with siMM (control) in cell lines. The LNPsiRE-mod treated cells accumulate more in the G0/G1 phase and proliferate less as indicated by the decrease in cells in the S- phase. B: Bar graph showing the senescent t(8;21) cells on day 6 stained by senescence-associated beta-galactosidase staining upon administration of a lipid nanoparticle according to the invention, loaded with siRE molecule (right) compared to a comparable lipid nanoparticle loaded with siMM (control; left) in cell lines. C: Bar graph showing the colony formation units in first (left) and second (right) platings upon administration of a lipid nanoparticle according to the invention, loaded with siRE molecule compared to a comparable lipid nanoparticle loaded with siMM (control) in t(8;21) cell lines.
Figure 19: A: Figure (left) showing the distribution of labelled LNPs (LNP/NIR) (top) or control mice treated with free NIR dye in PBS (bottom) as measured by fluorescence. Figure (right) showing the fluorescence of the indicated organs of mice treated with LNP/NIR (top) or control mice (bottom). B: Figures (left) showing the bioluminescence signal linked to tumour cells (left), the fluorescence signal linked to LNPs and overlay (overlay of bioluminescence and fluorescence signals) in mice. Figure (right) showing the fluorescence and bioluminescence signal co-localizing in harvested tumours from mice.
Figure 20: A: Western blot analysis showing reduction of RUNX1/ETO protein in cells harvested from mice after sequential addition of a lipid nanoparticle loaded with siRE RNA or siMM RNA. Each lane represents one animal. * denotes two tumours from the same animal. B: Bar graph showing the senescent t(8;21) cells stained by senescence-associated beta-galactosidase staining upon administration of a lipid nanoparticle according to the invention, loaded with siRE molecule (right) compared to a comparable lipid nanoparticle loaded with siMM (control; left) in cells harvested from mice. C: Bar graph showing the colony formation units upon administration of a lipid nanoparticle according to the invention, loaded with siRE molecule (right) compared to a comparable lipid nanoparticle loaded with siMM (control; left) in cells harvested from mice.
Figure 21: A: Graph showing the quantification of bioluminescence signal in mice upon administration of a lipid nanoparticle loaded with siRE molecule (round) compared to a comparable lipid nanoparticle loaded with siMM (control; square). B: Kaplan-Meier graph showing the survival curves of mice after sequential administration of lipid nanoparticles loaded with siRE molecule compared to lipid nanoparticles loaded with siMM (control). C: Graph showing the quantification of bioluminescence signal in mice that were transplanted with cells harvested from mice treated with a lipid nanoparticle loaded with siRE molecule (round) compared to a lipid nanoparticle loaded with siMM (control; square). D: Kaplan-Meier graph showing the survival curves of mice after transplantation of cells harvested from mice that were treated sequentially with a lipid nanoparticle loaded with siRE molecule compared to a comparable lipid nanoparticle loaded with siMM (control). E: Western blot analysis showing the RUNX1/ETO protein expression in cells harvested from the recipients upon administration of a lipid nanoparticle loaded with siRE molecule compared to a lipid nanoparticle loaded with siMM molecule (control).
Figure 22: A: siRNA targeting the unique breakpoint of the MLL/AF4 t(4; 11) fusion transcript. To improve the stability the siRNAs were modified by introducing 2’-deoxy- (2’-H), 2’-fluoro (2’-F) and 2’-methoxy (2’-OMe) ribose modifications and 3’-terminal phosphorothioate (PS) linkages. B: Bar graph showing reduction of expression of MLL/AF4 and its target gene HOXA7 over time (1 day, black columns; 3 days, grey columns; 6 days, white columns) in SEM cells as a result of administration of a lipid nanoparticle loaded with siMAG or siMAG- M2 RNA compared to a control lipid nanoparticles loaded with siMM or siMM-mod RNA, respectively. C: Graph showing proliferation of SEM cells over time after administration of a lipid nanoparticle loaded with siMA6-M2 (diamond), compared to lipid nanoparticles loaded with siMM (control; square), siMM-mod (modified control, triangle down), siMAG (unmodified, triangle up) and a mock sample (circles). D: Bar graph showing number of colony formation of SEM cells after administration of lipid nanoparticle loaded with siMA6-M2 compared to controls (striped bar) and loaded with siMM (black bar). Cells were seeded for colony formation 1 day (black columns) and 3 days (grey columns) after administration of lipid nanoparticles.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
The term “or” as used herein is defined as “and/or” unless specified otherwise.
The term “a” or “ an” as used herein is defined as “at least one” unless specified otherwise.
The term “substantially)” or “essentially)” is generally used herein to indicate that it has the general character or function of that which is specified. When referring to a quantifiable feature, these terms are in particular used to indicate that it is for at least 75 %, more in particular at least 90 %, even more in particular at least 95 % of the indicated feature.
When referring to a noun in the singular, the plural is meant to be included, unless it follows from the context that it should refer to the singular only.
The term “lipid nanoparticle” as used herein refers to a particle comprising a lipid outer layer at least partly encapsulating a lipid-therapeutic molecule complex. Said particle preferably is substantially spherical. The lipid outer layer typically comprises at least an ionizable or charged lipid comprising respectively an ionizable or charged portion and a hydrophobic portion. The ionizable portion is typically positively charged at low pH, i.e. a pH of below 5, and neutral at physiological pH. The charged portion is typically positively charged at physiological pH. The ionizable or cationic lipid further comprises a hydrophobic portion, such as an aliphatic portion of a fatty acid. In a lipid nanoparticle, the charged or ionizable portion is generally projected outwards, or, if present, projected towards a negatively charged therapeutic molecule, whereas the hydrophobic portion is generally projected inwards, or, if present, projected towards a hydrophobic portion of a therapeutic molecule. In solution, the ionizable or cationic portion is preferably capable of interacting with a liquid (polar) medium, such as water or, if present, with the negatively charged therapeutic molecule, and the hydrophobic portion is shielded therefrom. The lipid outer layer typically comprises one or more helper lipids, such as cholesterol, a phospholipid or a sphingolipid and may comprise one or more PEG-lipids. The lipid outer layer may further comprise a lipid monolayer, comprising a single layer of lipids, or a lipid bilayer wherein the outer layer is formed of two lipid layers. In such lipid bilayer, the hydrophobic portions of both layers are preferably facing each other.
The term “PEG-lipid” as used herein refers to a lipid comprising at least one poly(ethylene) glycol (PEG) moiety. A polyethylene glycol moiety has a molecular formula of C2nH4n+2On+1, wherein n is an integer typically varying from 5 to 100, preferably from 10 to 70, more preferably from 20 to 60, in particular around 40-50. The molecular weight of a PEG moiety may be calculated using the formula 44.05n+ 18.02 g/mol. Typically, a PEG molecule is referred to as PEG(average molecular weight). Hence a PEG(2000) has an average molecular weight of about 1900-2200 Da. A PEG-lipid may comprise one or more PEG moieties per lipid molecule, typically a PEG-lipid comprises between one and five PEG-moieties per lipid molecule, in particular around one PEG-moiety per lipid molecule.
The term “therapeutic molecule” as used herein refers to any molecule that has a pharmaceutical benefit in a subject in need thereof, when provided at a therapeutically effective amount.
The term “small interfering ribonucleic acid” (siRNA) as used herein refers to a short single or double stranded ribonucleic acid molecule that is capable of base pairing with a messenger RNA (mRNA) transcript of a target gene. Upon binding the mRNA said mRNA is typically degraded through a process commonly referred to in the art as RNA interference. Hereby, translation of the mRNA transcript into a functional protein is inhibited or prevented, thereby effectively inhibiting or preventing functional expression of said target gene. The length of an siRNA molecule typically is between 18 to 27 nucleotides.
The term “inhibiting”, “inhibition” or “inhibited” in the context of siRNA refers to the process of reducing the number of mRNA transcripts in a cell compared to the wildtype cell (wherein no siRNA is present), so that a reduced number of proteins will be generated in that cell. Generally, inhibition refers to a reduction of 50 mol% or more of protein or RNA that is produced in a cell, when compared to a wild-type cell wherein no siRNA is present.
As used herein, the term “target gene” refers to a section located on a chromosome comprising a protein-coding or an RNA-coding sequence. Typically, a target gene in the context of the present invention is involved in the onset or development of a disease or medical condition.
The term “fusion gene” as used herein refers to a combination of two or more genes which are normally located at distinct locations on a chromosome or on different chromosomes, but are joined as a result of a chromosomal aberration.
The term “chromosomal aberration” as used herein refers to an abnormality in one or more chromosomes, typically leading to a structural change or defect, such as translocation of a chromosome fragment. The chromosomal aberration may be genetic, or may be caused by external factors.
As used herein, the terms "encoding", "coding for", or "encoded" when used in the context of a specified nucleic acid mean that the nucleic acid comprises the requisite information to guide translation of the nucleotide sequence into a specified protein. The information by which a protein is encoded is specified by the use of codons. A nucleic acid encoding a protein may comprise a non-translated sequence, e.g. an intron, interspersed with translated regions of the nucleic acid or may lack such an intervening non-translated sequence.
As used herein, the term "protein" or "polypeptide" refers to a chain of amino acids arranged in a specific order determined by the coding sequence in a polynucleotide encoding the polypeptide.
As used herein, the term “Runt-Related Transcription Factor 1 (RUNX1) refers to a gene on chromosome 21q22 that encodes a transcription factor. The gene is characterized by HUGO Gene Nomenclature Committee accession number 10471; and by Ensembl accession number ENSG00000159216. The encoded protein is characterized by Uniprot accession number Q01196.
As used herein, the term “ETO”, or “RUNX1 Partner Transcriptional Co- Repressor 1” (RUNX1T1) refers to a gene on chromosome 8q21 that encodes a transcriptional corepressor. The gene is characterized by HUGO Gene Nomenclature Committee accession number 1535; and by Ensembl accession number ENSG00000079102. The encode protein is characterized by Uniprot accession number Q06455.
As used herein, the term “RUNX1/ETO” fusion gene refers to a chromosome (8;21)(q21;q22) translocation, which is one of the most frequent karyotypic abnormalities in acute myeloid leukemia. The translocation produces a chimeric gene made up of the 5'-region of the RUNX1 gene fused to the 3'-region of the ETO gene. The chimeric protein is thought to associate with the nuclear corepressor/histone deacetylase complex to block hematopoietic differentiation.
As used herein, the term “RUNX1/ETO” polypeptide refers to a polypeptide that is encoded by a RUNX1/ETO fusion gene.
As used herein, the term “functional expression”, in relation to a gene, refers to the expression of a gene product, such as a protein product, that is able to completely fulfil its function. A reduction of the functional expression of a gene may be accomplished by reduced expression of the encoded product such as protein product, by expression of a product, such as protein product, with reduced functional activity, or both reduced expression of a product with reduced activity. In the context of this invention, a reduction of functional expression is preferably accomplished by reduced expression of a gene product. The term “complementary” as used herein refers to a strand of RNA or DNA that is capable of hybridizing with another strand of RNA or DNA that it is complementary to, thereby forming a duplex structure. Said hybridizing typically occurs through the formation of one or more Watson-Crick base pairs. Not all nucleotides present in complementary RNA or DNA strands are required to form base pairs. The minimal number of base pairs is the number required to form a duplex structure. As is generally known, the formation of a duplex structure depends on the environmental conditions such as the temperature and/or salt concentration. Generally, at least 12 base pairs are required to form a duplex structure, in particular at least 15 base pairs. The maximum number of base pairs formed in complementary strands is the total number of nucleotides present in a strand.
The term “functional expression” as used herein refers to the transcription or translation of a nucleic acid into a functional RNA or polypeptide. The term “functional” as used herein refers to the ability of said RNA or polypeptide to perform its natural function, e.g. as observed in the wildtype.
The term “percentage sequence identity” as used herein refers to the percentage of sequence overlap when comparing a sequence with a reference sequence, i.e. bait sequence. For optimal alignment of the two sequences, the two sequences are preferably compared over the full length of the reference sequence. When a polypeptide or polynucleotide is compared to a reference sequence, i.e. a bait sequence, the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise an addition or a deletion (i.e. a gap) as compared to the reference sequence (which does not comprise an addition or a deletion). The percentage is calculated by determining the number of positions at which an identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.
When referring to “A”, “C”, “G”, “U”, “T” on a specific position in a sequence of siRNA unless specified otherwise, a nucleotide is meant comprising an unmodified ribose moiety attached to a nucleobase indicated with the letters A (adenine), C (cytosine), G (guanine), thymine (T), or uracil (U). For example, a “T” on a specific position in a sequence of siRNA indicates the presence of a nucleotide comprising a ribose moiety attached to a thymine, also referred to in the art as 5-methyluridine or ribothymidine. For example, a “T” on position 20 of the sequence of SEQ ID NO:1 indicates the presence of a ribothymidine, on position 20 of SEQ ID NO:1.
When referring to “dX”, “XPS”, “XoMe”, “XF”, or the like, on a specific position in a sequence of siRNA a nucleotide is meant comprising a modified ribose moiety attached to any nucleobase (X). For example, “dT” refers to a nucleotide comprising a modified ribose attached to a thymine, wherein the hydroxyl group on the 2- position of the ribose moiety is substituted for a hydrogen; “TPS” refers to a nucleotide attached to a thymine, comprising a modified ribose, wherein the phosphodiester group on the 3-position of the ribose moiety is substituted for a phosphorothioate; “CoMe” refers to a nucleotide comprising a modified ribose attached to a cytosine, wherein the hydroxyl group on the 2-position of the ribose moiety is substituted for a methoxy-group and “CF” refers to a nucleotide comprising a modified ribose attached to a cytosine, wherein the hydroxyl group on the 2-position of the ribose moiety is substituted for a fluoride.
The term “purifying'’ as used herein in the context of lipid nanoparticles, refers to at least partly separating a lipid nanoparticle from its environment. “Purifying” may encompass removing a water-miscible organic solvent from an aqueous solution comprising lipid nanoparticles. “Purifying'’ may further encompass removal of impurities and other substances until the solution comprising lipid nanoparticles is substantially pure. “Substantially pure” is used herein to indicate that the substance, e.g. a lipid nanoparticle or a lipid nanoparticle loaded with a therapeutic molecule has a purity of at least 95%, more preferably at least 96%, at least 97%, at least 98%, at least 99% or at least 100% pure, based on the total weight of the substance, e.g. lipid nanoparticle or lipid nanoparticle loaded with a therapeutic molecule, as determined with standard analytical techniques known in the art on the date of filing.
Lipid nanoparticles and compositions comprising said lipid nanoparticles
The invention relates to a lipid nanoparticle loaded with a therapeutic molecule, which lipid nanoparticle comprises a ligand on its outer surface capable of binding a receptor present on an outer surface of a target cell.
A lipid nanoparticle according to the invention advantageously comprises a ligand on its outer surface capable of binding a receptor present on an outer surface of a target cell. The presence of a ligand allows the targeting of specific cell types, expressing a receptor on an outer surface of said cell, and thus provides the possibility to target specific cell types (expressing said receptor on an outer surface), whereas other cell types (not expressing, or less abundantly expressing said receptor on an outer surface) are not or less targeted.
Such an approach advantageously allows more rapid and more efficient delivery of a therapeutic molecule to a target cell, because a therapeutic molecule may be specifically released in said target cell expressing said receptor on an outer surface. Further, such an approach may result in fewer side effects, because the therapeutic molecule is released to fewer or no cells that do not, or to a lesser extent, express a receptor on its outer surface.
In principle, any target cell may be suitable in the context of the present invention, but preferably, the target cell is a cell of the hematopoietic lineage. In a further preferred embodiment, the target cell is a cancer cell, more preferably an acute myeloid leukemia (AML) cell, such as a t(8;21) AML cell, including a Kasumi- 1 cell and a SKNO-1 cell.
Said target cell preferably comprises a unique RNA molecule that can be targeted with a negatively charged therapeutic molecule such as an oligonucleotide, in particular short interfering RNA (siRNA), that can specifically hybridize with said unique RNA molecule, thereby inducing RNA interference.
Lipids
A lipid nanoparticle according to the invention preferably comprises one or more, preferably two or more, in particular three or more, or even four or more lipid molecules that are assembled together to form a lipid nanoparticle.
Preferably, said lipid nanoparticle comprises at least one cationic or ionizable lipid.
A cationic or ionizable lipid is preferably present in a lipid nanoparticle according to the invention, if a lipid nanoparticle according to the invention is loaded with a negatively charged therapeutic molecule, preferably an oligonucleotide, in particular short interfering RNA (siRNA). In such a case, a cationic lipid was found to promote encapsulation of said negatively charged therapeutic molecule into said lipid nanoparticle, by forming a lipid-siRNA complex and stabilize a lipid nanoparticle loaded with said negatively charged therapeutic molecule.
A cationic lipid is a lipid that carries a net positive charge at physiological pH or lower, e.g. at pH of 7.5 or lower, such as 7.0 or lower.
Examples of cationic lipids include (N-[l-(2,3-Dioleoyloxy)propyl]-N,N,N- trimethylammonium (DOTAP), l,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), dioctadecylamidoglycylspermine (DOGS), 2,3-dioleyloxy-N-[2- (sperminecarboxamido)ethyl]-N,N-dimethyl -1-propanaminium trifluoroacetate (DOSPA), 3β- [N-(N',N'-dimethylamino-ethane)carbamoyl] -cholesterol (DC-Chol), N- [3-[2-(l,3-dioleoyloxy)propoxy-carbonyl]propyl]-N,N,N-trimethylammonium iodide (YKS-220), lipofectamine® 2000 (Invitrogen), Oligofectamine (Invitrogen).
An ionizable lipid is a lipid that is positively charged at a pH of 6.5 or lower, such as at a pH of 5 or lower, but is typically neutral at physiological pH. Inclusion of an ionizable lipid into a lipid nanoparticle according to the invention typically facilitates loading of lipid nanoparticles with negatively charged therapeutic molecules at acidic pH. Further, inclusion of an ionizable lipid in a lipid nanoparticle according to the invention may restore neutral or even negative surface charge of a lipid nanoparticle at physiological pH, which is associated with increased circulation times and lower aggregation (Zhao et al. 2011. Int J Nanomedicine 6:3087-3098).
Examples of ionizable lipids include (6Z,9Z,28Z,31Z)-heptatriacont-6,9,28,31- tetraene- 19-yl 4-(dimethylamino)butanoate (Dlin-MC3-DMA) or 2,2-dilinoleyl-4- dimethylaminoethyl- [ 1, 3] -dioxolane (DLin-KC2-DMA) .
Preferably, a lipid nanoparticle according to the invention comprises at least the ionizable lipid Dlin-MC3-DMA.
A lipid nanoparticle according to the invention further preferably comprises one or more helper lipids. The role of helper lipids in lipid nanoparticles has not been fully elucidated. However, without wishing to be bound by any theory, it is believed that helper lipids typically improve one or more physical properties of a lipid nanoparticle, such as, for example the stability of a lipid nanoparticle, in particular the in vivo stability of a lipid nanoparticle, intracellular delivery of a lipid nanoparticle, or release of a therapeutic molecule from a lipid nanoparticle into a target cell. Examples of helper lipids include cholesterol or a derivative thereof, β- sitosterol (Sito), fucosterol (Fuco), campesterol (Camp), stigmastanol (Stig), dioleoylphosphatidylethanolamine (DOPE), phosphatidylcholine, such as distearoylphosphatidylcholine (DSPC), l,2-dipalmitoyl-sn-glycero-3- phosphaoethanolamine, 1,2-dimyristoyl-rac-glycerol and sphingomyelin.
Examples of derivatives of cholesterol include cholestenol, cholestanone, cholestenone, coprostanol, cholesteryl- 2’-hydroxylethyl ether, cholesteryl-4’- hydroxybutyl ether, and mixtures thereof.
Preferably, a lipid nanoparticle according to the invention comprises one or more, preferably two or more, or all of cholesterol or a derivative thereof, sphingomyelin and DSPC.
Preferably, a lipid nanoparticle according to the invention comprises a PEG- lipid. A lipid nanoparticle comprising a PEG-lipid may improve colloidal stability of a lipid nanoparticle in vitro and may further improve circulation time of the lipid nanoparticle in vivo (Klibanov et al, 1990. FEES Lett. 268:235-7).
In principle, any PEG-lipid may be used, but preferably a PEG-lipid is used with a molecular weight of PEG in the range of 350 Da to 6000 Da, preferably between 550 Da and 5000 Da, more preferably between about 750 Da and about 4000 Da, even more preferably between about 1000 Da and about 3000 Da, in particular about 2000 Da.
Preferred PEG-lipids in a lipid nanoparticle according to the invention is DMG-PEG or DSPE-PEG, more preferably DMG-PEG1000-6000 or DSPE-PEG1000-6000, most preferably DMG-PEG2000 or DSPE-PEG2000.
A lipid nanoparticle according to the invention preferably comprises at least the ionizable lipid (6Z,9Z,28Z,31Z)-heptatriacont-6,9,28,31-tetraene-19-yl 4- (dimethylamino)butanoate (Dlin-MC3-DMA), at least one helper lipid selected from the group consisting of distearoylphosphatidylcholine (DSPC), cholesterol and sphingomyelin and at least one PEG-lipid selected from the group consisting of DMG-PEG and DSPE-PEG, preferably DMG-PEG1000-6000 or DSPE-PEG1000-6000, more preferably DMG-PEG(2000) and DSPE-PEG(2000).
The molar ratio between cationic and/or ionizable lipid, helper lipid and PEG- lipid in a nanoparticle according to the invention, is preferably between l:0.5:0 and 1:1.4:0.05, more preferably said ratio is between 1:0.8:0.01 and 1:1.2:0.03, in particular around 1:1:0.03 or 1:1:04. Preferably a lipid nanoparticle according to the invention comprises the lipids Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG, preferably DMG-PEG1000-6000 , more preferably DMG-PEG2000. Preferably herein, the molar ratio Dlin-MC3- DMA:DSPC:cholesterol:DMG-PEG is between about 1:0.1:0.5:0.02 and about 1:0.4:0.9:0.05, more preferably between about 1:0.2:0.7:0.03 and about 1:0.3:0.8:0.04.
In an alternative preferred embodiment, a lipid nanoparticle according to the invention comprises the lipids Dlin-MC3-DMA, DSPC, cholesterol and sphingomyelin. An advantage of such a lipid nanoparticle is that the presence of a PEG-lipid is not required in the lipid nanoparticle according to the invention. PEG- lipids may disadvantageously induce an immune response in a subject it is administered to. Therefore, it may be advantageous to provide a lipid nanoparticle that is essentially free of, or comprises a low content of a PEG-lipid, such as DSPE- PEG or DMG-PEG.
Preferably herein, the ratio Dlin-MC3-DMA:DSPC:cholesterol:spingomyelin is between about l:0.1:0.2:0.2 and about l:0.4:0.5:0.5, more preferably between about l:0.2:0.3:0.3 and about l:0.3:0.4:0.4.
In yet another preferred embodiment, a lipid nanoparticle according to the invention comprises the lipids Dlin-MC3-DMA, DSPC, cholesterol, sphingomyelin and a PEG-lipid preferably DMG-PEG or DSPE-PEG, more preferably DSPE- PEG1000-6000 or DMG-PEG1000-6000, even more preferably DSPE-PEG2000 or DMG- PEG2000. In such a lipid nanoparticle, the advantages of the presence of a PEG- lipid, such as a positive effect on the colloidal stability of a lipid nanoparticle in vitro and improvement of circulation time of the lipid nanoparticle in vivo are maintained, whilst the risk of inducing an undesired immune response is markedly reduced.
Preferably herein, the ratio Dlin-MC3- DMA:DSPC:cholesterol:sphingomyelin:DSPE-PEG is between about 1:0.1:0.2:0.2:0.001 and about 1:0.4:0.5:0.5:0.005, more preferably between about 1:0.2:0.3:0.3:0.002 and about 1:0.3:0.4:0.4:0.003.
Therapeutic molecule
The therapeutic molecule in a lipid nanoparticle according to the invention may be any molecule having a pharmaceutical benefit when administered in a therapeutically effective amount to a subject in need thereof. For example, the therapeutic molecule may be a small molecule, a natural product, such as Paclitaxel, known under the tradename Taxol®, a biomolecule, such as an oligonucleotide, for example a small interfering ribonucleic acid (siRNA), an antisense oligonucleotide (ON), messenger RNA (mRNA), or microRNA (miRNA) or a polypeptide, such as an antibody, a conjugate, or a combination thereof.
Preferably, said therapeutic molecule is a negatively charged molecule, more preferably an oligonucleotide, in particular an siRNA molecule. Said siRNA molecule may be single stranded or double stranded RNA preferably double stranded RNA.
Said siRNA molecule preferably comprises a first RNA strand which is complementary to an mRNA transcript of a target gene, or a portion of a target gene. Optionally, said siRNA molecule comprises a second RNA strand which is complementary to said first RNA strand.
Said first RNA strand may efficiently mediate RNA interference, by hybridizing to an mRNA transcript of a target gene, or a part thereof. Upon hybridizing, said siRNA molecule may direct an enzyme complex to degrade said mRNA transcript, thereby preventing translation of said mRNA transcript into a polypeptide and thus effectively inhibiting functional expression of said target gene.
Said target gene preferably expresses an unique RNA molecule that can be targeted with a negatively charged therapeutic molecule such as an oligonucleotide, in particular short interfering RNA (siRNA), that can specifically hybridize with said unique RNA molecule, thereby inducing RNA interference. Said unique RNA molecule may be the resultant of an alteration or rearrangement of a gene, for example a deletion or a duplication of a part of the RNA transcript of said gene, or the resultant of translocation resulting in a gene fusion by which part of a first gene becomes connected to a part of a second gene. The sequences of the gene at around the deletion, duplication or gene fusion break points provide an unique RNA molecule that can be targeted with a negatively charged therapeutic molecule such as an oligonucleotide.
Examples of such unique RNA molecules are provided by gene alterations including mutations in several epigenetic regulators such as ASXL1, DNMT3A EZH2, IDH1/2 and TET2.2, 3 DNMT3A IDH1/2 and TET2 (Brecqueville et al., 2011. Blood Cancer J 1, e33), IKZF1 (Stanulla et al., 2020. Blood 135: 252-260) and UBTF-TD (Umeda et al., 2022. Blood Cancer Discovery 2: 193-207).
Examples of such unique RNA molecules are provided by the BCR/ABL fusion gene product, the MLL/AF4 fusion gene product, the RUNX1/ETO fusion gene product, the CTLA4/CD28 fusion gene product, the NRIP1/MIR99AHG fusion gene product, the LATS2/ZMYM2 fusion gene product, the ATP11A/ING1 fusion gene product, the MBP/SLC66A2 fusion gene product, or the PRDM16/SKI fusion gene product. See, for example, Chen et al., 2021. Blood Cancer J 11: 112 https://doi.org/10.1038/s41408-021-00504-5.
Preferably said siRNA hybridizes to an mRNA transcript of the RUNX1/ETO gene, thereby inhibiting functional expression of the RUNX1/ETO fusion gene. By inhibiting function expression of a RUNX1/ETO fusion gene, translation of said fusion gene into the RUNX1/ETO protein is inhibited and/or silenced, which protein is believed to be involved in inhibition of hematopoietic differentiation. Thus, inhibiting and/or silencing of functional expression of the RUNX1/ETO fusion gene may enhance hematopoietic differentiation. Accordingly, inhibition or silencing of functional expression of the RUNX1/ETO fusion gene provides a fruitful approach towards treating leukemia, preferably amyloid myeloid leukemia (Agrawal et al., 2020. Leukemia 34: 630-634).
Preferably, functional expression of the RUNX1/ETO gene is inhibited by at least 25%, more preferably at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, most preferably about 100%.
Preferably, functional expression of the RUNX1/ETO gene is silenced, by inhibiting functional expression of the RUNX1/ETO gene such that its function is essentially lost. Typically, functional expression of the RUNX1/ETO gene is silenced, by inhibiting functional expression of the RUNX1/ETO gene by more than 70%, preferably more than 80%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, in particular 100%.
Accordingly, preferably, said siRNA comprises a first RNA strand complementary to an mRNA transcript of the RUNX1/ETO fusion gene, and optionally a second RNA strand complementary to the first RNA strand. In principle, said siRNA molecule may be any siRNA molecule that is capable of hybridizing with an mRNA transcript from the RUNX1/ETO fusion gene. Typically, an siRNA molecule is capable of hybridizing to an mRNA transcript of an RUNX1/ETO fusion gene when at least 50% of the nucleotides, preferably at least 75% of the nucleotides, more preferably at least 85% of the nucleotides are capable of forming a base pair.
Satisfactory results have been obtained with an siRNA molecule wherein said first RNA strand is represented by the sequence 5’- CCUCGAAAUCGUACUGAGAUU -3’ (SEQ ID NO:1). When present, said second RNA strand is preferably represented by the sequence 5’- UCUCAGUACGAUUUCGAGGUU-3’ (SEQ ID NO:2).
Accordingly, the inventors found that with a lipid nanoparticle according to the invention, comprising an siRNA molecule represented by SEQ ID NO:1 was effective in inhibiting functional expression of a RUNX1/ETO fusion gene when administered to (t8;21) AML cells.
Further, with a lipid nanoparticle according to the invention, comprising an siRNA molecule represented by SEQ ID NO:1, increased expression of Lysosomal Protein Transmembrane 5 (LAPTM5) and CCAAT Enhancer Binding Protein Alpha (CEBPA), and decreased expression levels of the adhesion molecule CD34, Angiopoietin 1 (ANGPT1) and Cyclin D2 (CCND2) in t(8;21) AML cells was observed. All these genes are target genes of RUNX1/ETO and are either repressed (LAPTM5, CEBPA) or activated (CD34, ANGPT1, CCND2)by RUNX1/ETO. The observed changes in gene expression provide functional prove for RUNX1/ETO loss.
Therefore, the invention also preferably relates to a lipid nanoparticle according to the invention, wherein the therapeutic molecule is an siRNA molecule comprising a first strand represented by the sequence as shown in SEQ ID NO:1, or a functional derivative having at least 70% sequence identity thereto, preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity thereto.
Optionally, said siRNA molecule comprises a second strand complementary to said first strand represented by the sequence as shown in SEQ ID NO:2 or a functional derivative having at least 70% sequence identity thereto, preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity thereto. The siRNA molecule preferably comprises between about 18 to about 27 nucleotides, more preferably between 19 and 25 nucleotides, between 20 and 23 nucleotides, in particular 21 nucleotides.
Preferably, the siRNA molecule comprises at least 18 nucleotides, more preferably at least 19 nucleotides, in particular at least 21 nucleotides.
Preferably, the siRNA molecule comprises at most 27 nucleotides, more preferably at most 25 nucleotides, in particular at most 24 nucleotides.
Said siRNA molecule may be modified to increase thermal stability of the siRNA molecule, to increase resistance of the siRNA molecule against nuclease degradation, and/or to increase the resistance of the siRNA molecule towards 2’- OH-dependent RNases including the RNase A family. Said modification may include one or more phosphorothioate (PS) bonds, substitution of one or more hydroxyl groups with one or more fluorides, substitution of one or more hydroxyl groups with one or more alkoxy groups including methoxy groups, substitution of one or more hydroxyl groups with hydrogen atoms and substitution of one or more uracil groups with one or more thymine groups.
Preferably said siRNA is modified, more preferably comprises one or more, more preferably two or more, even more preferably three or more, even more preferably four or more, in particular all five of a modification selected from the group consisting of a substitution of a hydroxyl group, preferably a 2-OH group of a ribose moiety of said siRNA with a methoxy group, a substitution of a hydroxyl group, preferably a 2-OH group of a ribose moiety of said siRNA with a fluoride or a substitution of a hydroxyl group, preferably a 2-OH group of a ribose moiety of said siRNA with a hydrogen, a substitution of a phosphodiester group, preferably a phosphodiester group on a 3-position of a ribose moiety of said siRNA, for a phosphorothioate group and a substitution of a uracil group with a thymine group.
A preferred siRNA molecule in a lipid nanoparticle according to the invention, preferably comprises at least 21 nucleotides, wherein the siRNA molecule comprises one or more, preferably two or more, more preferably three or more, even more preferably four or more, in particular all five of the following modifications:
(a) the sequence of SEQ ID NO:1 comprises a CFon position 1, aCF on position 2, anUr on position 3 and a CF on position 4, wherein CF and UF represent nucleotides wherein the hydroxyl group on the 2-position of the ribose moiety is substituted for a fluoride; (b) the sequence of SEQ ID NO:1 comprises dA on position 17, dG on position 18, dA on position 19, dT on position 20 and dT on position 21, wherein dA dG and dT represent nucleotides wherein the hydroxyl group on the 2-position of the ribose moiety is substituted for a hydrogen;
(c) the sequence of SEQ ID NO:1 comprises an UoMe on position 9, a CoMe on position 10, an UoMe on position 12, an CoMe on position 14 and a UoMe on position 15, wherein CoMe and UOME represent nucleotides wherein the hydroxyl group on the 2-position of the ribose moiety is substituted for a methoxy- group;
(d) the sequence of SEQ ID NO:1 comprises a TPS on position 20, and/or wherein the sequence of SEQ ID NO:2 comprises a TPS on position 20, wherein TPS represents a nucleotide wherein the phosphodiester group on the 3-position of the ribose moiety is substituted for a phosphorothioate.
(e) the sequence of SEQ ID NO: 1 comprises a T on position 20 and a T on position 21 and/or wherein the sequence of SEQ ID NO:2 comprises a T on position 20 and a T on position 21.
In a preferred embodiment, the sequence of SEQ ID NO:1 comprises a dTps on position 20 and a dT on position 21 and/or the sequence of SEQ ID NO:2 comprises a dTps on position 20 and a dT on position 21.
Without wishing to be bound by theory, it is believed that an siRNA molecule comprising modification a) renders the siRNA molecule more thermally stable. In addition, it is believed that modifications a) and b) independently or in combination, increase the resistance of the siRNA molecule towards 2’-OH- dependent RNases including the RNase A family (Sipa et al., 2007. RNA 13: 1301- 16; Khvorova et al., 2003. Cell 115: 209-16).
Further, it is envisaged that modification c) increases the resistance of said siRNA molecule against nuclease degradation (Selvam et al., 2017. Chem Biol Drug Des 90: 665-678). In addition, modification d) is envisaged to protect the siRNA molecule against exonuclease degradation (Stein, 1996. Chem Biol 3: 319- 23; Taylor et al., 1985. Nucleic Acids Res 13: 8765-85; Heidenreich et al., 1994. J Biol Chem 269: 2131-8).
Accordingly, the invention further relates to a lipid nanoparticle loaded with modified siRNA wherein said modified siRNA comprises a first RNA strand that is complementary to an mRNA transcript of the RUNX1/ETO gene and, optionally, a second RNA strand complementary to the first RNA strand, wherein said first RNA strand is preferably represented by the sequence as shown in SEQ ID NO:3 and wherein said second RNA strand, when present, is represented by the sequence as shown in SEQ ID NO:4.
Preferably, a modified siRNA molecule in a lipid nanoparticle according to the invention, comprises about 5% to about 100% of modified nucleotides, more preferably between 20% and 90%, between 30% and 85%, between 40% and 80%, between 50% and 75%, in particular between 60% and 75% of the nucleotides are modified nucleotides compared to a reference sequence, preferably compared to a sequence according to SEQ ID NO:1 and/or SEQ ID NO:2. Preferably, said modified nucleotides are nucleotides comprising a modified ribose moiety.
In particular, in a modified siRNA molecule in a lipid nanoparticle according to the invention, preferably about 15% to about 35% of the nucleotides are modified nucleotides, wherein a modified nucleotide comprises a modified ribose moiety, wherein the hydroxyl group on the 2-position of the ribose moiety is substituted for a fluoride. More preferably, about 20% to about 30% of the nucleotides are modified nucleotides, wherein the modified nucleotide comprises a modified ribose moiety, wherein the hydroxyl group on the 2-position of the ribose moiety is substituted for a fluoride.
In particular, in a modified siRNA molecule in a lipid nanoparticle according to the invention, preferably about 5% to about 30% of the nucleotides are modified nucleotides comprising a modified ribose moiety, wherein the hydroxyl group on the 2-position of the ribose moiety is substituted for a hydrogen. More preferably, about 10 to about 25% of the nucleotides are modified nucleotides, wherein the modified nucleotide comprises a modified ribose moiety wherein the hydroxyl group on the 2-position of the ribose moiety is substituted for a hydrogen.
In particular, in a modified siRNA molecule in a lipid nanoparticle according to the invention, preferably about 10% to about 30% of the nucleotides are modified nucleotides, wherein the modified nucleotide comprises a modified ribose moiety, wherein the hydroxyl group on the 2-position of the ribose moiety is substituted for a methoxy- group. More preferably, about 15% to about 25% of the nucleotides are modified nucleotides, wherein the modified nucleotide comprises a modified ribose moiety wherein the hydroxyl group on the 2-position of the ribose moiety is substituted for a methoxy- group. In particular, in a modified siRNA molecule in a lipid nanoparticle according to the invention, about 3% to about 8% of the nucleotides are modified nucleotides, wherein the modified nucleotide comprises a modified ribose moiety, wherein the phosphodiester group on the 3-position of the ribose moiety is substituted for a phosphorothioate. More preferably, wherein about 4% to about 6% of the nucleotides are modified nucleotides, wherein the modified nucleotide comprises a modified ribose moiety wherein the phosphodiester group on the 3-position of the ribose moiety is substituted for a phosphorothioate.
In particular, in a modified siRNA molecule in a lipid nanoparticle according to the invention, about 4% to about 11% of the nucleotides are modified nucleotides, wherein said modified nucleotide is a nucleotide comprising a ribose moiety attached to a thymine nucleobase.
In a lipid nanoparticle according to the invention, the molar ratio between lipid and therapeutic molecule is preferably between about 1:500 and about 1:150, more preferably between about 1:400 and about 1:250, in particular around 1:330.
Further, in a lipid nanoparticle according to the invention, the molar ratio between cationic or ionizable lipids and therapeutic molecule is preferably between 1:250 and 1: 75, more preferably between about 1:200 and about 1:130, in particular around 1:160.
In a lipid nanoparticle according to the invention, the ratio between positively charged groups, preferably amine groups (typically provided by a cationic or ionizable lipid) and negatively charged groups, preferably phosphate groups (typically provided by a therapeutic molecule) is preferably between 6:1 and 2:1, more preferably between 5:1 and 3:1, in particular about 4:1.
Receptor
The receptor present on an outer surface may be any suitable receptor that is present on a target cell. Preferably, the receptor is abundantly expressed on an outer surface of a target cell and/or undergoes endosomal recycling thus minimizing exposure of bound lipid nanoparticles to lysosomal degradation.
The receptor is preferably specifically expressed on an outer surface of a target cell, i.e. less abundantly or not expressed on a cell that are not a target cell. Such expression pattern allows more specific targeting of a target cell by a lipid nanoparticle according to the invention. Preferably, the concentration of the receptor on the outer surface of the target cell is twofold higher, more preferably fivefold higher, even more preferably tenfold higher, in particular one hundredfold higher compared to the concentration of the receptor on another cell that is not a target cell. In other words, the receptor is preferably twice as abundant on a target cell compared to another non-target cell, more preferably five times as abimdant, ten times as abundant, in particular one hundred times as abundant on a target cell compared to another non-target cell.
Examples of suitable receptors include integrin receptors, such as the Very Late Antigens VLA-4, VLA-5, VLA-6, LFA-1, CR3, and the transferrin receptor.
Preferably, the receptor is the VLA-4 receptor or a VLA-5 receptor, more preferably a VLA-4 receptor. Accordingly, the invention relates to a lipid nanoparticle loaded with a therapeutic molecule, which lipid nanoparticle comprises a ligand on its outer surface capable of binding to the VLA-4 receptor.
The VLA-4 receptor, or integrin a4Bl, is an integrin dimer composed of CD49d (a4) and CD29 (Bl). It is expressed on leukocytes, such as lymphocytes, eosinophils, monocytes, macrophages, natural killer cells, basophils and mast cells and mediates homing, trafficking, differentiation, activation, and survival of VLA-4 expressing cells.
By targeting the VLA-4 receptor, a nanoparticle according to the invention, comprising a ligand on its outer surface capable of binding to the VLA-4 receptor is taken up by leukocytes more rapidly compared to a comparable lipid nanoparticle that lacks a ligand capable of binding the VLA-4 receptor. Once taken up by the target cell, a lipid nanoparticle according to the invention may release a therapeutic molecule, having a therapeutic effect in said target cell. Hence, with a lipid nanoparticle according to the invention, a drug delivery approach specifically targeting leukocytes is provided.
Accordingly, a lipid nanoparticle according to the invention is particularly suitable for use in a method of treatment of diseases of leukocytes, including leukemia, such as amyloid myeloid leukemia.
Ligand
In principle, any ligand capable of binding the VLA-4 receptor is suitable to be present on the outer surface of a lipid nanoparticle according to the invention. Examples of natural ligands of VLA-4 include vascular cell adhesion molecule 1 (VCAM-1), mucosal vascular addressin cell adhesion molecule- 1 (MAdCAM-1), fibronectin and junctional adhesion molecule-B (JAM-B) (Imhof, 2004. Nat Rev Immunol 4: 432-444).
Preferably, the ligand comprises a structure according to Formula (I).
Figure imgf000031_0001
wherein R1 is preferably selected from a functional group selected from R1a to R1e,
Figure imgf000031_0002
wherein in R1c, the chiral carbon atom is in either in (R) or (S) configuration, preferably in (S) configuration; wherein in R1d, the chiral carbon atoms are independently in either (R) or (S) configuration, preferably both chiral carbon atoms are in (S) configuration; wherein X is preferably selected from a functional group selected from X1a and a peptide,
Figure imgf000032_0001
preferably wherein the peptide is a dipeptide, a tripeptide or a tetrapeptide, more preferably a tripeptide having the sequence leucine-asparagine-valine (Leu-Asp- Val or LDV), in particular L-Leu-L-Asp-L-Val; wherein W1 is an aliphatic group, preferably a benzyl, cyclohexyl, cyclobutyl, cyclopropyl, methyl cyclopropyl, methyl cyclobutyl, methyl cyclopentyl, methyl cyclohexyl, furan, methyl furan, pyrrole, methyl pyrrole, thiophene, methyl thiophene, methyl, ethyl, propyl, butyl or isopropyl group, more preferably wherein W1 is a cyclopropyl group or a benzyl group; wherein W2 is a hydrogen, a methyl, an ethyl, a propyl, a butyl, or a group having the following structure:
Figure imgf000032_0002
more preferably wherein W2 is a methyl or a phenyl; wherein Y is an aliphatic group, preferably a methyl group, an ethyl group, a propyl group, a butyl group, an isopropyl group, a cyclopropane group, a methyl cyclopropane group, an ethyl cyclopropane group, a cyclobutene group, a methyl cyclobutane group, an ethyl cyclobutene group, a cyclopentane group, a methyl cyclopentane group, an ethyl cyclopentane group, a cyclohexyl group, a methyl cyclohexyl group or an ethyl cyclohexyl group, more preferably wherein Y is isopropyl or cyclohexyl; wherein Z is an alkyl group, preferably a methyl group, an ethyl group, a propyl group, a butyl group, an isopropyl group, a methyl cyclopropane group, an ethyl cyclopropane group, a methyl cyclobutane group, an ethyl cyclobutene group, wherein V is an aliphatic or an aromatic group, preferably a methyl group, an ethyl group, a propyl group, a butyl group, an isopropyl group, a cyclopropane group a methyl cyclopropane group, an ethyl cyclopropane group, a cyclobutane group, a methyl cyclobutane group, an ethyl cyclobutene group, a cyclopentane group, a methyl cyclopentane group, a benzyl group, a phenyl group, a furan, a thiophene or a pyrrole.
With a lipid nanoparticle comprising a ligand comprising a structure according to formula (I), enhanced uptake said lipid nanoparticles in (8;21) AML cells was obtained compared to a comparable lipid nanoparticle lacking a ligand comprising a structure according to formula (I).
Further, a threefold and longer-lasting reduction of functional expression of the RUNX1/ETO gene was observed when a lipid nanoparticle comprising a ligand comprising a structure according to formula (I) was administered, compared to an approximate 1.5 fold reduction of functional expression when a comparable lipid nanoparticle, but lacking a ligand comprising a structure according to formula (I) was administered. Hence, a lipid nanoparticle according to the invention performs superiorly compared to comparable lipid nanoparticles in terms of uptake by VLA-4 expressing cells, such as (t8;21) AML cells and in terms of reducing functional expression of a target gene, such as RUNX1/ETO.
Further, it was demonstrated that administration of lipid nanoparticles comprising a ligand comprising a structure according to formula (I) caused a massive shift of subpopulations from immature CD34+ cells to more mature CD34- CD15+ cells indicating strong induction of myeloid differentiation by a lipid nanoparticle according to the invention. To the contrary, a comparable lipid nanoparticle lacking a ligand on its outer surface, did not significantly affect myeloid differentiation.
Excellent results have been obtained with a lipid nanoparticle according to the invention, wherein said ligand is a ligand according to formula (II). Therefore, the invention preferably relates to a lipid nanoparticle loaded with a therapeutic molecule, which lipid nanoparticle comprises a ligand comprising the structure according to formula (I), wherein R1 is X and wherein X is a peptide having the sequence LDV on its outer surface. Said ligand is represented by formula (II):
Figure imgf000034_0001
Alternatively or additionally, the ligand present on the outer surface of a lipid nanoparticle according to the invention, may comprise any of the structures shown in Table 1 of Baiula et al., 2019. Front Chem 7: 489.
Preferably, the ligand present on an outer surface of the nanoparticle according to the invention, has a KD value for the VLA-4 receptor between 1 pM and 100 nM, preferably between 2 pM and 20 nM as determinable with a suitable binding assay, for example, Jurkat cells in the presence of 2 mM MnCl2 at room temperature. A low KD value advantageously indicates strong binding to the VLA-4 receptor, and thus indicates rapid and efficient uptake of a lipid nanoparticle according to the invention by a target cell expressing the VLA-4 receptor on an outer surface.
The ligand is preferably coupled to a lipid molecule via a suitable linker to enable incorporation of the ligand into the lipid nanoparticle, such that the ligand is presented on the outer surface of the lipid nanoparticle.
Preferably said linker comprises a tetrazole, more preferably the linker comprises a structure as represented by Formula (III)
Figure imgf000034_0002
Formula (III), wherein R2 is an aliphatic group, preferably an -NHC(CH2)nCOOH, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, in particular wherein n is 4, such that R2 is - NHC(CH2)4COOH; wherein R3 is CO(CH2)mCONH, wherein m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, in particular wherein m is 2, such that R3 is CO(CH2)2CONH. Herein, the linker is connected to said lipid and said ligand via a suitable linkage, preferably via a covalent bond, more preferably a peptide bond.
Preferably said lipid attached via said linker to said ligand is selected from the group consisting of DSPE-PEG, DMG-PEG and Cholesterol- PEG, preferably DSPE-PEG1000-6000, DMG-PEG1000-6000 and Cholesterol-PEG1000-6000, more preferably DSPE-PEG(2000), DMG-PEG(2000) and Cholesterol-PEG(2000).
Physical properties and, detectable labels
A lipid nanoparticle according to the invention preferably has a hydrodynamic diameter of about 75 to about 180 nm, preferably about 78 to about 120 nm, more preferably between about 79 nm and about 90 nm as determinable with a Zetasizer Nano (Malvern Instruments, UK).
Preferably, the hydrodynamic diameter of a lipid nanoparticle according to the invention is at most 100% larger compared to a comparable lipid nanoparticle lacking a ligand on its outer surface, more preferably at most 80% larger, at most 60% larger, at most 40% larger, at most 35% larger, at most 30% larger, in particular at most 25% larger, such as at most 24% larger, or at most 23% larger compared to a comparable lipid nanoparticle lacking a ligand on its outer surface.
Accordingly, a lipid nanoparticle according to the invention is preferably between 23% and 100% larger compared to a comparable lipid nanoparticle lacking a ligand on its outer surface, more preferably between 24% and 80%, between 25% and 60%, between 30% and 50%, such as between 35% and 40% larger compared to a comparable lipid nanoparticle lacking a ligand on its outer surface.
It is advantageous that a lipid nanoparticle according to the invention is at most 100%, such as at most 23% larger than a comparable nanoparticle lacking a ligand on its outer surface, because a smaller lipid nanoparticle has a longer circulation time in the body, typically combined with superior tissue penetration properties and is therefore more likely to effectively target the target cell. (Widmer et al. 2018. Int J Pharm 535:444-451; Younis et al. 2021. J Control Release 331:335-349; Oussoren et al. 1997. Biochim Biophys Acta 1328:261-272).
A lipid nanoparticle according to the invention, preferably has a polydispersity index of about 0.15 to about 0.25, preferably about 0.18 to about 0.22 as determinable with a Zetasizer Nano (Malvern Instruments, UK).
Preferably, polydispersity index of a lipid nanoparticle according to the invention is at most 3 fold larger compared to a comparable lipid nanoparticle lacking a ligand on its outer surface, more preferably at most 2.8 fold, at most 2.5 fold larger, at most 2.0 fold larger, at most 1.9 fold larger, in particular at most 1.8 fold larger compared to a comparable lipid nanoparticle lacking a ligand on its outer surface.
Accordingly, a lipid nanoparticle according to the invention is preferably between 1.8 and 3.0 fold larger compared to a comparable lipid nanoparticle lacking a ligand on its outer surface, more preferably between 1.9 and 2.5 fold larger.
A lipid nanoparticle according to the invention, preferably comprises a detectable label, more preferably a detectable label on its outer surface.
In principle any detectable label suitable for in vivo use may be present in a lipid nanoparticle according to the invention, such as a fluorescent label, a radioactive label, a magnetic label, or a combination thereof. Preferably, the detectable label is a fluorescent label.
Examples of suitable fluorescent labels include Cy3, Cy5, Cy7, Cy9, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 561, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Bodipy Fl, coumarin, fluorescein, Oregon green, pacific blue, pacific green, pacific orange, PE-cyanine7, PerCP-Cyanine5.5, tetramethyl rhodamine and Texas red.
Preferably, the detectable label is a fluorescent label having an excitation wavelength in the range of 488 and 554 nm. Such a fluorescent label is typically excitable with a 532 nm laser line.
Preferably, the detectable label is a fluorescent label having an emission wavelength in the range of between 560 and 568 nm. Such a fluorescent label is typically well detectable in the context of the present invention. Pharmaceutical composition
The invention further relates to a composition comprising a lipid nanoparticle according to the invention and a pharmaceutically acceptable carrier.
Examples of suitable pharmaceutically acceptable carriers include liquid carriers such as water and aqueous buffer solutions, such as physiological saline, phosphate-buffered saline (PBS) or phosphate buffer and solid carriers such as lactose, calcium chloride, pectin and dextrin. Preferably said pharmaceutically acceptable carrier is a liquid carrier, preferably buffered water, more preferably PBS.
Said composition may optionally comprise one or more pharmaceutically acceptable excipients, such as salts, wetting agents, flavoring agents, texturing agents, and stabilizers.
Medical use
The invention further relates to a lipid nanoparticle or composition according to the invention for use in a method of treatment by therapy, preferably for use in a method of treatment of cancer.
For example, a lipid nanoparticle or composition according to the invention is for use in a method of treating lung cancer, colon cancer, rectal cancer, anal cancer, bile cancer, small intestine cancer, gastric cancer, gallbladder cancer, liver cancer, pancreatic cancer, breast cancer, ovarian cancer, endometrium cancer, cervical cancer, prostate cancer, renal cancer, glioblastoma, skin cancer, bone cancer, blood cancer, cancer of the central nervous system, head- and neck cancer and lymphoma.
Preferably, a lipid nanoparticle or composition according to the invention is for use in a method of treatment of leukemia, more preferably acute myeloid leukemia.
The RUNX1/ETO fusion gene described herein above is often observed in pediatric leukemia. Therefore, the invention preferably relates to a lipid nanoparticle or composition according to the invention for use in a method of treatment of leukemia, preferably acute myeloid leukemia, in a human, wherein the human has an age of 18 years or lower, preferably 15 years or lower, more preferably 12 years or lower, 10 years or lower, 8 years or lower, or 6 years or lower. Preferably, said human is a human having an age of at least 6 months, more preferably at least 1 year, at least 2 years, at least 3 years, at least 4 years, in particular at least 5 years of age.
Accordingly, the human is preferably between 6 months and 18 years of age, such as between 1 and 15 years of age, between 2 and 12 years of age, between 3 and 10 years of age, such as between 4 and 8 years of age.
Methods
Method for preparing a lipid nanoparticle according to the invention
The invention further pertains to a method for preparing a lipid nanoparticle according to the invention, comprising
-mixing at least one cationic or ionizable lipid and at least one helper lipid in a polar organic solvent to obtain a lipid mixture;
-mixing the lipid mixture with an aqueous solution comprising a therapeutic molecule at a pH between about 2 and about 6, preferably about 4, to form a mixture comprising lipid nanoparticles loaded with a therapeutic molecule;
-optionally at least partially purifying, preferably dialyzing, the mixture comprising lipid nanoparticles loaded with said therapeutic molecule; and
-contacting the mixture comprising the lipid nanoparticles loaded with the therapeutic molecule or the at least partially purified, preferably dialyzed, mixture comprising lipid nanoparticles loaded with the therapeutic molecule with a ligand coupled to a lipid to obtain a lipid nanoparticle according to the invention.
Mixing of said at least one cationic or ionizable lipid and said at least one helper lipid may be achieved using any suitable method known in the art, e.g. by pipetting or vortexing said lipids in a suitable holder, or by using a microfluidic mixer to obtain said lipid mixture.
Said lipid mixture may optionally comprise in addition to said at least one cationic or ionizable lipid and said at least one helper lipid, one or more PEG-lipids.
Said one or more cationic or ionizable lipid, one or more helper lipids and optionally said one or more PEG-lipids are as defined herein above. Preferably, said one or more cationic lipids comprises at least Dlin-MC3-DMA. Preferably, said one or more helper lipids comprises at least DSPC and cholesterol and optionally sphingomyelin. Preferably, a lipid nanoparticle according to the invention comprises one or more PEG-lipids, preferably DSPE-PEG1000-6000 or DMG-PEG1000- 6000, more preferably DSPE-PEG(2000) or DMG-PEG(2000). In principle, any water-miscible polar organic solvent is suitable for use in the method according to the invention. Examples include methanol, ethanol, propanol, isopropanol, butanol, isobutanol, acetonitrile, 1,4-dioxane, chloroform, ethyl acetate, acetone, dimethylformamide (DMF), dimethylsulfoxide (DMSO), tetrahydrofuran and mixtures thereof.
Preferably, the polar organic solvent is an alcohol, more preferably ethanol.
The ratio between the one or more cationic lipids, the one or more helper lipids and the optionally one or more PEG-lipids is preferably between l:0.5:0 and 1:1.4:0.05, more preferably said ratio is between 1:0.8:0.01 and 1:1.2:0.03, in particular around 1:1:0.03 or 1:1:0.04.
Preferably said lipid mixture comprises the lipids Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG, preferably DMG-PEG1000-6000, more preferably DMG- PEG2000. Preferably herein, the ratio Dlin-MC3-DMA:DSPC:cholesterol:DMG-PEG is between about 1:0.1:0.5:0.02 and about 1:0.4:0.9:0.05, more preferably between about 1:0.2:0.7:0.03 and about 1:0.3:0.8:0.04.
In an alternative preferred embodiment, said lipid mixture comprises the lipids Dlin-MC3-DMA, DSPC, cholesterol and sphingomyelin. Preferably herein, the ratio Dlin-MC3-DMA:DSPC:cholesterol:spingomyelin is between about l:0.1:0.2:0.2 and about l:0.4:0.5:0.5, more preferably between about l:0.2:0.3:0.3 and about l:0.3:0.4:0.4.
In yet another preferred embodiment, said lipid mixture comprises the lipids Dlin-MC3-DMA, DSPC, cholesterol, sphingomyelin and a PEG-lipid, preferably DSPE-PEG or DMG-PEG, more preferably DSPE1000-6000 or DMG-PEG1000-6000, more preferably DMG-PEG2000 or DSPE-PEG2000.
The lipid concentration in the lipid mixture is preferably between 2.5 and 25 mM, more preferably between 10 and 25 mM, in particular about 25 mM. Further, the concentration of cationic or ionizable lipids in the lipid mixture is preferably between about 1.25 mM and 12.5 mM, more preferably between 5 mM and 12.5 mM, in particular about 12.5 mM.
Said aqueous solution comprising said therapeutic molecule has a pH between about 2 and about 6, preferably between 3 and 5, more preferably around 4. Said pH may be determined using any suitable method known in the art, for example using a pH meter. At such pH, a therapeutic molecule, preferably a charged therapeutic molecule, is typically well soluble in an aqueous medium. Preferably, said aqueous solution is an aqueous buffer solution, such as an acetate buffer or a citric acid buffer, in particular an acetate buffer.
Preferably, the molar strength of said aqueous buffer solution is between 5 and 50 mM, more preferably between 10 and 40 mM, in particular around 25 mM. Most preferably, said aqueous solution is a 25 mM acetate buffer.
Said therapeutic molecule is as defined herein above, but is preferably an oligonucleotide, more preferably an siRNA molecule.
The concentration of therapeutic molecule in the aqueous solution is preferably between 5 and 50 μM, more preferably between 20 and 40 μM, in particular about 26 μM.
Mixing of said lipid mixture and said aqueous solution comprising said therapeutic molecule is preferably achieved using a microfluidic mixer, for example using a NanoAssemblr benchtop (Precision nanosystems). Herein, both lipid mixture and aqueous solution are preferably loaded in separate syringes and pumped through a microfluidic mixture to obtain a mixture comprising lipid nanoparticles loaded with said therapeutic molecule. Preferably, the total flow rate in the microfluidic mixer is between 1 ml/min and 12 ml/min, more preferably between 2 ml/min and 6 ml/min, in particular around 4 ml/min.
Preferably, the ratio between the volume of the aqueous mixture comprising a therapeutic molecule and the lipid mixture is between 1:1 and 5:1, more preferably between 2:1 and 4:1, in particular around 3:1.
Accordingly, the molar ratio between therapeutic molecule and lipid in the mixture comprising lipid nanoparticles loaded with a therapeutic molecule, is preferably between about 1:500 and about 1:150, more preferably between about 1:400 and about 1:250, in particular around 1:330.
Further, the molar ratio between therapeutic molecule and cationic lipid or ionizable lipid in the mixture comprising lipid nanoparticles loaded with a therapeutic molecule is preferably between 1:750 and 1:250, more preferably between about 1:200 and about 1:130, in particular around 1:160.
With the method according to the invention, the efficiency of encapsulating a therapeutic molecule in a lipid nanoparticle is relatively high. Preferably, the encapsulation efficiency is at least 70%, more preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, most preferably 100%.
The obtained lipid nanoparticles loaded with a therapeutic molecule, may be contacted directly with a ligand coupled to a PEG lipid, or may optionally be at least partly purified prior to said contacting.
Any method of purification that is capable of exchanging the organic solvent used to prepare said lipid mixture and the aqueous buffer used to prepare said aqueous solution comprising a therapeutic molecule may be used herein, such as dialysis or centrifugation over a suitable membrane.
Preferably, the mixture comprising lipid nanoparticles loaded with said therapeutic molecule is subjected to one or more dialysis steps. It has been found that when said lipid nanoparticles loaded with a therapeutic molecule have been at least partly purified prior to contacting with a ligand coupled to a lipid, a nanoparticle according to the invention exhibits superior physicochemical properties, in particular superior hydrodynamic diameter and superior polydispersity index, compared to a lipid nanoparticle that has been obtained without at least partly purification.
Preferably, said mixture comprising lipid nanoparticles loaded with said therapeutic molecule is dialyzed against an aqueous buffer having a pH of between 6 and 8, preferably around 7.
Examples of suitable buffers include 2,2-Bis(hydroxymethyl)-2,2’,2”- nitrilotriethanol (Bis-Tris), N-(2-Acetamido)iminodiacetic acid, (ADA), N-(2- Acetamido)-2-aminoethanesulfonic acid (ACES), Piperazine-N,N'-bis(2- ethanesulfonic acid) (PIPES), 3-Morpholino-2-hydroxypropanesulfonic acid (MOPS), 1,3-Bis(tris(hydroxymethyl)methylamino)propane (Bis-Tris propane), N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 2-{[l,3-Dihydroxy- (hydroxymethyl)propan-2-yl]amino}ethane-l-sulfonic acid (TES), 4-(2- Hydroxyethyl)-l-piperazineethanesulfonic acid (HEPES), 3-(N,N-Bis[2- hydroxyethyl]amino)-2-hydroxypropanesulfonic acid (DIPSO), 4-(N- Morpholino)butanesulfonic acid (MOBS), N-[Tris(hydroxymethyl)methyl]-3-amino- 2-hydroxypropanesulfonic acid (TAPSO), Tris(hydroxymethyl)aminomethane (Tris), 4-(2-Hydroxyethyl)piperazine-l-(2-hydroxypropanesulfonic acid)) (HEPPSO), Piperazine-N,N'-bis(2-hydroxypropanesulfonic acid) (POPSO), Phosphate-buffered saline (PBS), Tris-acetate-EDTA (TEA), {[l,3-Dihydroxy-2-(hydroxymethyl)propan- 2-yl] amino} acetic acid (Tricine), and combinations thereof. Preferably, said mixture comprising lipid nanoparticles loaded with said therapeutic molecule is dialyzed against PBS buffer.
Preferably, said dialysis takes place at a temperature between 0 °C and 20 C, preferably between 2 °C and 15 °C, in particular between 4 °C and 6 °C.
Said dialysis preferably takes place for at least 1 h, more preferably at least 2 h, at least 4 h, at least 8 h, in particular at least 12 h, or at least 16 h. Preferably, the dialysis is rim over night, e.g. between 12 h and 16 h.
Preferably, during dialysis, said aqueous buffer is replaced at least 1 time with fresh aqueous buffer, more preferably at least 2 times, most preferably at least 3 times.
The dialysis membrane preferably has a molecular weight cut off of at most 30 kDa, preferably at most 20 kDa, in particular at most 10 kDa.
Said mixture of lipid nanoparticles loaded with said therapeutic molecule is contacted with a ligand coupled to a lipid via a suitable linker.
Said ligand, said linker and said lipid are as defined herein above. Preferably said ligand comprises a structure according to formula (II). Preferably, said lipid is DSPE-PEG, more preferably DSPE-PEG1000-6000, even more preferably DSPE- PEG(2000).
Said contacting may be achieved using any suitable method known in the art, such as by pipetting, stirring or mixing of the mixture comprising lipid nanoparticles loaded with a therapeutic molecule and the ligand coupled to the lipid.
Preferably, the molar ratio between said mixture comprising the lipid nanoparticles loaded with the therapeutic molecule and said ligand coupled to said lipid is between 10000:1 and 500:1, more preferably between 5000:1 and 1000:1, in particular around 3000:1. With such a ratio, the molar fraction of ligand in a lipid nanoparticle according to the invention is between 0.01% and 0.2%, preferably between about 0.02% and about 0.1%, in particular around 0.03%.
Preferably, during contacting, the concentration of lipid nanoparticles loaded with the therapeutic molecule is between 5 and 40 mM, more preferably between 10 and 25 mM.
Preferably, the concentration of ligand coupled to lipid is between about 5 and about 40 μM, more preferably between about 10 and about 25 μM. The mixture comprising the lipid nanoparticles loaded with said therapeutic molecule and said ligand coupled to said lipid were contacted for at least 15 minutes, preferably at least 30 minutes, at least 1 h, in particular at least 2 h, to obtain a lipid nanoparticle according to the invention.
In the method according to the invention, said mixture comprising the lipid nanoparticles loaded with said therapeutic molecule and said ligand coupled to said lipid were preferably contacted at a temperature of between about 4 °C and about 80 °C, more preferably between about 10 °C and about 70 °C, between about 20 °C and about 60 °C, in particular between about 40 °C and about 50 °C, such as about 45 °C.
Said ligand is preferably coupled to said lipid via a linker comprising a triazole moiety to obtain a ligand coupled to a lipid via a triazole linkage. It was found that this molecule may be efficiently obtained using a copper-free click reaction protocol.
Method for coupling a ligand to a lipid
The invention further pertains to a method of coupling a ligand to a lipid, comprising
-providing a ligand according to general formula (I), preferably according to Formula (II) functionalized with a terminal azide moiety;
-providing a lipid functionalized with a strained alkyne moiety, preferably a dibenzocyclooctyne (DBCO) moiety;
-contacting the functionalized ligand with the functionalized lipid to obtain a ligand coupled to a lipid via a triazole linkage; and
-optionally isolating the ligand coupled to the lipid.
Advantageously, said method does not require the use of Cu2+ species, which is associated with copper-related toxicity when administered to animals, such as humans.
Preferably said ligand functionalized with a terminal azide moiety is a molecule comprising the structure of formula (IV).
Figure imgf000044_0001
Said ligand according to formula (I) functionalized with a terminal azide, preferably said molecule comprising the structure of formula (IV) may be prepared via a chemical synthesis protocol.
For example herein, a corresponding amine (lysine, preferably L-lysine) may be used as starting material. Said amine may be functionalized with a suitable leaving group, such as a sulfonamide group and subsequently substituting said sulfonamide with an azide group to obtain a ligand according to formula (I), preferably a ligand according to formula (II), functionalized with a terminal azide moiety.
Said molecule according to formula (IV) may also be obtained from a commercial source, for example from EMC Microcollections (Tubingen, Germany).
Said lipid functionalized with a strained alkyne molecule, is preferably a molecule according to formula (V).
Figure imgf000044_0002
Said lipid functionalized with a strained alkyne molecule, preferably said molecule according to formula (V) may be obtained via a chemical synthesis protocol.
Said molecule according to formula (V) may also be obtained from a commercial source, for example from Avanti Polar Lipids.
Said ligand functionalized with said azide and said lipid functionalized with said strained alkyne molecule, wherein said functionalized ligand is preferably the molecule according to formula (IV) and wherein said functionalized alkyne is preferably said molecule according to formula (V), are contacted using any suitable method known in the art, preferably by mixing, e.g. by pipetting, stirring or vortexing said functionalized ligand and said functionalized lipid.
Optionally said ligand functionalized with an azide and said lipid functionalized with a strained alkyne are dissolved in a suitable liquid medium to obtain a solution of said ligand functionalized with an azide and said lipid functionalized with a strained alkyne in a suitable liquid medium. Preferably, said suitable liquid medium comprises DMSO, an alcohol, preferably a lower alcohol such as methanol, ethanol and propanol, H2O, PBS or combinations thereof, more preferably PBS.
The ratio between said ligand functionalized with azide and said lipid functionalized with alkyne is preferably between 1:1 and 1:5, more preferably between 1:2 and 1:4, in particular about 1:3. Advantageously such a ratio promotes full conversion of said azide into the corresponding triazole.
The functionalized ligand and the functionalized lipid were contacted for an amount of time sufficient to convert at least 90% of the functionalized azide into the corresponding triazole. Typically, said functionalized ligand and said functionalized lipid were contacted for at least 1 h, preferably at least 2 h, at least 4 h, at least 8 h, at least 12 h, at least 16 h, at least 24 h. Conversion of the reaction may be monitored by following conversion of a fluorescently labelled mock substrate on a 1% agarose gel.
In the method according to the invention, the functionalized ligand the functionalized lipid were preferably contacted at a temperature of between 0 °C and 50 °C, more preferably between 4 °C and 37 °C, between 10 °C and 20 °C, in particular between 20 °C and 25 °C.
Said ligand coupled to said lipid is optionally isolated from the reaction mixture. Any method suitable for isolation known in the art may be used, for example size exclusion chromatography or column chromatography.
Method of treatment
The invention further pertains to a method of treating a subject suffering from cancer, preferably leukemia, more preferably acute myeloid leukemia, comprising administering a therapeutically effective amount of a lipid nanoparticle or pharmaceutical composition according to the invention to a subject in need thereof, preferably a human. Said human preferably is a human having an age of 18 years or lower, preferably 15 years or lower, more preferably 12 years or lower, 10 years or lower, 8 years or lower, or 6 years or lower. Preferably, said human is a human having an age of at least 6 months, more preferably at least 1 year, at least 2 years, at least 3 years, at least 4 years, in particular at least 5 years of age.
Accordingly, the human is preferably between 6 months and 18 years of age, such as between 1 and 15 years of age, between 2 and 12 years of age, between 3 and 10 years of age, such as between 4 and 8 years of age.
Said lipid nanoparticle or composition is as described herein above. Preferably, said lipid nanoparticle comprises a ligand according to formula (II). Further, said lipid nanoparticle is preferably loaded with an oligonucleotide, more preferably an siRNA molecule, most preferably an siRNA molecule comprising a first strand as represented by SEQ ID NO:1.
Said lipid nanoparticle or composition according to the invention may be administered via any suitable route of administration known in the art.
Examples of routes of administration include injection, oral administration, inhalation, transdermal application or rectal administration. Preferably, said lipid nanoparticle according to the invention is administered via injection.
Further, said lipid nanoparticle or said composition according to the invention may be administered parenterally, such as intraarticularly, intravenously, intraperitoneally, subcutaneously or intramuscularly. Preferably, said lipid nanoparticle or composition is administered intramuscularly.
The invention further relates to a use of a lipid nanoparticle according to the invention in the preparation of a medicament for the treatment of cancer, preferably leukemia, more preferably acute myeloid leukemia.
Examples
Example 1: siRNA linid nanon articles (LNP) components and preparation Preparation of lipid mixtures
50 mol% of (6Z,9Z,28Z,31Z)-heptatriacont-6,9,28,31-tetraene-19-yl 4- (dimethylamino)butanoate (Dlin-MC3-DMA), 10 mol% of distearoylphosphatidylcholine (DSPC), 38.5 mol% of cholesterol and 1.5 mol% of l,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG(2000)) were dissolved in ethanol to obtain a first lipid mixture of Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG(2000) at molar ratios of lipids of 50:10:38.5:1.5 having a final lipid concentration of 10 mM for ex vivo experiments and 25 mM for in vivo experiments.
To minimize potential immunogenic effects by the PEG component, a second lipid mixture was prepared by replacing DMG-PEG(2000) and part of the cholesterol by sphingomyelin, a major component of viral envelopes, mammalian membranes and exosomes. The lipid composition of the second lipid mixture had a molar ratio of 50:10:20:20 for Dlin-MC3-DMA:DSPC:cholesterol:sphingomyelin, respectively.
Preparation of loaded lipid LNP
For preparation of LNPs loaded with siRNA siRNA was diluted in 25 mM acetate buffer (pH = 4) to obtain a solution of siRNA in acetate buffer. The concentration of siRNA (csiRNA) in the stock solution was calculated according to formula (I):
Figure imgf000047_0001
wherein CDLin is the concentration of Dlin-MC3-DMA in the lipid mixture, equaling the concentration of positively charged amines, N/P is the ratio of positively chargeable amine groups to negatively charged nucleic acid phosphate groups (here 4, which was found to be optimal for siRNA LNP formulations), np defines the number of phosphate groups in the siRNA (here 40), VSiRNA and Vlipid are the volumes of stock solutions of siRNA (in 25 mM acetate buffer) and lipid mixture (in ethanol), respectively, with a Vlipid/VsiRNA ratio of 1:3. At a lipid concentration of 25 mM, cDlin is 12.5 mM and CsiRNA is 26 μM. The LNPs were prepared using a NanoAssemblr benchtop (Precision Nanosystems). Syringes were loaded with stock solutions comprising a lipid mixture and siRNA at a Vlipid/VsiRNA ratio of 1:3, and pumped through a microfluidic mixer at a total flow rate of 4 ml/min to obtain a solution comprising LNPs loaded with siRNA (siRNA LNPs).
To remove ethanol and neutralize the pH, the siRNA LNP solutions were dialysed against phosphate-buffered saline (PBS) overnight at 4 °C, using the Gamma- Irradiated Slide- A-Lyzers with lOkDa molecular weight cut-off (ThermoFisher Scientific). PBS was replaced after 1 and 2 hours, to obtain a solution of siRNA loaded LNPs in PBS buffer.
Example 2: Generation of decorated siRNA LNPs
To increase retention and LNP uptake in leukemia-relevant tissues, siRNA LNPs decorated with a ligand targeting the Very Late Antigen 4 (VLA-4), an integrin heterodimer of Integrin Subunit Alpha 4 (ITGA4) and Integrin Subunit Beta 1 (ITGB1), were prepared. The VLA-4 ligand is a small, modified peptide comprising the sequence LDV with structure as shown in formula (II).
Figure imgf000048_0001
In addition, siRNA LNPs were decorated with the fluorophore Cy3 to monitor cell association and uptake. To avoid copper-associated toxicity, a copper-free click- chemistry protocol using the restrained alkyne dibenzocyclooctyne (DBCO) that was linked to DSPE-PEG(2000) was developed. The ligand or fluorophore was functionalized with an azide moiety. The solvent was dependent on the hydrophobicity of the azide moiety and was most preferably H2O or DMSO. A solution of 1 molar eq. of ligand functionalized with an azide moiety dissolved in DMSO:H2O in a ratio of 3:2 was mixed with a solution of 3 molar eq. of DBCO- DSPE-PEG(2000) in ethanol at room temperature. The reaction is shown in Figure la. The reaction was confirmed by loading the product of a fluorescently labelled azide and DBCO-DSPE-PEG(2000) on a 1% agarose gel (Figure lb). The reaction was 90% effective as calculated based on the fluorescence intensity of the Cy3. siRNA LNPs were prepared with 10 or 25 mM lipid and dialysed. Ten or 25 μM ligand coupled to DSPE-PEG(2000) were subsequently post-inserted into the siRNA LNP at 45 °C for 2 hours. The resultant molar ratio of ligand coupled to DSPE- PEG(2000)to lipid was kept at 0.1% (figure 1c).
Example 3: Physicochemical characterization of siRNA LNPs
In this example the physicochemical parameters of siRNA LNP formulations were analysed. The siRNA LNPs with and without post-inserted ligands were analysed in terms of size (hydrodynamic diameter), polydispersity index (PDI) using a Zetasizer Nano (Malvern Instruments, UK) and encapsulation efficiency. The results are shown in table 1.
Table 1; Analysis of physicochemical parameters of siRNA LNPs.
Figure imgf000049_0001
* Abbreviations: LNP siRE; LNP loaded with siRNA comprising a first strand as represented by SEQ ID NO:3 and a second strand complementary to the first strand represented by SEQ ID NO:4; LNP siMM; Control siRNA comprising a first strand as represented by SEQ ID NO:5 and a second strand complementary to the first strand represented by SEQ ID NO:6. Ligand; ligand according to formula (II).
In table 2, the results of multiple batches of siRNA LNPs which were prepared with and without ligands inserted prior to or after dialysis. Post-insertion of the DBCO-DSPE-PEG(2000)-ligand product after dialysis at 0.1% molar ratio into pre- formed siRNA LNP containing 1.5% DMG-PEG(2000) gave a siRNA LNP decorated with a ligand, having a size of about 119 nm and a PDI of about 0.204 (entry 4). In these LNPs, the hydrodynamic diameter and PDI were increased by 100% compared to siRNA LNP free of ligand (entry 1), while post-insertion of the ligand prior to dialysis (entry 3) or post-insertion of DBCO-DSPE-PEG(2000) (entry 2) in the LNP caused a substantial increase in the hydrodynamic diameter by more than 140% (table 2; comparison entry 1 with entries 2 and 3).
The hydrodynamic diameter of the decorated ligand could be further reduced by post-insertion of DBCO-DSPE-PEG(2000)-ligand at 0.3% molar ratio into siRNA LNP containing 1.2% DMG-PEG(2000). The hereby obtained ligand-decorated siRNA LNP had a size of 79 nm, which corresponds to an increase of only 23% compared to non- decorated siRNA LNP. Further, the ligand-decorated siRNA LNP showed a PDI of 0.19, which corresponds to an increase in PDI of approximately 78% compared to non-decorated siRNA LNP (Table 2, entries 5 and 8).
Table 2; analysis of LNPs.
Figure imgf000050_0001
The non-decorated siRNA LNP were further analyzed in terms of stability over time (table 3). It was found that the size and PDI of siRNA LNP did not increase significantly when stored at 4 °C during an observation period of up to two months (table 3).
For size measurements machine settings were set to dispersant to water, incubation time 120 seconds and temperature of 25 °C. In addition, LNPs were visualized by cryo-electron microscopy (figure 2). Table 3; Analysis of stability of LNPs over time.
Figure imgf000051_0002
* Abbreviations: LNP siRE; LNP loaded with siRNA comprising a first strand as represented by SEQ ID NO:3 and a second strand complementary to the first strand represented by SEQ ID NO:4; LNP siMM; control siRNA comprising a first strand as represented by SEQ ID NO:5 and a second strand complementary to the first strand represented by SEQ ID NO:6.
Example 4: Uptake of fluorescent siRNA LNP in t(8:21) AML cell lines and patient cells
The following example shows the uptake of Cy3-labelled siRNA LNP in t(8;21) Kasumi-1 cells. Cy3-labelled DSPE-PEG(2000) was post-inserted together with or without a ligand coupled to a lipid according to Formula (VI) into siRE LNP and siMM LNP.
Figure imgf000051_0001
In figure 3, the uptake of siRE LNP containing 1.2% DMG-PEG(2000), 0.3% DBCO- DSPE-PEG(2000) and 0.03% Cy3-labelled DSPE-PEG(2000)) with (“targeted”) or without (“non-targeted”) 0.03% of the ligand coupled to lipid according to Formula (VI) in t(8;21) Kasumi-1 cells is demonstrated.
Further, in figure 4a the time course of uptake for both “targeted” and “non- targeted” siRE LNP and siMM LNP formulations is shown. Figures 3 and 4 both demonstrate that “targeted” siRNA LNPs showed higher and faster uptake compared to the “non-targeted” counterparts independent of the cargo siRNA.
Similarly, both patient-derived t(8;21) AML cells cultured in the presence (figure 4b) and absence (figure 4c) of mesenchymal stem cells (MSCs) show enhanced uptake of “targeted” siRNA LNPs compared to “non-targeted” siRNA LNPs. These findings demonstrate a negligible impact of MSCs on LNP uptake by AML cells. Accordingly, it is demonstrated that “targeted” siRNA LNPs show superior behavior in terms of uptake over “non-targeted” siRNA LNP in patient-derived AML cells that is not affected by bone marrow-derived MSCs.
Example 5: siRNA LNP efficacy in t(8;21) AML cell lines
This example shows the efficacy of “non-targeted” siRNA LNPs to knockdown RUNX1/ETO in the t(8;21) positive cell lines Kasumi-1 and SKNO-1.
Cells were treated with 2 pg/ml siRNA LNP for 24 hours followed by analysis of knockdown by qPCR between 24 hours and 9 days after addition of the siRNA LNPs.
In Figure 5 the reduction of RUNX1/ETO expression over time in Kasumi-1 and SKNO-1 cells is shown. Expression of the RUNX1/ETO transcript is reduced approximately three-fold after a single administration of siRE LNP when compared to the control siMM LNP.
Replacing the DMG-PEG(2000)with DSPE-PEG(2000) in the formulation resulted in decreased knockdown of the RUNX1/ETO transcript after 24 hours (Figure 6a). The effect of the knockdown was also investigated by clonogenicity assays.
Therefore the cells were diluted in 2.5 ml semi-solid medium (RPMI1640 with 10% FCS and 5% methylcellulose) and 0.5 ml of this solution was transferred to a 24- well plate, giving a concentration of about 3125 cells per well.
After 14 days pictures were taken using the Leica DMi8 and the colonies, defined as > 25 cells present, were counted (both manually as computationally). Reduced expression of RUNX1/ETO (figure 6a) was associated with impaired clonogenicity (figure 6b) and delayed proliferation (figure 6c).
In line with these phenotypic changes, siRE LNPs affected the expression of established RUNX1/ETO genes. We observed increased expression of Lysosomal Protein Transmembrane 5 (LAPTM5) (figure 7a) and (CCAAT Enhancer Binding Protein Alpha) CEBPA (figure 7b) and decreased expression levels of the adhesion molecule CD34, Angiopoietin 1 (ANGPT1) and Cyclin D2 (CCND2) (figure 8a-c respectively).
This data shows that administration of ligand-decorated siRE LNPs to t(8;21) AML cell fines lead to a reduction of expression of the RUNX1/ETO fusion gene.
Example 6: siRNA LNP efficacy in patient-derived t(8;21) AML cells
This example demonstrates superior RUNX1/ETO knockdown by the “targeted” siRNA LNPs in patient-derived AML cells compared to the non-decorated siRNA LNP.
While single administration of “non-targeted” siRE LNP led to less than two-fold reductions of the RUNX1/ETO transcript, single administration of “targeted” siRE LNP resulted in a threefold and longer-lasting reduction of expression of the RUNX1/ETO transcript (figure 9a).
Similarly, sequential treatment (indicated with a black arrow) of “targeted” siRE LNP reduced the transcript three-fold after 24 hours of treatment with knockdown remaining detectable up to 5 days after the final treatment (figure 9b, c). In contrast, siRE LNP and the two control formulations siMM LNP, both “targeted” and “non-targeted”, failed to achieve a reduced expression ofRUNXl/ETO (Figure 9b, c)
Western blot analysis also demonstrated two-fold reduction of RUNX1/ETO protein three and six days after addition of “targeted” siRE LNP (figure 10).
Furthermore, co-culture of patient-derived cells with MSCs did not affect RUNX1/ETO knockdown (figure 9c).
When comparing the impact of treatment of t(8;21) patient-derived AML cells, three sequential applications of “targeted” siRE LNPs (dosing at day 0, day 3 and day 6, indicated with a black arrow in figure 9) caused a massive shift of subpopulations from immature CD34+ cells to more mature CD34- CD 15+ cells indicating strong induction of myeloid differentiation by this formulation (Figure 11).
Notably, all other formulations tested (siRE LNP, “targeted” and “non-targeted” siMM LNPs) did not cause any substantial changes in cell differentiation when compared to untreated cells. In line with these phenotypic changes, only “targeted” siRE LNPs affected the expression of established RUNX1/ETO genes.
We observed increased expression of LAPTM5 and CEBPA and decreased expression levels of CD34, ANGPT1 and CCND2 (data not shown).
The combined data shows that treatment with “targeted” siRE LNPs disrupts the transcriptional network maintained by RUNX1/ETO. Therefore, administration of “targeted” siRE LNPs constitutes a promising approach in the treatment of AML.
Example 7: Tolerability of siRE LNPs decorated with an LDV ligand in BALB/cJRag2tm1 1Flv112rgtm1 1Flv/J mice
Toxicity of “targeted” siRE LNPs using BALB/cJ-Rag2tm1.1Flv Il2rgtm1 1FlvIJ in female mice was determined.
Dose escalation with three-day intervals starting with 0.5 mg/kg of “targeted” siRE LNPs were performed. The dosing scheme is shown in Figure 12a. The dose was increased with 0.5 mg/step until the maximum dose 20 ml/kg volume was reached in the mice, which corresponded with a dose of 2.3 mg/kg. Finally, mice received three dosages of this maximinn dose. No significant difference in weight between the treated and untreated mice was observed (Figure 12b).
Table 4; Spleen and liver weights of mice subjected to treatment with “targeted” LNP loaded with siRE, compared to control.
Figure imgf000054_0001
Slightly increased spleen sizes were observed in the treatment group (table 4 and figure 12C) but did not show any changes in morphology as confirmed by veterinary histopathological inspection of all three treated and two control animals (Synlab VPG Leeds UK).
Possible reasons might be an inflammatory response induced by the PEG lipids in the LNP or by mobilization of blood components. These data show that “targeted” siRNA LNPs can be safely applied in mice at the maximal dose tested without compromising animal welfare and overall health.
Example 8: Improved uptake of LNPs in AML primary cells
In Figure 13, the uptake of siRE LNP containing 1.5% DMG-PEG(2000) , 0.1% DBCG-DSPE-PEG(2000) and 0.02% Cy3-labelled DSPE-PEG(2000) ) with (“targeted”) or without 0.08% of the ligand coupled to lipid according to Formula (VI) in three different t(8;21) primary cells is demonstrated. This Figure demonstrates that “targeted” siRNA LNPs shows enhanced and faster uptake compared to non-targeted counterparts independent of the cargo siRNA in material directly isolated from t(8;21) patients.
Example 9: siRNA LNP efficacy in t(8:21) primary cells
Sequential treatment (indicated with a black arrow) of “targeted” siRE LNP reduced the RUNX1/ETO transcript in three different primary samples two-fold after 24 hours of treatment with knockdown remaining detectable up to 9 days (Figure 14A). In contrast, the control formulation “targeted” siMM LNP failed to achieve a reduced expression of RUNX1/ETO (Figure 14A) Western blot analysis also demonstrated complete loss of RUNX1/ETO protein twelve days after addition of “targeted” siRE LNP (Figure 14B). Furthermore, co- culture of primary cells with mesenchymal stem cells (MSG) did not affect RUNX1/ETO knockdown (Figure 14B left compared to Figure 14B right).
When comparing the impact of treatment of t(8;21) patient-derived AML cells, three sequential applications of “targeted” siRE LNPs (dosing at day 0, day 3 and day 6, indicated with a black arrow in Figure 14A) caused a massive shift of subpopulations from immature CD34+ cells to other cell populations (Figure 15A) as determined by a multiparameter flow analysis. It is important to realize that cells that are clustering together have the same parameter values. This reduction of CD34 and shift in cell population after sequential addition of “targeted” siRE LNP was confirmed by single-cell RNAseq (Figure 15B).
After permeabilization patient-derived cells were intracellularly stained with an anti-ETO polyclonal antibody, followed by conjugation of this antibody to a fluorophore (AF555) for visualization. Incorporating this staining into our differentiation panel showed on multiparameter flow analysis a shift of RUNX1/ETO positive cells towards RUNX1/ETO negative cells upon administration of a lipid nanoparticle according to the invention, loaded with siRE molecule (Figure 15C, right) compared to a comparable lipid nanoparticle loaded with siMM (control; Figure 15C, left). This indicates the loss of the leukaemic fusion protein as the ETO protein is only present in the leukaemic cells.
Immunohistochemistry of primary t(8;21) cells showed the loss of the RUNX1/ETO protein upon administration of a lipid nanoparticle according to the invention, loaded with siRE molecule (Figure 15D, middle) compared to a comparable lipid nanoparticle loaded with siMM (control; Figure 15D, bottom) and untreated primary cells (control; Figure 15D, top). This indicates the loss of the leukaemic fusion protein and confirms the finding in Figure 14B and Figure 15C.
Example 10: LNPs without DMG-PEG are still functionally active in patient- derived material
Sequential treatment (indicated with a black arrow) of “targeted” siRE LNP comprising either 1.5% DMG-PEG and 38.5% cholesterol or 20% sphingomyelin and 20% cholesterol reduced the RUNX1/ETO transcript in patient-derived material for both the lipid nanoparticle formulations to a similar extent (Figure 16). In contrast, the control formulations “targeted” siMM LNP failed to achieve a reduced expression of RUNX1/ETO (Figure 14A)
Example 11: Modified siRE has high efficacy in t(8:21) cell lines
Figure 17A shows the siRNAs that were designed specifically targeting the unique breakpoint of the RUNX1/ETO t(8;21) fusion transcript (siRE) and the mismatch control (siMM) generated by swapping two nucleotides in the sequence. To improve their stability siRNAs were modified by site-specifically introducing 2’-deoxy- (2’- H), 2’-fluoro (2’-F) and 2’-methoxy (2’-OMe) ribose modifications and 3’-terminal phosphorothioate (PS) backbones (Figure 17B).
Administration of these siRNA LNPs in t(8;21) cell lines show an altered cell cycle profile (Figure 18A) with more cells accumulating in the G0/G1 phase when comparing the siRE LNP with siMM LNP. Accordingly, the administration of the siRE LNP also resulted in an increase in senescent cells as determined by senescence-associated beta galactosidase staining (Figure 18B). The colony formation units were decreased in t(8;21) cells after administration of the siRE LNP (Figure 18C). This decrease was also observed for the second replating (Figure 18C, right).
Example 12: Lipid composition reaches the bone-marrow and tumour-site in vivo
Administration of labelled LNPs (LNP/NIR) or free NIR dye in PBS resulted in an accumulation of the LNP/NIR in the tibia, femur, brain, spleen and liver as shown in Figure 19A Co-localisation on the overlay of the bioluminescence signal of the tumour cells and the fluorescent signal of the LNPs showed that the LNP/NIR reaches the tumour cells in vivo (Figure 19B). This resulted in a decrease of the RUNX1/ETO protein expression as shown by Western Blot analysis on cells harvested from mice that were treated with the LNP siRE compared to the LNP siMM (Figure 20A). The percentage of senescent cells was also increased in the LNP siRE treated group (Figure 20B) and the colony forming units were decreased (Figure 20C). Overall, the treatment of the LNP encapsulating siRE resulted in a decrease in measured tumour signal (Figure 21 A) and improved median and overall survival (Figure 2 IB). Proof of the loss of the sternness of these treated cells, was given by retransplantation experiments. Secondary recipients of the LNP siRE treated cells showed less tumour growth (Figure 21C) and improved survival (Figure 21D) after transplantation compared to the recipients that received the cells from the LNP siMM treated group. The cells that were harvested from the secondary recipients were not resistant to LNP siRE treatment as shown by Western Blot analysis were the RUNX1/ETO protein expression decreased in the cells that were treated with the siRE LNP, even the cells that were harvested from the secondary recipients treated with the siRE LNPs (Figure 2 IE). This indicates that the cells remained dependent on RUNX1/ETO and are still susceptible to repeated LNPs treatment.
Example 13: siRNA LNP efficacy in a t(4:11) ALL cell line
This example shows the efficacy of “non-targeted” siRNA LNPs to knockdown fusions of mixed lineage leukemia gene 1 (MLL; Lysine Methyltransferase 2A; KMT2A) with ALF Transcription Elongation Factor 1 (AF4 or AFF1) in the t(4; 11) positive cell line SEM (Greil et al., 1994. Br J Haematol 86: 275-283). The siRNA sequences are shown in Figure 22A. Cells were treated with 2 μg/ml siRNA LNP for 24 hours followed by analysis of knockdown by qPCR between 24 hours and 6 days after addition of the siRNA LNPs.
In Figure 22AB the reduction of expression of MLL/AF4 and of its target gene Homeobox A7 (H0XA7) over time in SEM cells is shown. Expression of the MLL/AF4 transcript is reduced approximately two-fold after a single administration of siMA6 LNP and three-fold by siMAl-Ml LNPs when compared to the control siMM and siMM-mod LNPs, respectively, while expression of H0XA7 is reduced two-fold.
Two sequential applications of siMA6-Ml LNPs at day 1 and day 3 blocked proliferation of SEM stronger than control LNP formulations (Figure 22C). The effect of the knockdown was also investigated in clonogenicity assays. Therefore SEM cells were seeded in semi-solid medium (RPMI1640 with 10% FCS and 5% methylcellulose) at a cell concentration of 2000 cells/ml. After 14 days pictures were taken using the Leica DMi8 and the colonies, defined as > 25 cells present, were counted (both manually as computationally). siMA6-Ml LNPs reduced clonogenic growth more than 7 fold when compared to siMM-mod LNPs (Figure 22D).
Sequences:
Figure imgf000058_0001
Figure imgf000059_0001

Claims

Claims
1. A lipid nanoparticle (LNP) loaded with a therapeutic molecule, said LNP comprising a ligand on its outer surface capable of binding to the Very Late Antigen 4 (VLA-4) receptor, wherein the ligand comprises the structure according to formula (II):
Figure imgf000060_0001
2. The LNP according to claim 1, wherein the therapeutic molecule is a small interfering ribonucleic acid (siRNA), preferably a modified siRNA.
3. The LNP according to claim 2, wherein the siRNA inhibits functional expression of a RUNX1/ETO fusion gene.
4. The LNP according to any one of the preceding claims, wherein the siRNA comprises a first RNA strand complementary to an mRNA transcript of the RUNX1/ETO gene and, optionally, a second RNA strand complementary to the first RNA strand, wherein said first RNA strand is preferably represented by the sequence as shown in SEQ ID NO:1 and wherein said second RNA strand, when present, is represented by the sequence as shown in SEQ ID NO:2.
5. The LNP according to any one of claims 2-4, wherein the siRNA comprises at least 21 nucleotides and which siRNA comprises one or more of the following modifications:
(a) the sequence of SEQ ID NO:1 comprises a CFon position 1, a CFon position 2, an UF on position 3, and aCF on position 4, wherein CF and UF represent nucleotides wherein the hydroxyl group on the 2-position of the ribose moiety is substituted for a fluoride;
(b) the sequence of SEQ ID NO:1 comprises dA on position 17, dG on position 18, dA on position 19, dT on position 20 and dT on position 21, wherein dA dG and dT represent nucleotides wherein the hydroxyl group on the 2-position of the ribose moiety is substituted for a hydrogen;
(c) the sequence of SEQ ID NO:1 comprises an UoMe on position 9, a CoMe on position 10, an UoMe on position 12, an CoMe on position 14 and a UoMe on position 15, wherein CoMe and UOME represent nucleotides wherein the hydroxyl group on the 2-position of the ribose moiety is substituted for a methoxy group;
(d) the sequence of SEQ ID NO:1 comprises a TPS on position 20, and/or wherein the sequence of SEQ ID NO:2 comprises a TPS on position 20, wherein TPS represents a nucleotide wherein the phosphodiester group on the 3-position of the ribose moiety is substituted for a phosphorothioate;
(e) the sequence of SEQ ID NO: 1 comprises a T on position 20 and a T on position 21 and/or wherein the sequence of SEQ ID NO:2 comprises a T on position 20 and a T on position 21.
6. The LNP according to any one of the preceding claims, wherein the LNP comprises at least one cationic or ionizable lipid, at least one helper lipid and preferably at least one PEG-lipid.
7. The LNP according to claim 6, wherein the at least one cationic or ionizable lipid comprises (6Z,9Z,28Z,31Z)-heptatriacont-6,9,28,31-tetraene-19-yl 4- (dimethylamino)butanoate (Dlin-MC3-DMA), wherein the at least one helper lipids is selected from the group consisting of distearoylphosphatidylcholine (DSPC), cholesterol and sphingomyelin and wherein the LNP preferably comprises at least one PEG-lipid selected from a l,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol (DMG-PEG) and a l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [amino(polyethylene glycol)] salt (DSPE-PEG), preferably DMG-PEG1000-6000 or DSPE-PEG1000-6000, more preferably DMG-PEG(2000)or DSPE-PEG(2000).
8. The LNP according to any one of the preceding claims, wherein the LNP has a hydrodynamic diameter of about 75 to about 130 nm, preferably about 78 to about 90 nm and/or a polydispersity index of about 0.15 to about 0.25, preferably about 0.18 to about 0.22, as determinable with a Zetasizer Nano (Malvern Instruments, UK).
9. The LNP according to any one of the preceding claims, wherein the LNP further comprises a detectable label, preferably on its outer surface.
10. The LNP according to any one of the preceding claims, for use in a method of treatment by therapy.
11. The LNP according to any one of claims 1-9, for use in a method of treatment of acute myeloid leukemia.
12. A pharmaceutical composition comprising the LNP according to any one of the claims 1-9 and a pharmaceutically acceptable carrier.
13. A method of coupling a ligand to a lipid, comprising
-providing a ligand according to general Formula (II) functionalized with a terminal azide moiety;
-providing a lipid functionalized with a strained alkyne moiety, preferably a dibenzocyclooctyne (DBCO) moiety;
-contacting the functionalized ligand with the functionalized lipid, to obtain a ligand coupled to a lipid via a triazole linkage; and
-optionally isolating the ligand coupled to the lipid.
14. A method for preparing an LNP according to any one of claims 1-9, comprising
-mixing at least one cationic or ionizable lipid and at least one helper lipid in a polar organic solvent to obtain a lipid mixture;
-mixing the lipid mixture with an aqueous solution comprising a therapeutic molecule at a pH between about 2 and about 6, preferably about 4, to form a mixture comprising LNPs loaded with a therapeutic molecule;
-optionally at least partially purifying, preferably dialyzing, the mixture comprising LNPs loaded with said therapeutic molecule; and
-contacting the mixture comprising the LNPs loaded with the therapeutic molecule or the at least partially purified, preferably dialyzed, mixture comprising LNPs loaded with the therapeutic molecule with a ligand coupled to a lipid as described in claim 13 to obtain an LNP according to any one of claims 1-9.
15. The method according to claim 14, wherein the at least one helper lipid comprises DSPE-PEG, preferably DSPE-PEG(2000).
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