WO2014005596A1 - Molécules cargo modifiées, leurs interactions et leurs utilisations - Google Patents

Molécules cargo modifiées, leurs interactions et leurs utilisations Download PDF

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WO2014005596A1
WO2014005596A1 PCT/DK2013/050223 DK2013050223W WO2014005596A1 WO 2014005596 A1 WO2014005596 A1 WO 2014005596A1 DK 2013050223 W DK2013050223 W DK 2013050223W WO 2014005596 A1 WO2014005596 A1 WO 2014005596A1
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albumin
sirna
binding
sina
composition according
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PCT/DK2013/050223
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Kenneth Alan Howard
Konrad BIENK
Ulrich KRAGH-HANSEN
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Aarhus Universitet
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • 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
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3515—Lipophilic moiety, e.g. cholesterol
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • the present invention relates to modified payload molecules such as short interfering nucleic acids (siNAs) and compositions comprising payload molecules such as a siNAs in combination with natural or synthetic variants of Human Serum Albumin (HSA) as well as the uses of such payload molecules and compositions as medicaments.
  • siNAs short interfering nucleic acids
  • HSA Human Serum Albumin
  • Effective drug delivery should address one or both of: 1) circulatory half-life control and 2) specific tissue targeting, with an aim to have a zero order drug release profile (Figure 1) in the tissue of interest. This has led to the development of drug delivery technologies to improve the efficacy of existing drugs and enable the use of new therapeutics such as peptides and oligonucleotides.
  • Drug delivery carrier systems incorporating the therapeutic can be composed of polymers that include poly (lactide-co-glycolide) (PLGA), chitosan, polyethyleneimine (PEI), lipids and fatty acids. These systems can control the rate of drug release, reduce renal clearance and have the possibility for targeted delivery.
  • PLGA poly (lactide-co-glycolide)
  • PEI polyethyleneimine
  • nanoparticles due to size and spherical nature.
  • Nanoparticles can be targeted to tissues by passive or active targeting.
  • EPR-Effect Enhanced Permeability and Retention Effect
  • Synthetic lipid and polymer-based particles have been shown to accumulate in tumours by transport across disrupted endothelium, which has resulted in marketed systems such as Doxil ® and DaunoXome ® for the delivery of anticancer agents.
  • Active targeting can be achieved by incorporation of a targeting moiety to the drug or carrier system.
  • a targeting moiety to the drug or carrier system.
  • Common targeting molecules are: antibodies and antibody fragments, proteins, peptides, carbohydrates and aptamers.
  • targeting moieties can lead to non-specific accumulation or clearance if the targeting molecule is recognized as foreign, and can, therefore, compromise the effect of the carrier system.
  • Targeting can also be achieved by triggered release, meaning that the therapeutic molecule is encapsulated in a liposome, polymer, gel or particle, and released from the carrier in the presence of certain stimuli.
  • the release trigger can be internal e.g. driven by pH or enzymes, or external e.g. application of heat or magnetism to the site of release.
  • Nanoparticles reduce renal clearance because of their size, and can, therefore, reduce the clearance of the incorporated therapeutic.
  • the particles can, however, be recognized by the Mononuclear Phagocyte System (MPS) composed of circulatory monocytes and fixed tissue macrophages that capture foreign material.
  • MPS Mononuclear Phagocyte System
  • Nanoparticles can be functionalized with hydrophilic polymers such as poly ethylene glycol (PEG) to install "stealth” characteristics.
  • PEG poly ethylene glycol
  • Surface PEG results in a highly hydrated surface, that reduces phagocyte recognition and capture.
  • albumin is a natural carrier with prolonged half-life, which avoids renal clearance and accumulates in tumours.
  • the protein is biocompatible and biodegradable and, therefore, an attractive candidate for a drug carrier.
  • modified short interfering nucleic acids siNAs
  • albumin as carrier molecule
  • An object of the present invention relates to a composition
  • a composition comprising a payload of one or more (several) molecules such as a short interfering nucleic acid (siNA) alone or in combination with albumin or variant of albumin or fragment thereof, or fusion polypeptide comprising albumin or variant albumin or fragment thereof.
  • severe short interfering nucleic acid
  • Another object of the present invention relates to a payload of one or more (several) molecules such as a siNA conjugated with one or more cholesteryl and/or one or more fatty acids.
  • a further object of the present invention relates to a composition of the one or more (several) payload molecules of the present invention for use as a medicament.
  • Yet another object of the present invention relates to one or more (several) payload molecules such as a siNA or the composition of the present invention for use in regulating a genetic expression of a transcript or protein associated with a disease.
  • a further object of the present invention relates to a method of treating a disease comprising administration of the of one or more (several) payload molecules such as a siNA or the composition of the present invention to a person in need thereof.
  • the siNA comprises a sense strand and an antisense strand, and wherein said sense strand is conjugated with said one or more cholesteryl and/or one or more fatty acid.
  • the siNA comprises a sense strand and an antisense strand, and wherein said antisense strand is conjugated with said one or more cholesteryl and/or one or more fatty acid.
  • the of one or more (several) payload molecules such as a siNA comprises at least one terminal nucleic acid conjugation to said one or more cholesteryl and/or one or more fatty acid.
  • the of one or more (several) payload molecules such as a siNA comprises at least two terminal nucleic acid conjugations to said one or more cholesteryl and/or one or more fatty acid.
  • the siNA comprises at least one mid- sequence conjugation to said one or more cholesteryl and/or one or more fatty acid.
  • the siNA comprises at least two mid- sequence conjugations to said one or more cholesteryl and/or one or more fatty acid.
  • the siNA comprises a combination of at least one mid-sequence conjugation and at least one terminal nucleic acid conjugation to said one or more cholesteryl and/or one or more fatty acid.
  • this fatty acid derived from a carboxylic acid selected from the group consisting of saturated monocarboxylic acids of length clO-18, being decanoic acid, dodecanoic acid, tetradecanoic acid, hexadecanoic acid and octadecoanoic acid.
  • the siNA has the siNA a nucleic acid length selected from the group consisting of, 8-250, 8-120 , 16-50, 18-35, 18-25, 18-21, 18, 21, and 27.
  • the siNA is a Dicer substrate.
  • the of one or more (several) payload molecules such as a siNA further has a linker between the cholesteryl and/or one or more fatty acid and the payload molecule.
  • the linker is selected from the group consisting of disulfide moiety, amide, phosphate, phosphate ester,
  • the payload molecule comprises further modifications selected from the group consisting of a dye label, a radioactive label, PET label, OMe, and drugs such as short peptides or proteins.
  • the albumin is wild-type Human Serum Albumin (wtHSA), SEQ ID No. 2 or a Human Serum Albumin (HSA) with at least 70% sequence identity to SEQ ID No. 2.
  • wtHSA Human Serum Albumin
  • HSA Human Serum Albumin
  • albumin or variant albumin or fragment thereof, or fusion polypeptide comprising albumin or variant albumin or fragment thereof has a binding affinity to FcRn, and/or serum half-life, which is altered relative to a reference albumin, e.g. parent albumin, such as HSA (SEQ ID No: 2) or fragment thereof or to a parent fusion polypeptide, e.g. comprising wild-type albumin or a fragment thereof.
  • the binding affinity of the albumin to FcRn (and/or serum half- life) may be stronger (longer) or weaker (shorter) than that of the reference albumin.
  • the binding affinity to FcRn may be at least 2, 5, 10, 20, 30, 40, 50, 100, 500 or 1000 fold stronger than that of the reference albumin to FcRn.
  • the binding affinity to FcRn may be at most 50%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0.2, 0.1 % of the binding affinity of the reference albumin to FcRn. Binding affinity may be measured according to the Surface Plasmon Resonance (SPR) procedure described in WO 2011/051489, incorporated herein by reference in its entirety.
  • SPR Surface Plasmon Resonance
  • the albumin is albumin or variant albumin or fragment thereof, or fusion polypeptide comprising albumin or variant albumin or fragment thereof comprising an alteration at one or more (several) positions corresponding to positions 417, 440, 464, 490, 492, 493, 494, 495, 496, 499, 500, 501, 503, 504, 505, 506, 510, 535, 536, 537, 538, 540, 541, 542, 550, 573, 574, 575, 577, 578, 579, 580, 581, 582 and 584 of the mature polypeptide of SEQ ID NO: 2.
  • the variant is not the variant consisting of SEQ ID NO: 2 with the
  • Corresponding positions in other albumins may be identified by aligning the other albumin(s) with SEQ ID NO: 2 and identifying the equivalent (or corresponding) amino acid position.
  • suitable alignment algorithms One suitable alignment algorithm is the Needleman-Wunsch algorithm as described herein.
  • the albumin variant has an alteration at position 573 or 500 such as K573A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, Y or K500A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, Y, particularly K573P, K573Y, K573W, K500A, K500D or K500G.
  • an alteration at position 573 or 500 such as K573A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, Y, particularly K573P, K573Y, K573W, K500A, K500D or K500G.
  • the albumin variant has an alteration selected from the group consisting of K573P, K573Y, and K573W.
  • the albumin variant has a K573P alteration. In a further embodiment of the present invention the albumin variant has a K573W alteration. In a further embodiment of the present invention the albumin variant has a K573Y alteration. In a further preferred embodiment, the albumin variant comprises alterations selected from or corresponding to those of EP12191856.9, particularly the alterations which relate to the 'high binders' of SEQ ID NO: 123, SEQ ID NO: 113, SEQ ID NO: 111; more preferred SEQ ID NO: 3 i.e.
  • HSA with substitution K573P 117, 124; even more preferred SEQ ID NO: 131, 110, 128, 134, 108; most preferred SEQ ID NO: 115 or SEQ ID NO: 114.
  • the alterations are in HSA (SEQ ID NO: 2), however since they may be at equivalent (corresponding) positions in other species of albumin.
  • the albumin variant comprises alterations selected from or corresponding to those of SEQ ID NO: 123 of EP12191856.9.
  • the albumin variant comprises alterations selected from or corresponding to those of SEQ ID NO: 113 of EP12191856.9.
  • the albumin variant comprises alterations selected from or corresponding to those of SEQ ID NO: 111 of EP12191856.9.
  • the albumin variant comprises alterations selected from or corresponding to those of SEQ ID NO: 3 of EP12191856.9.
  • the albumin variant comprises alterations selected from or corresponding to those of SEQ ID NO: 117 of EP12191856.9.
  • the albumin variant comprises alterations selected from or corresponding to those of SEQ ID NO: 124 of EP12191856.9.
  • the albumin variant comprises alterations selected from or corresponding to those of SEQ ID NO: 131 of EP12191856.9.
  • the albumin variant comprises alterations selected from or corresponding to those of SEQ ID NO: 110 of EP12191856.9.
  • the albumin variant comprises alterations selected from or corresponding to those of SEQ ID NO: 128 of EP12191856.9.
  • the albumin variant comprises alterations selected from or corresponding to those of SEQ ID NO: 134 of EP12191856.9.
  • the albumin variant comprises alterations selected from or corresponding to those of SEQ ID NO: 108 of EP12191856.9.
  • the albumin variant comprises alterations selected from or corresponding to those of SEQ ID NO: 115 of EP12191856.9. In a most preferred embodiment, the albumin variant comprises alterations selected from or corresponding to those of SEQ ID NO: 114 of EP12191856.9.
  • albumin variants comprising K573P, Y or W have a stronger binding affinity to FcRn and therefore are understood to have a longer half-life than wild-type albumin.
  • albumin variants comprising K500A, D or G have a weaker binding affinity to FcRn and therefore are understood to have a shorter half-life than wild-type albumin.
  • the payload molecule such as a siNA optionally in combination with albumin is a payload albumin conjugate such as a siNA albumin conjugate.
  • the payload optionally in combination with albumin are associated through electrostatic forces, hydrophobic forces or hydrogen bonds.
  • composition comprises one or more further therapeutic molecules.
  • composition comprises one or more excipients, diluents and/or carriers.
  • composition is a pharmaceutical composition.
  • a further embodiment of the present invention relates to a payload molecule modified by two or more cholesteryls.
  • Figure 3 The 3-dimensional structure of human serum albumin. The subdomains of the protein are colored in gray scale. Figure was made in PyMol from the Protein Data Bank entry 1A06.
  • FIG. 4 The secondary structure of human serum albumin. Helical structures are represented as boxes, and numbered according to the subunit location. The figure illustrates the homologous domains and the presence of characteristic double disulfide bridges in each subdomain.
  • FIG. 5 The structure of albumin with warfarin (dark gray) and diazepam (light gray). The location of these ligands represents the location of Sudlows Site I (warfarin) and Site II (diazepam). The ligands are enlarged to emphasize their location. Only the backbone of the protein is shown. The image was made in PyMol from the Protein Data Bank entries 2BXF (diazepam) and 2BXF (warfarin).
  • FIG. 6 The figure shows the 10 binding sites for decanoic acid and 7 for stearic acid revealed by x-ray diffraction studies. The numeration of binding sites is also done according to this publication. Only the backbone of the protein is represented for transparency. The sites 6 and 6 ' facilitate one decanoic acid molecule each, but one stearic acid together. The image was made in PyMol from the Protein Data Bank entry 1E7E (decanoic acid) and 1E7I (stearic acid).
  • Figure 7 The distribution of albumin between plasma (IV) and extravascular spaces (EV), and the rates of synthesis, exchange and loss.
  • TER Transcapillary Escape Rate.
  • FIG. 8 Schematic representation of the FcRn recycling pathway, which is highly important for maintaining the high physiological levels and long half-life of albumin. As albumin enters the early endosomes the acidic environment facilitates binding between albumin and FcRn. Unbound albumin in destined for lysosomal degradation, whereas FcRn bound albumin is returned to the cell membrane, and released to the
  • FIG. 9 Plasma insulin levels after administration. Due to interaction with albumin, insulin Detemir exhibits prolonged circulation compared to regular insulin. Half-life of other insulin types is controlled by other means.
  • FIG. 10 Illustration of the tumour and cell uptake of Abraxane ® paclitaxel protein- bound particles for injectable suspension) (albumin-bound).
  • the albumin bound drug complex is transcytosed into the tumour interstitium by the gp60 receptor, where it is bound and internalized by the SPARC protein, overexpressed by the tumour.
  • Figure 11 The INNO-206 prodrug, consisting of a Cys-34 binding maleimide linker, and pH sensitive N-N bond and the Doxorubicin drug. The low pH of solid tumours facilitates the release of free Doxorubicin.
  • Figure 12. Schematic representation of the albumin fusion protein Albinterferon-alpha- 2b. With an appropriate linker both protein fold properly, and the interferon benefits from the long circulatory half-life of albumin. Image created from Protein Data Base entries 1E7E and 1ITF.
  • Figure 13 Schematic representation of a siRNA molecule. The double stranded part of 21-23 residues is flanked by 2 residue overhangs at each 3 ' terminal.
  • RNAi is thought to have evolved as a defense mechanism against foreign RNA such as viruses.
  • An alternate microRNA (miRNA) pathway exists that regulates cellular processes by expression of endogenous miRNA. These miRNA enter the RNAi pathway using Dicer and RISC, and control mRNA levels by enzymatic cleavage or translational repression. It is possible to study cellular functional genomics by the use of synthetic RNA.
  • FIG 14. Examples of chemical modifications in an siRNA backbone, A, and in single nucleotides, B-E.
  • the modifications can decrease serum degradation (A, B, C) or immune response (D), and improve the gene silencing efficiency (E). Modifications can be inserted in discrete positions in the siRNA, and therefore combined.
  • FIG. 15 Representation of the functionalized siRNA designs in this project. Circle palmitoyi, square: cholesteryl. These representations will be used throughout the text. All the siRNA described in this figure are of the same length and sequence.
  • FIG. 16 Structures of the hydrophobically modified nucleotides;
  • A Cholesteryl modification, with a triethylene glycol linker between cholesteryl and phosphate. The bend in the molecule does not represent the actual structure, instead it allows the structure to be visualized on one page.
  • B Palmitoyi modification. The palmitoyi is conjugated to a LNA-thymidine, and the cholesteryl to an unmodified cytosine.
  • FIG. 17 Mobility shift assay binding experiment carried out with single stranded RNA 7 to 14. First well of each design contains RNA alone, the second well contains RNA and rAlb. All experiments were done using rAlb. siRNA: rAlb ratio was 1 : 8. All wells contained 2pmol siRNA.
  • FIG. 18 Mobility shift binding experiments carried out with siRNA 15-26.
  • Well 1 contained siRNA alone, well 2 contained siRNA and rAlb. All experiments were done using rAlb.
  • Figure 19 A) Assessment of the double bands appearing in the binding experiments. siRNA 24, compared to its components: ssRNA 8 and 10, next to a 25BP Ladder. B) siRNA 18 before (left well) and after adjustment of annealing concentrations. C) siRNA 20 after annealing (left) and after four freeze-thaw cycles. The amount of single stranded material has increased.
  • Figure 20 Comparison of the binding capacity of rAlb and sAlb. All wells contained 2pmol siRNA 26, and the amount of albumin indicated below the gel.
  • Figure 21 Results of the competition mobility shift experiment.
  • the following siRNA were used : A: 23; B: 24; C: 25 and D: 26.
  • the first well contained no albumin, the remaining contained albumin and siRNA.
  • Identities of the competing ligands are given above the figure.
  • Figure 22 Schematic representation of an ITC instrument.
  • Figure 25 ITC single injection experiments determining the enthalpy of the siRNA- Albumin interaction.
  • Figure 26 The raw data from a representative ITC binding experiment with siRNA 26 and albumin.
  • the curve represents the baseline reference power.
  • Each downward peak is an injection of siRNA 26 into albumin.
  • Sample cell albumin concentration 5 ⁇
  • syringe siRNA concentration 50 ⁇ .
  • FIG. 27 ITC curve for the binding of siRNA 26 to albumin, calculated from the raw data shown in Figure 25.
  • Figure 28 Western blot assessment of albumin antibody efficiency. Lane 1 contained 10 ng rAlb, lane 2 100 ng rAlb. Blot was stained with A) Alexa 488 conjugated secondary antibody, or B) HRP conjugated secondary antibody. The HRP blot was overstained in this case, but clearly shows the presence of albumin.
  • Figure 29 Confocal microscopy of HepG2 cells (A and B) stained with Hoechst, (dark) and albumin antibodies (bright). Reference experiment using OK cells (C) and the same albumin staining procedure revealed no unspecific staining.
  • FIG. 31 Cellular albumin uptake in Caco-2 monolayer.
  • FIG. 33 Evaluation of siRNA knockdown efficiency. Experiments were four hour transfections with siRNA concentrations of 50nM. All values have been normalized to non-treated samples. Untr: Untreated, Mism : Mismatch. The experiments were carried out in triplicate, using standard protocol.
  • FIG. 34 Optimization of albumin transfection using siRNA 19. Samples were normalized to an untreated sample, and siRNA transfected with TransIT-TKO was included as a control.
  • Figure 35 50nM of siRNA was added without (bright) or with (dark) 200nM albumin to cells using the transfection conditions described above.
  • Figure 36 Quantified fluorescence from serum, measured in terminal blood samples at 24, 72 and 120 hours after injection.
  • Figure 37 Fluorescence in serum samples from blood samples taken at 4, 24 and 48 hours after injection.
  • Figure 38 Total signal from the collected organs at 24 hours after injection.
  • Figure 39 Total signal from the remaining carcass of the animals at 24, 72 and 120 hours after injection. Note the logarithmic axis.
  • Figure 42 Peripheral tissue fluorescence at 24 hours, normalized to fluorescence per gram of tissue.
  • FIG 45 Albumin is detected in rat large intestine after 24 hours.
  • arrows indicate accumulated albumin (gray); dark gray-DAPI (nucleus), light gray-ATTO 488-phalloidin (actin); gray - Alexa 680 conjugated albumin. The color coding is done in gray scale.
  • Figure 46 Albumin is detected in rat small intestine after 24 hours.
  • Figure 47 HB albumin is detected in rat small intestine after 24 hours.
  • Figure 48 Animals were divided into 3 groups injected with siRNA 56, 57 and 58 shown in the table. The square is cholesteryl, the star is Cy7.
  • Figure 49 Blood fluorescence following intravenous injection.
  • Figure 50 Scan of the organs from the 3 groups, A) animals dosed with siRNA 56, B) siRNA 57 and C) siRNA 58. Left animal in each caption is the PBS control. The three captions are depicted on the same intensity scale.
  • Figure 51 IVIS scans of live animals A) 0.5 hours after injection, from left PBS, LB ("Low Binder”), WT (Wild-Type), HB ("High Binder”) B) 2 hours after injection, from left LB, WT, HB C) 4 hours after injection, from left PBS, LB, WT, HB D) 24 hours after injection, from left PBS, LB, WT, HB.
  • the data are represented after spectral unmixing, and represent Alexa 680 signal only. The images are provided at different scales.
  • Figure 52 Organs at 1 hour and 2 hours after injection, from the left animals dosed with PBS, LB, WT, HB.
  • Figure 53 Fluorescence in the organs. After the successful unmixing, no signal is detected in the PBS animal.
  • Figure 54 Unmixed Alexa 680 signal. From top, the organs are: Stomach, upper small intestine, lower small intestine, cecum and colon.
  • Figure 55 The table shows the siRNA used in Phase II, containing various combinations of siRNA modification. Squares indicate cholesteryl modification.
  • Figure 56 Results of binding studies for the single stranded siRNA 30 and 31, and double stranded siRNA 33, 34, 35 and 36, with estimated Kd values. Squares indicate cholesteryl modification.
  • Figure 57 Schematic representation of the LNA cholesteryl oligoes. Site of modification by cholesteryl is indicated. Squares indicate cholesteryl modification.
  • Figure 58 Binding of LNA oligoes to albumin.
  • Figure 59 schematic representations of Cy7 labeled oligoes.
  • Figure 60 Albumin-siRNA complex formation. Increasing the number of modifications increased the amount of dimers and trimers compared to monomers.
  • Figure 61 Dimer formation with single stranded siRNA. Left lane: one cholesteryl, right lane: two cholesteryl.
  • FIG. 62 siRNA knockdown efficiency. siRNA 18 is non-modified.
  • Figure 63 Binding of siRNA 34 to albumin from different species. All the binding gels have been run where the siRNA amount is constant and the albumin amount decreases from right to left. The more clear bands present in the top left, the higher the affinity. Due to the solubility issues with the cholesteryl siRNA, the free siRNA is less visible in the gel than the albumin-bound. Different albumin types are shown.
  • Figure 64 Assessment of binding of cholesteryl siRNA to albumin variants.
  • Figure 65 Competition study with three albumin variants LB, WT and HB and siRNA 34.
  • Figure 66 Serum degradation experiment. Top pane: Non-cholesteryl siRNA, bottom pane: 3X cholesteryl siRNA.
  • the arrows show the following : 1) Albumin-siRNA complexes 2) Free siRNA 3) Degraded siRNA.
  • the circle shows the albumin-siRNA complex.
  • FIG. 67 The serum degradation was using the LNA modified siRNA 52, 53 and 54, to determine whether the siRNA: albumin complex stays together over time when incubated in serum.
  • the arrows show the following : 1) Albumin-siRNA complexes 2) Free siRNA 3) Degraded siRNA.
  • the circle shows the albumin-siRNA complex
  • Figure 68 TNF-a response towards siRNA either with or without albumin.
  • cholesteryls and 35 (3 cholesteryl) B) LNA modified siRNA, from left siRNA 52 (0 cholesteryl), 54 (1 cholesteryl), 53 (2 cholesteryls)
  • Figure 69 Examples of gel assessment of annealing. The arrows indicate the anneling method.
  • Figure 70 Gel of albumin annealing. A) Cy7 Fluorescence and B) SYBR Gold stain
  • Figure 71 Competition experiment to compare behaviour of siRNA 34 annealed with/without albumin.
  • siRNA 57 was used in this experiment.
  • the amount of stearic acid is in each well : 1) 0 2) 2048 pmol 3) 1024 pmol 4) 512 pmol 5) 256 pmol 6) 128 pmol 7) 64 pmol 8) 32 pmol 9) 16 pmol 10) 8 pmol 11) 4pmol
  • Figure 73 A competition experiment with siRNA 58 annealed with albumin was done. The control experiment without albumin was not included because the siRNA 58 cannot be annealed efficiently without.
  • Figure 75 Binding of siRNA 34 to A) Alexa 680 labeled LB albumin and B)Alexa 680 labeled HB albumin.
  • Figure 76 Schematic representation of SMCC coupling of siRNA and albumin.
  • the present invention relates to one or more (several) payload molecules in particular small interfering nucleic acids (siNAs) or plasmids that have been modified and compositions comprising such siNAs in combination with natural or synthetic variants of Human Serum Albumin (HSA).
  • siNAs small interfering nucleic acids
  • HSA Human Serum Albumin
  • a payload molecule can be any molecule that would be suitable for a composition comprising HSA.
  • a payload molecule can also be any molecule that is capable of being modified with cholesteryl and/or fatty acids.
  • An object of the present invention relates to composition
  • composition comprising a payload molecule that is less than 25 kDa in size and one or more (several) albumins or fragments thereof, or fusion polypeptides comprising albumin or fragments thereof, wherein the albumin is a wild-type albumin or a variant albumin such as a variant of a parent albumin.
  • the size of the payload molecule may also be less than 10 MDa, less than 5 MDa, less than 20 kDa or even less than 10 kDa.
  • the size of the payload molecule may also be more than 10 kDa, more than 100 kDa or more than 1 MDa.
  • the payload molecule can also be a plasmid DNA or a vector.
  • the payload molecule is selected from the group consisting of a small molecule, a synthetic small molecule, a peptide or a siNA.
  • a composition comprising a short interfering nucleic acid (siNA) and one or several albumins or fragments thereof, or fusion polypeptides comprising albumin or fragments thereof, wherein the albumin is a wild-type albumin or a variant albumin such as a variant of a parent albumin.
  • compositions comprising a payload molecule and one or several albumins or fragments thereof, or fusion polypeptides comprising albumin or fragments thereof, wherein the albumin is a wild-type albumin or a variant albumin such as a variant of a parent albumin.
  • the small molecules of the present invention are selected from the group consisting of peptides, small molecules, small synthetic molecules, plasmids, vectors and small nucleic acids.
  • the small molecule can, for example, also be a dye such as Cy7as exemplified in the present examples.
  • a small molecule In the fields of pharmacology and biochemistry, a small molecule is a low molecular weight ( ⁇ 1000 Daltons) organic compound that may serve as an enzyme substrate or regulator of biological processes, with a size in the order of 10 "9 m .
  • the term 'small molecule' is usually restricted to a molecule that binds to a biopolymer such as protein, nucleic acid, or polysaccharide and acts as an effector, altering the activity or function of the biopolymer. Small molecules may function across a variety of cell types and species (e.g. mice and humans) and their study can lead to the development of new therapeutic agents. Some can inhibit a specific function of a multifunctional protein or disrupt protein-protein interactions.
  • compositions comprising a peptide and one or several albumins or fragments thereof, or fusion polypeptides comprising albumin or fragments thereof, wherein the albumin is a wild-type albumin or a variant albumin such as a variant of a parent albumin.
  • the small molecule is a GLP-1 analogue or a GLP-2 analogue.
  • the peptide may be any peptide of interest that can be used as a payload for albumin facilitated delivery.
  • the peptide is insulin, e.g. an insulin comprising an A and/or B chain, preferably comprising both an A chain and a B chain.
  • the peptide is less than 300 kDa in size, such as less than 200 kDa in size.
  • the peptide is less than 70 kDa in size.
  • the peptide is more than 3 kDa in size, such as more than 5 kDa in size, such as more than 10 kDa in size, such as more than 20 kDa in size.
  • the peptide in another embodiment of the present invention is the peptide more than 10 kDa in size.
  • Another object of the present invention relates to the payload molecule such as modified siNAs described herein which is not fused or conjugated to albumin.
  • siNAs of the invention are single or double stranded RNA or DNA oligonucleotides capable of binding target RNA or DNA sequences, including endogenous regulatory sequences, thereby inhibiting gene expression.
  • blockmir miRNA sponges as used herein refers to any nucleic acid molecule capable of inhibiting or down regulating gene expression or viral replication, for example by mediating RNA interference "RNAi” or gene silencing in a sequence-specific manner.
  • RNAi RNA interference
  • siNA can be a double-stranded polynucleotide molecule comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof.
  • the siNA can be assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self-complementary i.e. each strand comprises nucleotide sequence that is complementary to nucleotide sequence in the other strand; such as where the antisense strand and sense strand form a duplex or double stranded structure, for example wherein the double stranded region is about 19 base pairs); the antisense strand comprises a nucleotide sequence that is complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense strand comprises nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof.
  • the siNA can be a polynucleotide with a hairpin secondary structure, having self- complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a separate target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof.
  • the siNA is a short hairpin RNA (shRNA).
  • the siNA is a siRNA composed of an intact antisense strand complemented with two shorter 10-12 nucleotide sense strands. This three-stranded construct is termed small internally segmented interfering RNA (sisiRNA).
  • the siNA can be a circular single-stranded polynucleotide having two or more loop structures and a stem comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active siNA molecule capable of mediating RNAi.
  • siNAs examples include pre-miRNAs and miRNAs.
  • the siNA can also comprise a single stranded polynucleotide having nucleotide sequence complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof (for example, where such siNA molecule does not require the presence within the siNA molecule of nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof), wherein the single stranded polynucleotide can further comprise a terminal phosphate group, such as a 5'-phosphate or 5', 3'- diphosphate.
  • the siNA molecules of the invention comprise a nucleotide sequence that is complementary to a nucleotide sequence of a target gene.
  • the siNA molecules of the invention interact with a nucleotide sequence of a target gene in a manner that causes inhibition of expression of the target gene.
  • siNAs examples include antagomirs or blockmirs.
  • An antagomir or blockmir is a small synthetic RNA or DNA that is perfectly
  • antagomirs have some sort of modification, such as 2' methoxi groups and phosphothioates, to make it more resistant to degradation. It is unclear how antagomirization (the process by which an antagomir inhibits miRNA activity) operates, but it is believed to inhibit by irreversibly binding the miRNA.
  • siNA short interfering RNA
  • dsRNA double-stranded RNA
  • miRNA micro-RNA
  • shRNA short hairpin RNA
  • siRNA short interfering oligonucleotide
  • ptgsRNA post- transcriptional gene silencing RNA
  • antagomirs are now used as a method to constitutively inhibit the activity of specific miRNAs.
  • siNA short interfering RNA
  • dsRNA double-stranded RNA
  • miRNA micro-RNA
  • shRNA short hairpin RNA
  • ptgsRNA post- transcriptional gene silencing RNA
  • ptgsRNA post- transcriptional gene silencing RNA
  • antagomirs or others.
  • the siNA is a Dicer substrate.
  • a Dicer substrate refers to any siNA that is recognisable by Dicer and that can be processed by Dicer. Usual lengths are 27mers but longer substrate such as 40mers or 70mers could also work. Thus is one embodiment of the present invention siNAs that are recognisable by Dicer with a length of 27 to 40, such as 40 to 70 nucleotides.
  • RNAi is meant to be equivalent to other terms used to describe sequence specific RNA interference, such as post transcriptional gene silencing, epigenetics, or antagomirization.
  • siNA molecules of the invention can be used to epigenetically silence genes at both the post-transcriptional level or the pre-transcriptional level.
  • epigenetic regulation of gene expression by siNA molecules of the invention can result from siNA mediated modification of chromatin structure to alter gene expression.
  • nucleic acid molecule refers to a molecule having
  • the nucleic acid can be single, double, or multiple stranded and can comprise modified or unmodified nucleotides or non-nucleotides or various mixtures and combinations thereof.
  • inhibit or “down-regulate” it is meant that the expression of the gene, or level of RNAs or equivalent RNAs encoding one or more protein subunits, or activity of one or more protein subunits, such as pathogenic protein, viral protein or cancer related protein subunit(s), is reduced below that observed in the absence of the compounds or combination of compounds of the invention.
  • inhibition or down-regulation with an enzymatic nucleic acid molecule preferably is below that level observed in the presence of an enzymatically inactive or attenuated molecule that is able to bind to the same site on the target RNA, but is unable to cleave that RNA.
  • inhibition or down-regulation with antisense oligonucleotides is preferably below such as 10, 20, 30, 40, 50, 60, 70, 80, 90 % or, 10, 1, 0.1, 0.01% below that level observed in the presence of, for example, an oligonucleotide with scrambled sequence or with mismatches.
  • inhibition or down-regulation of viral or oncogenic RNA, protein, or protein subunits with a compound of the instant invention is greatersuch as 10, 20, 30, 40, 50, 60, 70, 80, 90 % greater or 2, 5, 10, 25, 50, 100, 1000 times in the presence of the compound than in its absence.
  • siNAs of the invention are antisense
  • Antisense oligonucleotides encompass single-stranded DNA or RNA that is
  • RNA sequence complementary to a portion of a specific RNA sequence, or alternatively the
  • Non-limiting examples of antisense oligonucleotides include RNA sequences
  • RNA duplex resulting in reduced levels of translation
  • antisense oligonucleotides may encompass a DNA sequence
  • Ribozymes are another form of siNAs having potential as a therapeutic. Ribozymes are ribonucleic acids having catalytic activity that can specifically cleave other RNA molecules.
  • the siNAs of the invention may be derived from any number of sources, including genomic DNA, cDNA, mRNA, and synthetic oligonucleotides.
  • siNAs of the present invention can have different lengths.
  • siNA has a nucleic acid length selected from the group consisting of 8-250, 8-120 , 16-50, 18-35, 18-25, 18- 21, 18, 21, and 27.
  • the payload molecules of the present invention such as siNAs of the present invention can be linked to further therapeutic molecules or protein such as albumin through linking groups.
  • the payload molecule may also be linked to further therapeutic molecules without any link to albumin.
  • the siNA further has a linker between the cholesteryl and/or one or more fatty acid and the siNA.
  • Linking groups of the invention are chemical moieties that link or conjugate reactive groups to siNAs.
  • the linking groups typically contain between four and twelve carbon atoms, saturated or unsaturated and optionally branched.
  • Linking groups include, but are not limited to, one or more alkyl groups such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, alkoxy groups, alkenyl groups, alkynyl groups or amino group substituted by alkyl groups, cycloalkyl groups, polycyclic groups, aryl groups, polyaryl groups, substituted aryl groups, heterocyclic groups, and substituted heterocyclic groups.
  • alkyl groups such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, alkoxy groups, alkenyl groups, alkynyl groups or amino group substituted by alkyl groups, cycloalky
  • Linking groups may also comprise polyethoxy amino acids such as AEA ((2-amino) ethoxy acetic acid) or a preferred linking group AEEA ([2-(2-amino)ethoxy)] ethoxy acetic acid).
  • AEA ((2-amino) ethoxy acetic acid)
  • AEEA [2-(2-amino)ethoxy)] ethoxy acetic acid.
  • the linking group is a "biodegradable nucleic acid linker molecule”.
  • biodegradable nucleic acid linker molecule refers to a nucleic acid molecule that is designed as a biodegradable linker to connect one molecule to another molecule, for example, a biologically active molecule.
  • the stability of the biodegradable nucleic acid linker molecule can be modulated by using various combinations of ribonucleotides, deoxyribonucleotides, and chemically modified nucleotides, for example 2'-0-methyl, 2'-fluoro, 2'-amino, 2'-0-amino, 2'-0- allyl, 2'-0-allyl, and other 2'-modified or base modified nucleotides.
  • Such linkers can also be disulphide linkers and acid sensitive linkers.
  • the linker may also be selected from the group consisting of Poly ( ⁇ -amino ester), diorthoester, vinylether, phosphoramidate, hydrazone, beta-thiopropionate, which are pH sensitive linkages and peptides that are protease sensitive that are to the cleavable spacer parts.
  • the biodegradable nucleic acid linker molecule can be a dimer, trimer, tetramer or longer nucleic acid molecule, for example an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length, or can comprise a single nucleotide with a phosphorus based linkage, for example a phosphoramidate or phosphodiester linkage.
  • the biodegradable nucleic acid linker molecule can also comprise nucleic acid backbone, nucleic acid sugar, or nucleic acid base modifications.
  • linkers of interest include linkers that utilize external activation such as physical cleavage exemplified by magnetism or by dicer processing to cleave siNA or sisi processing.
  • the linkers can be of any length, it can for example include 200 atoms, such as 100 atoms, such as 50 atoms, such as 20 atoms e.g. from 10, 20, 50, 100, 200 to 20, 50, 100, 200, 250.
  • the linker may be selected from the group consisting of disulfide moiety, amide, phosphate, phosphate ester, phosphoramidate, polyethylene glycol (PEG), 2N-hydroxy-propyl-methacrylamide (HPMA), or thiophosphate ester linkers.
  • An object of the present invention relates to a siNA conjugated with one or more cholesteryl and/or one or more fatty acids.
  • a fatty acid is a carboxylic acid with a long aliphatic tail (chain), which is either saturated or unsaturated.
  • the carboxylic acid is selected from the group of mono-, di- and poly-unsaturated carboxylic acids of length cl6-20, being palmitoleic acid, oleic acid, linoleic acid, linolenic acid and arachinodic acid.
  • the two groups of carboxylic acids above contain a single, terminal carboxyl group, which is used as a reactive group for siRNA conjugation.
  • the carboxyl is therefore consumed in the reaction, resulting in an aliphatic chain modification of the siRNA.
  • the carboxylic acid may be selected from the group of dicarboxylic acids of length clO-18, being decanedioic acid, dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid and octadecanedioic acid.
  • This group of carboxylic acids contains 2 carboxyl groups, one in each terminal of the molecule. Terminal may be the terminal nucleotide or the second nucleotide from the terminal nucleotide. During the conjugation, one carboxyl group is consumed, but the other remains intact. This results in an aliphatic chain with a terminal carboxyl group, which can improve binding.
  • Cholesteryl refers the radical of cholesterol, formed by removal of the hydroxyl group and is thus the cholesterol substituted to a target molecule.
  • the cholesteryl may be any variant of cholesteryl for example PEGylated cholesteryl or 2-hydroxypropyl)methacrylamide (HP A) cholesteryl.
  • the siNAs are siRNAs
  • the siNAs can be modified with two different types of single strand cholesteryl modifications; 3 ' modification or 3 ' within or mid sequence modification which are modification that are not 3. modifications.
  • siNA it is possible to anneal siNA with up to 3 cholesteryls or even more cholesteryls.
  • the siNA is modified by at least two cholesteryls, such as from two to five cholesteryls.
  • the siNA is modified by at least two cholesteryls.
  • the siNA is modified by at least three cholesteryls.
  • the siNA is modified by at least four cholesteryls.
  • the siNA is modified by four cholesteryls. In an embodiment of the present invention the siNA is modified by five cholesteryls.
  • the siNA is modified by six cholesteryls.
  • the siNA comprises a sense strand and an antisense strand, and wherein said sense strand is conjugated with said one or more cholesteryl and/or one or more fatty acid.
  • the siNA comprises a sense strand and an antisense strand, and wherein said antisense strand is conjugated with said one or more cholesteryl and/or one or more fatty acid.
  • the siNA comprises at least one terminal nucleic acid conjugation to said one or more cholesteryl and/or one or more fatty acid.
  • the terminal nucleic acid conjugation is a 3' conjugation.
  • the siNA comprises at least two terminal nucleic acid conjugations to said one or more cholesteryl and/or one or more fatty acid.
  • the siNA comprises at least one mid- sequence conjugation to said one or more cholesteryl and/or one or more fatty acid.
  • the siNA comprises at least two mid- sequence conjugations to said one or more cholesteryl and/or one or more fatty acid.
  • the siNA comprises a combination of at least one mid-sequence conjugation and at least one terminal nucleic acid conjugation to said one or more cholesteryl and/or one or more fatty acid.
  • a mid-sequence or within-sequence conjugation is defined as a conjugation that is nonterminal i.e. modification of any nucleic acid other than the terminal nucleic acid.
  • Preferably a mid-sequence conjugation is located at least 20 nucleic acids, such as 10 nucleic acids, such as 5 nucleic acids, such as 3 nucleic acids from the terminal nucleic acid.
  • the mid-sequence conjugation is located in the first 50 % of the siNA, such as the first 40 %, such as the first 30 %, such as the first 20 %, such as the first 10 %, wherein "the first" is defined as the 5' end of the nucleic acid.
  • the mid-sequence conjugation is located in the last 50 % of the siNA, such as the last 40 %, such as the last 30 %, such as the last 20 %, such as the last 10 %, wherein "the first" is defined as the 5' end of the nucleic acid.
  • a mid-sequence conjugation is located at from 1 to 50 nucleic acids such as 1 to 20 nucleic acids, such as 1 to 10 nucleic acids such as 10 to 20 nucleic acids such as 10 to 20 nucleic acids such as 5 to 15 nucleic acids, from another mid-sequence conjugation.
  • the term "one or more” includes an integer selected from the group consisting of 1, 2, 3, 4, 5,6 or at least one, such as at least one two, such as at least three.
  • the payload molecules of the present invention can also be modified by at least one cholesteryl, such as from one to six cholesteryls. In one embodiment of the present invention the payload molecule of the present invention is modified by at least a single cholesteryl.
  • the payload molecule of the present invention is modified by at least two cholesteryls.
  • the payload molecule of the present invention is modified by at least three cholesteryls. In one embodiment of the present invention the payload molecule of the present invention is modified by at least four cholesteryls.
  • the payload molecule of the present invention is modified by at least five cholesteryls.
  • the peptides of the present invention can similarly be modified by cholesteryls.
  • the peptide of the present invention is modified by at least two cholesteryls.
  • the peptide of the present invention is modified by at least three cholesteryls.
  • the peptide of the present invention is modified by at least four cholesteryls.
  • the peptide of the present invention is modified by two cholesteryls. In one embodiment of the present invention the peptide of the present invention is modified by three cholesteryls.
  • the peptide of the present invention is modified by four cholesteryls. Modifications
  • short interfering nucleic acids that do not require the presence of nucleotides having a 2'-hydroxy group for mediating RNAi and as such, short interfering nucleic acid molecules of the invention optionally do not include any ribonucleotides (e.g., nucleotides having a 2'-OH group).
  • siNA molecules that do not require the presence of ribonucleotides within the siNA molecule to support RNAi can however have an attached linker or linkers or other attached or associated groups, moieties, or chains containing one or more nucleotides with 2'-OH groups.
  • siNA molecules can comprise ribonucleotides at about 5, 10, 20, 30, 40, or 50% of the nucleotide positions.
  • the siNA molecules can also comprise deoxyribonucleotides at about 5, 10, 20, 30, 40, or 50% of the nucleotide positions. Chemically synthesizing nucleic acid molecules with modifications (base, sugar and/or phosphate) that prevent their degradation by serum ribonucleases can increase their potency.
  • the nucleic acid molecules comprise a 5' and/or a 3'-cap structure.
  • the 3'-cap includes, for example 4',5'-methylene nucleotide; 1- (beta-D-erythrofuranosyl) nucleotide; 4'-thio nucleotide, carbocyclic nucleotide; 5'- amino-alkyl phosphate; l,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate; 6- aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5- anhydrohexitol nucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide; phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleot
  • siNAs are designed to contain locked nucleic acids (LNAs), phosphorothiorates (PS), TINAs and/or unlocked nucleic acids (UNAs).
  • LNAs locked nucleic acids
  • PS phosphorothiorates
  • UINAs unlocked nucleic acids
  • SiNAs containing LNAs may have, among other attributes, improved affinity for complementary sequences and increased melting temperatures (hereinafter "Tm").
  • siNA molecules need not be limited to those molecules containing only RNA, but further encompasses chemically-modified nucleotides and non-nucleotides.
  • the short interfering nucleic acid molecules of the invention lack 2'-hydroxy (2'-OH) containing nucleotides.
  • the siNA comprises further modifications selected from the group consisting of a dye label, a radioactive label for example for PET, SPECT, optimal imaging or MRI, OMe, and drugs such as short peptides or proteins.
  • PET radionucleotide agents can be such as n C, 13 N, 15 0, 18 F, rubidum- 82 and
  • SPECT tracers can be such as 131 I, 67 Ga, m In, 123 I, "mTC.
  • Optical imaging tracers can be such as Quantum dots and fluorophores.
  • Modifications may be anywhere in the molecule, for example in a 5' overhang, a 3' overhang or within the molecule at the positions described above in connection with mid-sequence modifications.
  • Modifications in a 3' overhang are preferred. Particularly preferred modifications are addition of one or more (several) such as at least 1, 2, 3, 4, or 6 palmitoyl modifications.
  • Human serum albumin (HSA) Human serum albumin
  • the payload molecules such as the siNAs, the peptides and the small molecules according to the present invention are particularly useful when combined with natural or synthetic variants of an albumin such as a serum albumin, in particular Human Serum Albumin (HSA).
  • HSA Human Serum Albumin
  • Albumins are proteins and constitute the most abundant protein in plasma in mammals and albumins from a long number of mammals have been characterized by biochemical methods and/or by sequence information.
  • albumins e.g., human serum albumin (HSA)
  • HSA human serum albumin
  • HSA is a preferred albumin according to the invention and is a protein consisting of 585 amino acid residues and has a molecular weight of 67 kDa. In its natural form it is not glycosylated.
  • HSA The amino acid sequence of HSA is shown in SEQ ID NO: 2.
  • natural alleles may exist having essentially the same properties as HSA but having one or more amino acid changes compared to SEQ ID NO: 2, and the inventors also contemplate the use of such natural alleles as parent albumin according to the invention.
  • Albumins have generally a long plasma half-life of approximately 20 days or longer, e.g., HSA has a plasma half-life of 19 days. It is known that the long plasma half-life of HSA is mediated via interaction with its receptor FcRn, however, an understanding or knowledge of the exact mechanism behind the long half-life of HSA is not essential for the present invention. According to the invention the term "albumin" means a protein having the same, or very similar three dimensional structure as HSA and having a long plasma half-life.
  • albumin proteins can be mentioned human serum albumin, primate serum albumin, (such as chimpanzee serum albumin, gorilla serum albumin), rodent serum albumin (such as hamster serum albumin, guinea pig serum albumin, mouse albumin and rat serum albumin), bovine serum albumin, equine serum albumin, donkey serum albumin, rabbit serum albumin, goat serum albumin, sheep serum albumin, dog serum albumin, chicken serum albumin and pig serum albumin.
  • HSA as disclosed in SEQ ID NO: 2 or any naturally occurring allele thereof, is the preferred albumin according to the invention.
  • the parent albumin, a fragment thereof, or albumin part of a fusion polypeptide comprising albumin or a fragment thereof according to the invention has generally a sequence identity to the sequence of HSA shown in SEQ ID NO: 2 of at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 85%, preferably at least 86%, preferably at least 87%, preferably at least 88%, preferably at least 89%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, more preferred at least 96%, more preferred at least 97%, more preferred at least 98% and most preferred at least 99%.
  • sequence identity The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity”.
  • the degree of sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm
  • Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453 as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 3.0.0 or later.
  • the optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.
  • the output of Needle labelled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:
  • the degree of sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 3.0.0 or later.
  • the optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix.
  • the output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:
  • the albumin variant is a HSA with a sequence identity to SEQ ID No. 2 of at least 70%, such as at least 75, such as at least 80, such as at least 85, such as at least 90, such as at least 95, such as at least 96, such as at least 97, such as at least 98, such as at least 99, such as at least 99.2, such as at least 99.4, such as at least 99.6, such as at least 99.8, such as 100% to wtHSA (SEQ ID No. 2).
  • the parent preferably comprises or consists of the amino acid sequence of SEQ ID NO: 2. In another aspect, the parent comprises or consists of the mature polypeptide of SEQ ID NO: 2.
  • the parent is an allelic variant of the mature polypeptide of SEQ ID NO: 2.
  • the albumin is wild-type albumin or variant albumin or fragment thereof, or fusion polypeptides comprising wild-type albumin or variant albumin or fragment thereof.
  • a variant albumin may comprise an alteration at one or more (several) positions corresponding to positions 417, 440, 464, 490, 492, 493, 494, 495, 496, 499, 500, 501, 503, 504, 505, 506, 510, 535, 536, 537, 538, 540, 541, 542, 550, 573, 574, 575, 577, 578, 579, 580, 581, 582 and 584 of an albumin such as the mature polypeptide of SEQ ID NO: 2.
  • the variant does not consist of SEQ ID NO: 2 with the substitution H464A, D494N, E501K, E503K, E505K, H510A, H535A, H536A, K536E, I537N, K541E, D550G,A, K573E, K574N or K584E.
  • the albumin variant has an alteration at position 573 or 500, particularly K573P, K573Y, K573W, K500A, K500D or K500G.
  • the albumin variant has an alteration selected from the group consisting of K573P, K573Y, and K573W.
  • the albumin variant has a K573P alteration.
  • the albumin variant has a K573Y alteration.
  • the albumin variant has a K573W alteration.
  • the albumin variant has an alteration selected from the group consisting of K500A, K500D or K500G. In a further embodiment of the present invention the albumin variant has a K500A alteration.
  • the albumin variant has a K500D alteration.
  • the albumin variant has a K500G alteration.
  • albumin or variant albumin or fragment thereof, or fusion polypeptide comprising albumin or variant albumin or fragment thereof has a binding affinity to FcRn, and/or serum half-life, which is altered relative to a reference albumin, e.g. parent albumin, such as HSA (SEQ ID No: 2) or fragment thereof or to a parent fusion polypeptide, e.g. comprising wild-type albumin or a fragment thereof.
  • the binding affinity of the albumin to FcRn (and/or serum half- life) may be stronger (longer) or weaker (shorter) than that of the reference albumin.
  • the binding affinity to FcRn may be at least 2, 5, 10, 20, 30, 40, 50, 100, 500 or 1000 fold stronger than that of the reference albumin to FcRn.
  • the binding affinity to FcRn may be at most 50%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0.2, 0.1 % of the binding affinity of the reference albumin to FcRn. Binding affinity may be measured according to the Surface Plasmon Resonance (SPR) procedure described in WO 2011/051489, incorporated herein by reference in its entirety.
  • SPR Surface Plasmon Resonance
  • the albumin variant comprises alterations selected from or corresponding to those of EP12191856.9, particularly the alterations of SEQ ID NO: 123, SEQ ID NO: 113, SEQ ID NO: 111; more preferred SEQ ID NO: 3 (i.e. HSA with substitution K573P) , 117, 124 ; even more preferred SEQ ID NO: 131, 110, 128, 134, 108 ; most preferred SEQ ID NO: 115 or SEQ ID NO: 114. It is preferred that the alterations are in HSA (SEQ ID NO: 2), however they may be at equivalent
  • the albumin variant comprises alterations selected from or corresponding to those of SEQ ID NO: 123 of EP12191856.9.
  • the albumin variant comprises alterations selected from or corresponding to those of SEQ ID NO: 113 of EP12191856.9.
  • the albumin variant comprises alterations selected from or corresponding to those of SEQ ID NO: 111 of EP12191856.9.
  • the albumin variant comprises alterations selected from or corresponding to those of SEQ ID NO: 3 of EP12191856.9.
  • the albumin variant comprises alterations selected from or corresponding to those of SEQ ID NO: 117 of EP12191856.9. In a more preferred embodiment, the albumin variant comprises alterations selected from or corresponding to those of SEQ ID NO: 124 of EP12191856.9.
  • the albumin variant comprises alterations selected from or corresponding to those of SEQ ID NO: 131 of EP12191856.9. In an even more preferred embodiment, the albumin variant comprises alterations selected from or corresponding to those of SEQ ID NO: 110 of EP12191856.9.
  • the albumin variant comprises alterations selected from or corresponding to those of SEQ ID NO: 128 of EP12191856.9.
  • the albumin variant comprises alterations selected from or corresponding to those of SEQ ID NO: 134 of EP12191856.9. In an even more preferred embodiment, the albumin variant comprises alterations selected from or corresponding to those of SEQ ID NO: 108 of EP12191856.9.
  • the albumin variant comprises alterations selected from or corresponding to those of SEQ ID NO: 115 of EP12191856.9.
  • the albumin variant comprises alterations selected from or corresponding to those of SEQ ID NO: 114 of EP12191856.9.
  • fragment means a polypeptide having one or more (several) amino acids deleted from the amino and/or carboxyl terminus of an albumin and/or an internal region of albumin that has retained the ability to bind to FcRn. Fragments may consist of one uninterrupted sequence derived from HSA or may comprise two or more sequences derived from HSA.
  • the fragments according to the invention have a size of more than approximately 20 amino acid residues, preferably more than 30 amino acid residues, more preferred more than 40 amino acid residues, more preferred more than 50 amino acid residues, more preferred more than 75 amino acid residues, more preferred more than 100 amino acid residues, more preferred more than 200 amino acid residues, more preferred more than 300 amino acid residues, even more preferred more than 400 amino acid residues and most preferred more than 500 amino acid residues.
  • Useful fragments of HSA are domains e.g. Dl, D2, D3, D1+D2, D2+D3, D1+D3, D3+D3, D1+D3+D3, D1+D2+D3+D3. Fragments may or may not comprise one or more of the amino acid sequence alterations described herein. Such fragments and other variants as well as fusion variants are described in WO 2011/124718 which hereby is incorporated by reference in its entirety.
  • a particularly preferred method is: SPR analyses were performed on a Biacore 3000 instrument (GE Healthcare). Immobilisation was carried out on CM5 chips coupled with shFcRn (GeneArt lot#1177525) using GE Healthcare amine coupling chemistry as per the manufacturer's instructions. Immobilised levels of shFcRn-HIS (shFcRn with a 6-His tail on the C-terminus of beta-2-microglobulin) were 1200 - 2500RU and achieved by injecting 20Mg/ml_ shFcRn diluted using sodium acetate pH4.5 (G E Healthcare). Chip surface was left to stabilize with a constant flow ( ⁇ ) of running buffer - Di- basic/Mono-basic phosphate buffer pH5.5 at 25 °C overnight.
  • the chip surface was conditioned by injecting 5-12 x 45 ⁇ _ Di-basic/Mono-basic phosphate buffer at 30 ⁇ _/ ⁇ - ⁇ followed by HBS_EP (0.01 M HEPES, 0.15 M NaCI, 3mM EDTA, 0.005% surfactant P20) at pH 7.4 (GE Healthcare)) regeneration steps (12s) in between each injection. Surfaces were then checked for activity by injecting 3 ⁇ 45 ⁇ _ positive control at 30 ⁇ " ⁇ , followed by 12s regeneration pulse. Kinetic measurements were performed by injecting dilutions ( ⁇ - ⁇ . ⁇ ) of HSA and HSA variants at 30 ⁇ " ⁇ over immobilised shFcRn, at 25°C.
  • the reference cell value was then subtracted and Biaevaluation software 4.1 used to obtain kinetic data and confirm KD values.
  • the variants were wild-type albumin (SEQ ID NO: 2) and variant albumins. The variants were analysed by SPR to determine their binding response (RU) to shFcRn. Some variants were further characterized to determine KD values.
  • the relative binding affinity of albumin variants compared to wild-type albumin was calculated by calculating the mean of duplicate measurements for each variant and for wild-type albumin (SEQ ID NO: 2).
  • An embodiment of the present invention relates to the compositions or payload molecules of the present invention that have an enhanced or weaker binding to the FcRn receptor.
  • the variants of albumin or fragments thereof according to the invention may also be fused with a non-albumin polypeptide fusion partner.
  • the term 'fusion' means a genetic fusion to a fusion partner.
  • the fusion partner may in principle be any polypeptide but generally it is preferred that the fusion partner is a polypeptide having therapeutic or diagnostic properties.
  • Fusion polypeptides comprising albumin or fragments thereof are known in the art. It has been found that such fusion polypeptide comprising albumin or a fragment thereof and a fusion partner polypeptide have a longer plasma half-life compared to the unfused fusion partner polypeptide. According to the invention it is possible to alter the plasma half-life of the fusion polypeptides according to the invention compared to the corresponding fusion polypeptides of the prior art.
  • One or more therapeutic polypeptides may be fused to the N-terminus, the C-terminus of albumin, inserted into a loop in the albumin structure or any combination thereof. It may or it may not comprise linker sequences separating the various components of the fusion polypeptide.
  • conjugation' means a chemical conjugation to a conjugation partner.
  • the term 'linked' covers both genetic fusions and chemical conjugations.
  • Allelic variant means any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences.
  • allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.
  • the alteration at one or more position may independently be selected among
  • the albumin is a recombinant albumin.
  • the variants of albumin or fragments thereof according to the invention may be conjugated to a second molecule such as the siNAs of the present invention using techniques known within the art.
  • Said second molecule may comprise a diagnostic moiety, and in this embodiment the conjugate may be useful as a diagnostic tool such as in imaging; or the second molecule may be a therapeutic compound and in this embodiment the conjugate may be used for therapeutic purposes where the conjugate will have the therapeutic properties of the therapeutic compound as well as the long plasma half-life of the albumin.
  • Conjugates of albumin and a therapeutic molecule are known in the art and it has been verified that such conjugates have long plasma half-life compared with the non- conjugated, free therapeutic molecule as such.
  • the conjugates may conveniently be linked via a free thiol group present on the surface of HSA (amino acid residue 34 of mature HSA, or corresponding amino acid residue in other species of albumin) using well known chemistry.
  • the payload molecule such as a siNA optionally in combination with albumin is a payload albumin conjugate such as a siNA albumin conjugate.
  • variants of albumin or fragments thereof may further be used in form of
  • sociates In this connection the term “associate” is intended to mean a compound comprising a variant of albumin or a fragment thereof and another compound bound or associated to the variant albumin or fragment thereof by non-covalent binding.
  • the payload molecule such as an siNA optionally in combination with albumin can be non-covalently linked by ionic interactions, hydrogen bonding, van der waals
  • the payload molecule man be in combination with another molecule in either covalent or non-covalent links.
  • the payload molecule such as a siNA optionally in combination with albumin is associated through electrostatic forces.
  • a further embodiment of the present invention relates to a composition comprising several albumins, such as two or more albumins that are linked using a linker as described above and payload molecule such as a siNA of the present invention.
  • the albumin is a recombinant albumin such as from a fungus such as a yeast, preferably from Pichia or Saccharomyces, most preferably from Saccharomyces cerevisiae.
  • the albumin has a lower level of post-translational covalent modification, such as glycation, than serum derived albumin.
  • the albumin has, prior to conjugation or association with payload molecule such as a siNA, low levels of covalently bound molecules to Cys34.
  • payload molecule such as a siNA
  • a payload molecule such as a small molecule, a peptide or a siNA
  • a payload molecule such as a small molecule, a peptide or a siNA
  • dimers and trimmers can be seen in the present examples.
  • the result of the conjugation is a dimer. In another embodiment of the present invention the result of the conjugation is a trimer.
  • the payload molecules of the present invention have the unique property that they are able to form soluble conjugates with albumin. Specifically have the present inventors in the example "Albumin annealing of siRNA” shown that siRNAs with two cholesterols and a Cy7 dye are capable of annealing in the presence of albumin, see figures 71 and 72, whereas the molecule precipitates out due to hydrophobicity without albumin presence, resulting in poor annealing efficiency, seee figures 69 and 70.
  • one aspect of the present invention relates to a method for forming a conjugate comprising a highly hydrophobic payload molecule, for example the two cholesteryl and Cy7 modified siRNA described in the example, and an albumin, where the formulation with albumin increases the solubility and stability of the payload molecule.”
  • a highly hydrophobic payload molecule for example the two cholesteryl and Cy7 modified siRNA described in the example, and an albumin, where the formulation with albumin increases the solubility and stability of the payload molecule.
  • a further aspect of the present invention relates to the use of an albumin as disclosed herein for the formation of a conjugate comprising the albumin and a payload molecule modified by at least two cholesterols.
  • composition comprises one or more further therapeutic molecules.
  • Such molecules can be other payload molecules, siNAs, natural or synthetic proteins, small molecules, hormones or similar.
  • Such further therapeutics can be conjugated directly to the siNAs of the present invention and can also be included in the compositions of the present invention either by conjugation to a siNA or the albumin(s).
  • compositions comprising the payload molecules such as siNAs such as alone or optionally in combination with albumin may be administered in a
  • physiologically acceptable medium e.g., deionized water, phosphate buffered saline (PBS), saline, aqueous ethanol or other alcohol, plasma, proteinaceous solutions, mannitol, aqueous glucose, vegetable oil, or the like.
  • PBS phosphate buffered saline
  • aqueous ethanol or other alcohol plasma, proteinaceous solutions, mannitol, aqueous glucose, vegetable oil, or the like.
  • Buffers may also be included, particularly where the media are generally buffered at a pH in the range of about 5 to 10, where the buffer will generally range in concentration from about 50 to 250 mM salt, where the concentration of salt will generally range from about 5 to 500 mM, physiologically acceptable stabilizers, and the like.
  • the compounds may be lyophilized for convenient storage and transport.
  • composition comprises one or more excipients, diluents and/or carriers.
  • Aqueous suspensions may contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions.
  • Such excipients include suspending agents, for example sodium
  • dispersing or wetting agents can be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or
  • condensation products of ethylene oxide with long chain aliphatic alcohols for example heptadecaethyleneoxycetanol
  • condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate
  • condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides for example polyethylene sorbitan monooleate.
  • the aqueous suspensions can also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.
  • preservatives for example ethyl, or n-propyl p-hydroxybenzoate
  • coloring agents for example ethyl, or n-propyl p-hydroxybenzoate
  • flavoring agents for example ethyl, or n-propyl p-hydroxybenzoate
  • sweetening agents such as sucrose or saccharin.
  • Oily suspensions can be formulated by suspending the active ingredients in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin.
  • the oily suspensions can contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol.
  • Sweetening agents and flavoring agents can be added to provide palatable oral preparations.
  • These compositions can be preserved by the addition of an anti-oxidant such as ascorbic acid.
  • Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives.
  • a dispersing or wetting agent e.g., glycerol, glycerol, glycerol, glycerol, glycerol, glycerol, glycerin, glycerin, glycerin, glycerin, glycerin, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, glycerol, glycerol, glycerol, glycerol, glycerol, glycerol, glycerol, glycerol, glycerol
  • compositions of the invention can also be in the form of oil-in-water emulsions.
  • the oily phase can be a vegetable oil or a mineral oil or mixtures of these.
  • Suitable emulsifying agents can be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example, sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate.
  • the emulsions can also contain sweetening and flavoring agents.
  • Compounds of the invention can be administered parenterally in a sterile medium.
  • siNA alone or in combination with albumin, depending on the vehicle and concentration used, can either be suspended or dissolved in the vehicle.
  • adjuvants such as local anesthetics, preservatives and buffering agents can be dissolved in the vehicle.
  • the payload molecules such as a siNAs, peptides, small molecules or compositions comprising siNAs, peptides or small molecules optionally in combination with albumin are generally administered parenterally, such as intravascularly (IV), intraarterial ⁇ (LA), intramuscularly (LM), subcutaneously (SC), mucosally, orally or the like.
  • IV intravascularly
  • LA intraarterial ⁇
  • LM intramuscularly
  • SC subcutaneously
  • mucosally orally or the like.
  • Administration may also be made by transfusion, or it may be mucosal, oral, nasal, rectal, transdermal or aerosol, where the nature of the conjugate allows for transfer to the vascular system.
  • the payload molecule such as siNAs, peptides or small molecules or compositions comprising siNAs peptides or small molecules optionally in combination with albumin may be administered by any convenient means, including syringe, trocar, catheter, or the like.
  • the administration can be intravascularly, where the site of introduction is not critical to this invention, preferably at a site where there is rapid blood flow, (e.g., intravenously, peripheral or central vein).
  • the route of administration is mucosal or oral.
  • Other administration routes may be useful, e.g. where the administration is coupled with slow release techniques or a protective matrix.
  • siNAs or compositions are effectively distributed in the blood, so as to be able to react with the mobile proteins.
  • Dosage levels of the order of from about 0.1 mg to about 140 mg per kilogram of body weight per day are useful in the treatment of the described conditions (about 0.5 mg to about 7 g per patient per day).
  • the amount of active ingredient that can be combined with the carrier materials to produce a single dosage form may vary depending upon the host treated and the particular mode of administration.
  • the concentration of the modified siNA for administration may vary, generally ranging from about 1 pg/ml to 100 mg/ml, pre-administration.
  • the total amount administered intravascularly will generally be in the range of about 0.1 mg to about 500 mg, more usually about 1 mg to about 250 mg.
  • composition of the present invention may be formulated for the intended use.
  • composition of the present invention that is formulated for oral or mucosal administration.
  • the payload molecule such as siNAs, peptides or small molecules and compositions of the invention may be used to treat diseases in a mammal in which inhibition of gene expression of a particular gene is beneficial.
  • “Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, or sports, animals, such as dogs, horses, cats, cows, etc.
  • the mammal is human.
  • the diseases include, but are not limited to, cancer, autoimmune diseases, viral and bacterial infections, endocrine system disorders, neural disorders including central and peripheral nervous system disorders, cardiovascular disorders, pulmonary disorders, and reproductive system disorders.
  • a payload molecule such as a siNA, peptides or small molecules or the composition of the present invention for use as a medicament.
  • the payload molecule such as siNAs, peptides or small molecules and compositions of the invention are useful for the amelioration and/or treatment of cancers and other hyperproliferative disorders.
  • Cancer cells are usually characterized by aberrant expression of a gene.
  • Cancers and other hyperproliferative disorders for which this invention provides therapy include, but are not limited to, neoplasms associated with connective and
  • musculoskeletal system tissues such as fibrosarcoma, rhabdomyosarcoma,
  • myxosarcoma, chondro sarcoma, osteogenic sarcoma, chordoma, and liposarcoma neoplasms located in the abdomen, bone, brain, breast, colon, digestive system, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, liver, lymphatic system, nervous system (central and peripheral), pancreas, pelvis, peritoneum, skin, soft tissue, spleen, thorax, and urogenital tract, leukemias (including acute promyelocytic, acute lymphocytic leukemia, acute
  • myelocytic leukemia myeloblasts, promyelocytic, myelomonocytic, monocytic, erythroleukemia), lymphomas (including Hodgkins and non-Hodgkins lymphomas), multiple myeloma, colon carcinoma, prostate cancer, lung cancer, small cell lung carcinoma, bronchogenic carcinoma, testicular cancer, cervical cancer, ovarian cancer, breast cancer, angiosarcoma, lymphangiosarcoma, endotheliosarcoma,
  • lymphangioendotheliosarcoma synovioma
  • mesothelioma mesothelioma
  • Ewing's sarcoma
  • leiomyosarcoma squamous cell carcinoma, basal cell carcinoma, pancreatic cancer, renal cell carcinoma, Wilm's tumor, hepatoma, bile duct carcinoma, adenocarcinoma, epithelial carcinoma, melanoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, emangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, neuroblastoma, retinoblastoma, bladder carcinoma, embryonal carcinoma, cystadenocarcinoma, medullary carcinoma, choriocarcinoma, and seminoma.
  • an aspect of the present invention relates to the use of a payload molecule such as a siNA or siNA-albumin conjugate as descried herein for treatment of diseases that benefit from intestinal delivery e.g. cancer, inflammatory disease as described above.
  • a payload molecule such as a siNA or siNA-albumin conjugate as descried herein for treatment of diseases that benefit from intestinal delivery e.g. cancer, inflammatory disease as described above.
  • Another aspect of the present invention relates to the use of a payload molecule such as a siNA or siNA-albumin conjugate as descried herein for intestinal delivery of a drug.
  • a payload molecule such as a siNA or the composition of the present invention for use as in regulating a genetic expression of a transcript or protein associated with a disease.
  • a further object of the present invention relates to a method of treating a disease comprising administration of the payload molecule such as a siNA or the composition of the present invention to a mammal in need thereof.
  • Drug delivery carrier systems incorporating the therapeutic can be composed of polymers that include poly (lactide-co-glucolide) (PLGA), chitosan, polyethyleneimine (PEI) or lipid-based. These systems can control the rate of drug release, reduce renal clearance and have the possibility for targeted delivery. They can be referred to as nanoparticles due to size and spherical nature.
  • Nanoparticles can be targeted to tissues by passive or active targeting. Passive accumulation by the Enhanced Permeability and Retention Effect (EPR-Effect) has been utilized for delivery to tumours. Proteins greater than 40-50kDa accumulate in the tumour, due to the leaky vasculature and lack of lymphatic drainage, and function as a source of nutrients. Synthetic lipid and polymer-based particles have been shown to accumulate in tumours by transport across disrupted endothelium, which has resulted in marketed systems such as Doxil ® and DaunoXome ® for the delivery of anticancer agents. Active targeting can be achieved by incorporation of a targeting moiety to the drug or carrier system. Common targeting molecules are: antibodies and antibody fragments, proteins, peptides, carbohydrates and aptamers. The addition of targeting moieties can lead to non-specific accumulation or clearance if the targeting molecule is recognized as foreign, and can therefore compromise the effect of the carrier system.
  • EPR-Effect Enhanced Permeability and Retention Effect
  • Targeting can also be achieved by triggered release, meaning that the therapeutic molecule is encapsulated in a liposome, polymer, gel or particle, and released from the carrier in the presence of certain stimuli.
  • the release trigger can be internal; driven by pH or enzymes, or external; application of heat or magnetism to the site of release. This results in an increased concentration of the drug in the desired tissue.
  • Nanoparticles reduce renal clearance because of their size, and can therefore reduce the clearance of the incorporated therapeutic.
  • the particles can, however, be
  • MPS Mononuclear Phagocyte System
  • Nanoparticles can be functionalized with hydrophilic polymers such as poly ethylene glycol (PEG) to install "stealth" characteristics.
  • PEG poly ethylene glycol
  • Surface PEG results in a highly hydrated surface, that reduces phagocyte recognition and capture.
  • Studies have shown, however, an increased immune response after repeated administration of PEG coated particles, that may result in an accumulation in liver and spleen over time. There is, therefore, a necessity to investigate alternative methods for improving circulatory half- life of delivery systems.
  • HSA Human Serum Albumin
  • HSA has an extraordinary long circulatory half-life of 19-23 days due to its high stability, interaction with recycling receptors and rescue from renal clearance.
  • the protein is present in extravascular tissues and organs in addition to plasma.
  • the properties of albumin make the protein an attractive candidate for drug delivery. Many conventional drugs interact with the binding domains of albumin with
  • the transport and depot properties of albumin can be attributed to the unique binding capacity of the protein.
  • the hydrophilic and charged surface of the protein makes it very soluble in water and hydrophobic binding pockets facilitate binding of hydrophobic ligands.
  • HSA improves the solubility and transports natural ligands such as fatty acids, bile salts, steroids, metal ions and bilirubin.
  • HSA interacts with many known drugs such as Diazepam, Digitoxin, Warfarin, Cisplatin and Taxol.
  • Dependent on the drug type over 99 % of the drug can be bound to serum albumin, thereby significantly lowering the amount of free drug in circulation.
  • the next sections will describe the binding sites, which enable these interactions. Subdomains
  • Albumin consists of 3 homologous domains; I, II and III (sometimes referred to as 1, 2 and 3), further divided into subdomains A and B (see Figure 3). These domains have predominantly helical structure, (67 % of entire protein) with a total of 17 disulfide bonds between helices (see Figure 4).
  • the 3 homological domains are not only similar in helical arrangement, but also in 3-dimensional structure. Due to the arrangement of the domains, however, albumin is a highly asymmetric protein. Domains I and II are perpendicular to each other, and have a large contact region with hydrophobic interactions between IIA and IA-IB interfaces. In contrast, domain III interacts only with IIB, and domains I and III are separated by a large hydrophobic cleft. Due to the flexible intradomain connections the entire molecule is a very adaptable structure, adjusting and changing shape depending on surroundings.
  • the albumin molecule contains a total of 35 cysteines, 34 of which are involved in formation of 17 disulfide bridges. Sixteen of these form characteristic double bridges, where two adjoining cysteines form disulfide bridges to two different helices. Due to this arrangement, each subdomain has a rigid and stable structure ( Figure 4). The remaining Cys-34 is located on a loop in subdomain IA, close to the protein surface, but with the sulfur-atom pointing inward and somewhat sterically hindered from interaction with large molecules. In the bloodstream 30-40% of HSA is oxidized by cysteine or glutathione.
  • Cys-34 is a potent antioxidant, because it can form a sulfenic acid by accepting oxygen when subjected to oxidative pressure, and plays an important role in binding and trafficking of NO and SH-containing compounds. Despite these interactions, Cys-34 is located some distance away from the hydrophobic pockets of albumin, and plays no role in binding of the most common ligands and drugs. This has made the cysteine a common target for covalent modifications of albumin for labeling, surface modification and other purposes. Binding sites Albumin interacts with many ligands via a panel of binding sites distributed over the protein surface.
  • albumin Compared to other proteins, albumin has high content of the ionic amino acids glutamic acid and lysine, resulting in ⁇ 185 ions per albumin molecule, and a net negative charge at pH 7 of -17. This makes the protein very soluble in water, and solutions up to 50% w/v can be prepared. Most of these hydrophilic residues are on the surface of the protein, and the core is generally hydrophobic. There are a number of clefts, cavities and pockets of different size and shape, through which hydrophobic ligands can interact with the hydrophobic core and bind.
  • Sudlow's site I is a pocket comprising all six helices of subdomain IIA, with the entrance to the pocket restricted by subdomain IIIA ( Figure 5).
  • the pocket is predominantly hydrophobic, with a few hydrophilic residues that further stabilize binding of ligands, and positive residues close to the protein surface.
  • the best-known ligand for this site is warfarin, but many other ligands have been identified to bind to this site (see Table 1).
  • Ligands of sizes smaller than 310 Da do not induce changes in the binding site, however, binding of larger ligands promotes dislocation of residues around the binding site.
  • Site II is similar to site I in structure, but smaller. It facilitates binding for many ligands (see Table 1), most notable Ibuprofen, but no drug ligands are shared with site I.
  • the heme cleft In close proximity of the FA binding site in subdomain IB, there is an additional binding pocket called the heme cleft. According to crystal log raphic data, this is the binding site for heme, bilirubin and fusidic acid, and is the only binding site for these ligands.
  • the flexibility of the intradomain regions enables conformational changes to facilitate binding.
  • the long loops between subdomains allow large movements of the regions relative to each other.
  • Large structural changes occur in the protein during pH changes, where 5 different shapes have been observed.
  • the Normal (N), heart shaped form of the protein is present at pH 4.3-8.
  • the Fast (F) conformation below pH 4.3 and the Basic (B) above pH 8 show decreased helical content and some degree of denaturation.
  • the B conformation increases the binding strength of warfarin, indicating that the structure of the binding site is only partially altered and not destroyed by this pH.
  • the protein is further denatured.
  • the binding of fatty acids induces conformational changes in the overall protein structure, as well as local changes within the binding sites. In this way, FA binding can regulate binding of ligands to other sites.
  • One of the high affinity sites of myristate overlaps to some extent with Sudlows site I, and myristate regulates drug binding to this site both allosterically and competitively.
  • binding of heme to the heme site inhibits binding of drugs to site I.
  • X-ray diffraction data of bound ligands confirm changes in the conformation, and ligand co-binding studies indicate that the conditions surrounding the binding sites are also altered by these conformational changes.
  • Most known ligands, which have been crystalized, show allosteric changes in the protein, indicating that the allostery is a sensitive and highly regulated system, which continuously modulates the binding properties of albumin. It is a difficult task to compose a comprehensive description of ligand interactions and binding sites. Crystal log raphic data provide precise information about binding sites and their location, but the information is static and far from physiological conditions. In contrast, binding experiments such as equilibrium dialysis reveal thermodynamic data but no information about binding site location.
  • albumin is synthesized by the liver as a single peptide chain containing a signal peptide, a pro-peptide and the albumin itself.
  • the signal peptide is cleaved of directly after translation, but ensures that the protein is transferred into the endoplasmatic reticulum (ER).
  • ER endoplasmatic reticulum
  • the resulting protein, proalbumin is transported via the Golgi apparatus to secretory vesicles, where the 6 amino acid large propeptide is cleaved off before secretion of the mature albumin from the cell.
  • albumin In a healthy adult, approximately 13.6 g albumin is synthesized and secreted daily.
  • Figure 7 shows the amount of albumin present in the bloodstream (IV) and in the extravascular spaces (EV), and the steady state exchange rates between the
  • the extravascular albumin is mainly localized in skin, muscle, liver, gut and in subcutaneous space.
  • Albumin has a wide tissue distribution, and there is a dynamic exchange of protein between different organs and compartments
  • the mechanisms of transcapillary escape are less studied, however, it is clear that several mechanisms exist and contribute to the albumin biodistribution.
  • the increased vascular permeability accounts for most of the albumin escape to EV.
  • the albumin transport is mediated by active, receptor-mediated transcytosis.
  • SPARC Secreted Protein Acidic and Rich in Cysteine
  • osteonectin is usually present in bone, promoting mineralization.
  • the role of SPARC in albumin transport in healthy tissue is unclear.
  • the protein is, however, upregulated in many cancers, where it promotes uptake and degradation of albumin for nutrition.
  • gp30 and gpl8 have been associated with scavenger receptors, and have never been shown to transport albumin. Altered and distorted conformations of albumin increase binding to these proteins compared to native albumin and it has, therefore, been proposed that they are responsible for degradation of non-functioning albumin, and not transcytosis.
  • gp60 is proposed as the main transport mediator for albumin where gp60 binds albumin and subsequently binds to caveolin-1 to initiate invagination of the cell membrane.
  • the consequent clustering of the gp60-albumin complex during vesicle formation reduces receptor affinity for albumin, which permits the release of albumin and any bound ligands to the abluminal side of the cell (John et al, 2003, Am. J. Physiol. Lung Cell Mol. Physiol. 284: L187-L196.
  • This protein is expressed in many different endothelial tissues, and blocking of the protein with antibodies lowers the albumin transport in endothelial cell monolayers by 90 %.
  • the affinity of the albumin-gp60 interaction is regulated by allosteric changes in albumin.
  • albumin When albumin is saturated with fatty acids, the albumin transport across endothelium can be increased as much as 200 %. This indicates a preference for transcytosis of albumin carrying cargo, and further improves the ability of albumin to transport fatty acids to tissues.
  • the gp60 receptor has never been sequenced or crystallized, and little is known about the mechanism of binding and uptake. It is possible that other proteins or pathways are involved in the transport of albumin across endothelium.
  • Albumin exhibits an extended half-life of ⁇ 20 days. This can be attributed to the stability of the protein and to two receptor-mediated pathways, which promote the rescue of the protein from renal excretion or degradation: 1) Reabsorption of albumin by megalin-cubilin from renal proximal tubules and 2) recycling from lysosomal degradation by the neonatal Fc receptor (FcRn).
  • FcRn neonatal Fc receptor
  • Megalin is a transmembrane transport protein expressed in the renal proximal tubules. It is associated with the formation of coated pits and the internalization of materials. Cubilin interacts with megalin, and has binding sites for apolipoproteins, immunoglobulin light chain and albumin. Albumin filtered into the proximal tubuli binds to cubilin, is internalized and released back into the bloodstream via the megalin- mediated pathway, thereby avoiding urinal excretion.
  • FcRn is an intracellular receptor complex expressed in a wide range of tissues, including most endothelial cells. The complex plays a role in maternofetal protein transfer, and also protects
  • albumin plasma levels are generally stable, and levels below 30 g/L (hypoalbuminemia) or above 55 g/L (hyperalbuminemia) are considered
  • Hyperalbuminemia is normally related to dehydration, but can also be linked to high blood pressure, high body weight and an unhealthy diet.
  • hypoalbuminemia can result from lower synthesis rate, loss of albumin or a
  • albumin concentration is used as a diagnostic screening tool.
  • Less acute hypoalbuminemia can be related to hepatitis, rheumatoid arthritis and other infections, which lower the rate of albumin synthesis.
  • hypoalbuminemia is a marker of many cancers, and some tumours utilize albumin as a source of energy and nutrients.
  • the albumin is accumulated in tumour tissue passively by the EPR effect and actively by binding to the SPARC protein receptor. In contrast to healthy tissue, the albumin is not recycled but internalized and degraded, resulting in albumin loss.
  • the accumulation of albumin in tumours makes albumin an attractive drug carrier to target cancer.
  • analbuminemia In rare cases of analbuminemia, very little or no albumin is present. The condition is caused by mutations in the albumin gene. When a stop-codon is introduced, the resulting truncated protein does not exhibit the same circulatory or binding properties as native albumin and is, therefore, rapidly degraded. Despite the role of albumin in trafficking and transport, analbuminemia is generally tolerated as other transport proteins are upregulated and take over the transport role of albumin. Improved half-life and passive targeting of drugs with albumin
  • albumin The long circulatory half-life of albumin can be utilized to improve the circulatory time of therapeutic molecules.
  • a drug cargo By attachment of a drug cargo to albumin, the drug benefits from the same recycling and rescuing pathways as albumin, and the circulation time is prolonged.
  • the choice of approach depends on the mode of action of the therapeutic and target tissue.
  • the hydrophobic interaction approach features reversible attachment facilitated by hydrophobic interactions with the albumin binding sites.
  • the cargo can, therefore, be released into the free form over time or from binding competition by other endogenous ligands.
  • the covalent coupling and co-expression approaches are less versatile due to irreversible attachment that may require albumin degradation for drug release. These covalent approaches can, therefore, only be used if the molecule can fulfill the therapeutic role whilst attached to albumin, or a release mechanism can be
  • a drug is not a natural ligand of albumin, it may be functionalized with a compound that interacts reversibly with the hydrophobic binding sites of albumin.
  • the most notable example is the insulin analog Detemir, produced by Novo Nordisk under the trade name Levemir. This insulin is functionalized with a myristic acid moiety protruding from the B chain of the insulin. Detemir is formulated and injected in the same manner as standard insulin, but it binds reversibly to subcutaneous albumin via the myristic acid after injection. As the binding is reversible, clearance of free insulin changes the equilibrium of binding and releases bound insulin. Consequently, bound insulin is slowly released into the free, active form, giving an extended period of activity (Figure 9).
  • the ligand binds to endogenous albumin after injection, so there is no requirement for preformulation with albumin prior to injection. There is no overall depletion of albumin, since the drug dissociates over time and causes no albumin degradation. This enables multiple administrations and high drug concentrations without compromising albumin concentrations, making the approach ideal for insulin.
  • Albumin micro- and nanoparticles can be prepared by covalent crosslinking or by ionic or hydrophobic interactions under appropriate conditions. Chemically cross-linked albumin microspheres (1-5 ⁇ " ⁇ ) encapsulating a drug were extensively researched in the 1980 ' s, but none were commercialized . Current systems focus on smaller nanoparticles (50-200nm) produced without covalent cross-linking. Albumin particles containing 99Technetium or other radioactive compounds have been used for in vivo cancer detection. Albumin particle formulations have also been used for drug delivery. The most successful has been Nanoparticle Albumin Bound (NAB) Paclitaxel, sold under the name Abraxane ® . Without chemical crosslinking the anticancer drug Paclitaxel is emulsified under pressure with albumin to form particles ranging from 100-200nm (mean size is 130nm).
  • NAB Nanoparticle Albumin Bound
  • Paclitaxel in free form is water insoluble, but is soluble in this formulation without the necessity to include toxic excipients.
  • Abraxane ® is proposed to improve targeting to tumours. Endothelial binding and transcytosis of Abraxane ® is higher than that of free paclitaxel, and this difference can be negated by inhibiting the gp60 albumin uptake pathway, indicating an albumin specificity. It is also suggested that the particles accumulate in tumour tissue in a non-receptor mediated way by the EPR effect.
  • the albumin particles bind to the overexpressed SPARC protein, and are internalized into cells (Figure 10). It is not known whether the entire particles are taken up, or the particles dissociate to albumin-paclitaxel conjugates before internalization, but it is has been shown that the particles dissociate over time.
  • the superior performance of Abraxane over other paclitaxel formulations is attributed to albumin properties.
  • the NAB technology is proposed as a general anticancer drug formulation method to lower adverse effects and improve efficiency.
  • Abraxane ® no other NAB-based drugs have been commercialized.
  • the therapeutic molecule is covalently coupled to albumin.
  • One common method is conjugation through the SH group of Cys-34. Since there is only one unpaired cysteine, the conjugation product is homogeneous and reproducible. Cys- 34 is not located in the direct proximity of any ligand binding site, nor does covalent attachment alter the binding capabilities of the important drug binding sites. Due to the irreversibility of the covalent attachment, the drug is not readily released from the albumin requiring a cleavable spacer such as pH and enzyme degradable for drug release. A single prodrug of this type, INNO-206, has been through phase I clinical trials.
  • This prodrug consists of a maleimide derived albumin binding domain, an acid sensitive linker and the anticancer-drug doxorubicin (Figure 11).
  • the prodrug reduces systemic adverse effects whilst increasing the concentration of the drug at the tumour site, where the pH sensitive bond is cleaved.
  • the product is currently under phase II clinical evaluation.
  • the albumin is able to fold correctly, and thus retain its functions and long half-life.
  • a notable example is the fusion protein
  • Albinterferon-a-2b which is a fusion protein of albumin and interferon-a-2b.
  • Human Genome Sciences developed this molecule for the treatment of hepatitis C.
  • the fusion protein has a half-life of around 6 days, which is lower than that of native albumin, but substantially higher than that of interferon (2-5 hours).
  • lactosylated albumin is accumulated in hepatocytes and albumin modified with cyclic peptides localizes to the stellate cells of the liver. It is, therefore, possible to modulate albumin delivery to certain cell types by surface modifications. However, modifications can result in the loss of albumin extended circulatory half-life and recycling capacity.
  • Albumin is an attractive yet still filedging drug carrier with huge potential.
  • the interest for the application of albumin for drug delivery is steadily increasing, and improving methods of characterization and engineering make it possible to further improve and develop this potential.
  • Short interfering RNA is an emerging class of nucleic acid-based drug. Delivery, however, is key to the clinical translation of these drugs. The next section will outline the mechanism of action of siNA and highlight delivery challenges to development of an albumin-based delivery solution.
  • RNA interference RNA interference
  • RNA interference is a post-transcriptional gene silencing (PTGS) process mediated by double stranded RNA.
  • RISC protein complex complex
  • Diicer RNA-induced silencing complex
  • Dicer endonuclease Dicer
  • the RISC complex recognizes these siRNA fragments, and one strand, the guide strand or antisense strand (AS), is assembled into the RISC complex, leading to degradation of the other strand, sense strand (SS), by the RISC component
  • Argonaute 2 (Ago 2).
  • the AS After the degradation of the SS, the AS functions as a guide or template for recognition of mRNA targets by the RISC complex, by complementary base-pairing between the guide strand and the mRNA. After engagement into RISC the mRNA is degraded by Ago2, resulting in gene silencing.
  • siRNA Small interfering RNA
  • siRNA is a class of synthetic, double stranded RNAs of less than 30 base-pairs, which are able to enter the RNAi pathway. They can be designed as 27-mer Diicer substrates, that are recognized by Dicer and cleaved, or shorter 21 to 23-mers, which directly enter RISC.
  • siRNA Despite of the enormous potential of siRNA therapeutics, systemic delivery and cellular uptake pose a great challenge. Renal clearance, serum degradation and nonspecific accumulation are associated with systemic delivery of siRNA.
  • Naked, non-modified siRNA has a serum half-life of minutes.
  • Extensive modification can lead to increased sterical hindrance that reduces the silencing efficiency. This can be overcome by introduction of UNA that exhibit less steric hindrance than native RNA. Chemical modifications can be introduced in discrete positions during synthesis, and combined to achieve desired properties. Chemical modifications can significantly increase the serum stability, but they cannot alone solve the problem of renal clearance and cellular delivery.
  • nanoparticle-based carrier systems for siRNA have been proposed, including nanoparticles functionalized with PEG. Some of these approaches have proven successful; however, many show accumulation in liver, possibly as a consequence of MPS capture, which restricts the clinical application.
  • albumin may act as an effective systemic carrier for siRNA due to its long circulatory half-life, renal clearance avoidance, and cellular uptake properties.
  • the inventors have developed albumin as a natural drug carrier for systemic delivery of drugs using siRNA as the model therapeutic molecule.
  • the stepwise synthesis of siRNA enables a high degree of functionalization on the molecular level, meaning that siRNA can be modified and designed reproducibly and homogenously.
  • the great therapeutic potential of siRNA makes it a relevant molecule to investigate.
  • the inventors propose that the challenges of siRNA delivery; renal clearance, degradation and non-specific accumulation can be overcome with albumin.
  • the aim of this work is to utilize albumin for siRNA delivery.
  • the work can be divided into two categories 1) development of albumin binding siRNA and 2) the investigation of albumin uptake and trafficking in cells. 1.
  • siRNA containing fatty acid and/or cholesterol modifications were designed. Binding of these modified siRNAs to albumin was investigated using gel mobility shift assay and isothermal titration calorimetry. The goal was modification of siRNA in such a way, that a desired albumin binding strength was obtained.
  • Albumin uptake and trafficking in cells was investigated. Fluorescently labeled albumin in combination with confocal microscopy or flow cytometry was used to evaluate the uptake of the protein, and, therefore, the potential as a cellular drug delivery system. This platform can also include labeled siRNA, to visualize both carrier and cargo.
  • albumin as a versatile, customizable drug carrier for siRNA.
  • the design rules established with siRNA forms the basis of a generic albumin delivery platform for a range of
  • siRNA for interaction with the ligand binding sites of albumin designed.
  • Hydrophilic siRNA is not a natural ligand for albumin and was, therefore, functionalized with hydrophobic moieties.
  • Two different hydrophobic modifications, palmitoyl and cholesteryl were used.
  • the palmitoyl functionalization targets the fatty acid binding sites of the albumin.
  • Cholesterol itself does not bind to albumin, but cholesteryl modified siRNA has been reported to increase circulatory half-life, which can possibly be attributed to albumin interaction.
  • the effect of the following was also studied :
  • siRNA molecules presented in Figure 15 were designed. All the siRNA molecules mentioned above contained the same sequence and length:
  • This sequence (SEQ ID NO: 3) has been shown to provide efficient knockdown of EGFP (Enhanced Green Fluorescent Protein) in cells expressing this protein.
  • the first part includes characterization of albumin-siRNA interactions using mobility shift assay and isothermal titration calorimetry (ITC).
  • the second part includes cellular uptake of albumin and EGFP knockdown studies.
  • the electrophoretic mobility shift assay is a simple and sensitive method of separating proteins and nucleic acids by size or charge. EMSA was used as the primary technique to study the interactions between modified siRNA and albumin. The method was chosen because it allows experiments on native, non-labeled and non-modified protein and siRNA and requires small amounts of material.
  • siRNA migration is retarded when siRNA binds to albumin, resulting in a significant shift in siRNA localization.
  • RNA is complementary in sequence to RNA 7, and the weak interaction can be caused by sequence specificity of this single stranded RNA.
  • AS RNA 12 and SS RNA 13 contain a terminal 3 ' palmitoyl, and both interacted weakly with albumin.
  • SS RNA 8 with the palmitoyl placed within the sequence of the RNA, showed no binding, indicating that this design can sterically hinder the interaction with albumin compared to terminal modifications.
  • SS RNA 9 When the number of palmitoyl modifications was increased to two in the same strand (SS RNA 9), an almost complete displacement of siRNA was observed, which suggests strong albumin binding.
  • the second band observed in SS RNA 7 and 9 of this RNA is probably caused by RNA self-annealing, which is sequence specific and can be observed in ssRNA.
  • RNA 17 and 20 The insertion of the palmitoyi within the RNA sequence (siRNA 17 and 20) may cause in steric hindrance from the RNA, which results in the observed lower albumin binding.
  • Table 4 ssRNA concentration measurements for ssRNA 7-14. The experiments were performed in triplicate using both machines. The expected concentrations were 200 ⁇ .
  • siRNA containing ssRNA 7 and 10 were initially annealed in ratios different than 1 : 1, accounting for the additional bands present in the binding studies. Reannealing in the appropriate ratios provided a single band on the gel ( Figure 19 B). Another possibility for the double bands is the decreased stability of the siRNA due to the hydrophobic modifications present. The amount of single stranded RNA increased after four freeze-thaw cycles ( Figure 19 C). The additional bands can, therefore, be avoided by preparing new aliquots of siRNA when experiments are carried out, and by carefully controlling the concentration prior to annealing. The presence of the double bands does not change the results of the qualitative mobility shift binding experiments, but will change results of quantitative studies and should be avoided.
  • siRNA is surrounded by albumin molecules, it is better protected against recognition and degradation.
  • albumin as a carrier for functionalized siRNA can be utilized in two ways, by:
  • L-Thyroxine Warfarin and octanoate bind with high affinity to Sudlow's site I and II, respectively (see Table 1).
  • Fusidic acid binds to the heme binding cleft.
  • Myristate has two binding sites of similar affinity, FA site 1 and 5, respectively (see Figure 6).
  • Stearic acid is thought to bind with high affinity to the same two sites as myristic acid.
  • Crystal log raphic data has revealed four binding sites for thyroxine, but it has not been determined which site has the highest affinity. Two of these sites are in subdomain IIIB near FA site 5, and the remaining are Sudlow's site I and II. Finally it is proposed that the high affinity site for salicylate is located in domain II, but is distinct from the warfarin binding site.
  • Cholesterol has not been reported to bind to albumin, but was relevant for this study as a control for siRNA modified with cholesteryl.
  • Lithocholic acid is a bile salt of similar structure to cholesterol, but less hydrophobic. Because of the similar structure, lithocholic acid was included in the experiment. It is reported to bind to albumin, but the binding sites are unknown.
  • the ligands described above cover the important high affinity binding sites of albumin. By competition studies with these ligands the inventors aimed to determine the binding site for the cholesteryl modified siRNA.
  • stearic acid is the ligand most effectively displacing cholesteryl modified siRNA.
  • Myristic acid and myristate were also able to displace the siRNA, but not as efficiently. This is consistent with the lower affinity of myristic acid than stearic acid for albumin.
  • Thyroxine is able to displace the siRNA 24 and 26, but not 23 and 25. Thyroxine has two binding sites close to FA site 5, and it was found that it can displace siRNA from this site. siRNA 23 and 25 are not displaced by thyroxine, which can be related to the SS 3 ' palmitoyl modification in both these siRNA. It is possible that this modification either 1 : further improves the binding strength to FA site 5, making displacement more difficult, or 2: allows binding with lower affinity to a secondary site, where the siRNA is not displaced by thyroxine.
  • the pattern of displacement is similar whether the siRNA contains palmitoyl
  • ITC Isothermal Titration Calorimetry
  • the ITC instrument consists of two identical cells (sample cell and reference cell) and an injection syringe ( Figure 22).
  • the sample cell contains dissolved albumin
  • the reference cell contains buffer and the siRNA is loaded into the syringe.
  • Each cell has a heater, and the heaters are controlled so that the temperatures in both cells are equal.
  • a constant power (reference power) is added to the reference cell, and the sample heater supplies a similar power (measured power) to keep temperatures equal.
  • the two cells are surrounded by a thermostated jacket, which absorbs the excess heat from the heaters, keeping temperature constant.
  • ITC equipment manufactured by MicroCal and software supplied by MicroCal LLC ITC for Origin 7, Microcal was used to carry out calculations and analysis of the data.
  • the work was carried out on three identical VP-ITC instruments (one in Daniel Otzen Lab, Aarhus University; two at Peter Westh Lab, Roskilde University), and preliminary testing was carried out on an ITC200 (Medical Biochemistry, Aarhus University).
  • the VP-ITC is the most commonly used ITC, and is considered the golden standard of ITC measurement. There can be very small variations between specific ITC machines, but it is generally considered that data is reproducible.
  • the critical parameter which, determines the shape of the binding curve in an ITC experiment, is the unitless constant c defined as:
  • Albumin-Octanoate binding was used as a control binding system to establish the ITC method.
  • Figure 24 shows a representative binding curve for albumin- octanoate binding.
  • a sample cell albumin concentration of 10 ⁇ and a syringe octanoate concentration of 100 ⁇ were used for this experiment.
  • siRNA can be designed to interact with albumin with varying strength depending on the location and type of modification.
  • the inventors have confirmed that the cholesteryl modified siRNA is a high affinity ligand for albumin binding.
  • the inventors also show that albumin binding can be adjusted and controlled by different combinations of modifications.
  • HepG2 cells are known to express and secrete albumin and they have also been shown to bind the protein. They were used to assess albumin antibody specificity for immunocytochemistry (ICC) using fluorescently labeled secondary antibodies for labeling.
  • Opossum Kidney (OK) cells are derived from proximal tubuli, express the megalin and cubilin proteins, and are reported to bind albumin. Further experiments were carried out using the colorectal Caco-2 cell line, which is utilized to evaluate intestinal epithelial permeability. The aim was to investigate the potential of albumin to cross intestinal epithelium and, therefore, the potential of the albumin-based delivery system for oral delivery.
  • the cellular uptake of albumin was investigated by two different approaches 1) uptake of fluorescent albumin and 2) immunocytochemistry (ICC). Studies with fluorescently labeled albumin were carried out using FITC labeled rAlbumin (rAlb, Novozymes Biopharma). Prior to the uptake experiments, the efficiency of the antibodies used for cellular albumin staining was evaluated.
  • the ICC experiments show specific albumin staining in the albumin expressing HepG2 cells (Figure 29 A and B), and no staining unspecific staining in the control experiment using OK cells ( Figure 29 C).
  • the localization and shape of albumin containing compartments in HepG2 cells suggests Golgi apparatus and secretory pathways, where endogenous albumin is present prior to secretion.
  • FITC labeled albumin uptake (Figure 31 B) revealed presence of albumin, however, the signal was weak and the localization of albumin could not be determined.
  • the antibody labeled albumin uptake revealed a clear presence of albumin in certain cells ( Figure 31 C).
  • the albumin was present within the actin-lined borders, indicating an intracellular localization. It was noted, however, that only a subpopulation of cells showed this pattern of albumin uptake.
  • siRNA modifications especially in the antisense strand, can reduce incorporation into RISC and, therefore, the knockdown efficiency of siRNA.
  • An initial screen was, therefore, conducted with the modified siRNAs 15-26 to determine whether the hydrophobic modifications had altered siRNA silencing efficiency.
  • two different commercially available transfection agents were used to transfect the cells: TransIT-TKO Transfection Reagent (Mirus) and Lipofectamine 2000 (Invitrogen). Both are widely and routinely used transfection agents and have been used to transfect cell lines of human origin. The transfections were done according to manufacturers guidelines. Cells were transfected for four hours in serum free media using 50 nM siRNA. A mismatch siRNA with unspecific sequence was included as negative control.
  • the non-modified siRNA 18 resulted in potent knockdown.
  • the presence of the mid- sequence palmitoyl (siRNA 16-17, 20-21 and 24-25) reduced knockdown significantly, possibly due to steric hindrance of RISC interactions of in-sequence modifications compared to 3 ' terminal modifications.
  • siRNA and albumin complexes were mixed in 20 ⁇ _ nuclease free water (Ambion) and incubated for 4 hours before addition to the cells. Cells were transfected for 16 hours in serum free media, cultured for additional 32 hours with growth media, collected and prepared for flow cytometry.
  • albumin did not facilitate silencing of the non-modified siRNA 18, which was expected, as no binding was shown in the binding experiments for this siRNA. There was a slight increase in knockdown in the presence of albumin in the remaining siRNA, except 17. siRNA 21, with 3 palmitoyl modifications, showed most knockdown activity. This siRNA showed knockdown without albumin, but presence of albumin clearly improved transfection efficiency. For the cholesteryl modified siRNA 23-26, the presence of albumin lowered the EGFP expression between 10-15%. This is a moderate knockdown, which may reflect insufficient release of siRNA due to high albumin affinity or ineffective incorporation into RISC.
  • siRNA is not a natural ligand for albumin, but binding was achieved by functionalization with palmitoyl and/or cholesteryl modifications. Binding was analyzed using electrophoretic mobility shift assay (EMSA) and isothermal titration calorimetry (ITC).
  • EMSA electrophoretic mobility shift assay
  • ITC isothermal titration calorimetry
  • the inventors have, therefore, successfully designed functionalized siRNA, which bind to albumin with high affinity.
  • the inventors propose that by interacting with albumin, the siRNA will utilize the trafficking properties of albumin to avoid renal clearance, thereby improving the half-life of the siRNA.
  • Albumin was used as a transfection agent for the functionalized siRNA.
  • the knockdown efficiency was in the range of 10-15 %, indicating the need of further optimization, but nonetheless, these experiments show that albumin has an improving effect on the knockdown efficiency.
  • albumin-based siRNA delivery system This work provides a novel albumin-based siRNA delivery system.
  • the inventors have successfully designed siRNA with high affinity for albumin, and shown that albumin can improve the cellular uptake of such siRNA.
  • the inventors therefore conclude that albumin can be utilized as a carrier for siRNA in the in vitro settings used in this project. Due to the success of existing albumin-based drug delivery systems for other drugs, it is clear this system can be applied to in vivo applications for siRNA delivery.
  • siRNA used in this project are described in Figure 15.
  • ssRNA were dissolved in Nuclease-Free water (Ambion) to 200 ⁇ solutions, evaluated using GeneQuant II (Pharmacia Biotech) or Implen NanoDrop (AH Diagnostics).
  • GeneQuant II Pharmacia Biotech
  • Implen NanoDrop AH Diagnostics
  • ssRNAs were mixed 1 : 1, incubated for 1 min at 95°C for lmin, 1 hour at 37°C, and stored at -20°C.
  • Recombinant albumin was provided by Novozymes Biopharma.
  • the non-stripped rAlb (lOOmg/mL) contains 145 mM sodium chloride and 8 mM sodium octanoate.
  • Recombumin® Prime (formerly Recombumin ® ), Recombumin ® Alpha (formerly
  • the defatted rAlb is also referred to as stripped rAlb, because excipients are removed from the rAlb formulation. All binding and competition experiments are carried out using the defatted Novozymes Biopharma rAlb material.
  • the recombinant albumin may have one or more of the following characteristics: Sodium : preferably 100 to 200 mM, more preferably 120 to 160 mM or 140 to 150 mM, most preferably about 145 mM.
  • Fatty acid such as Octanoate: preferably 2 to 40 mM, more preferably 4 to 36 mM such as 28 to 36 mM or 4 to 12 mM.
  • Detergent such as Polysorbate 80: preferably 5 to 50 mg/L, more preferably 10 to 50 mg/L such as 10 to 20 mg/L or about 50 mg/L.
  • Endotoxin preferably less than 0.5 EU/ml (for example, as measured by LAL) pH : preferably pH 5.5 to 8.0, more preferably 6.0 to 7.8, most preferably 6.4 to 7.4.
  • Purity preferably at least 90% pure, more preferably at least 95% pure, most preferably at least 99.0% pure (e.g. as measured by native PAGE).
  • Polymer content preferably less than or equal to 5% (w/w), more preferably less than or equal 2% (w/w), most preferably less than or equal 1% (w/w) (e.g. as measured by GP.HPLC).
  • Host cell protein preferably less than or equal to 150 ng/g, 15 ng/g, 5 ug/g, 0.3 ug/g protein, most preferably less than or equal to 0.15 ug/g protein (e.g. as measured by ELISA).
  • Sodium octanoate (Sigma, #C5083), fusidic acid (Sigma, #F0881), sodium myristate (Sigma, #M8005), sodium salicylate (Merck, #106600), lithocholic acid (Sigma, #L6250, cholesterol (Sigma, #C8667), myristic acid (Sigma, #M3128), stearic acid (Fluka, #85680), warfarin (Sigma, #A2250) and L-thyroxine (Sigma, #T2376).
  • Sodium octanoate (Sigma, #C5083), fusidic acid (Sigma, #F0881), sodium myristate (Sigma, #M8005), sodium salicylate (Merck, #106600), lithocholic acid (Sigma, #L6250, cholesterol (Sigma, #C8667), myristic acid (Sigma, #M3128), stearic acid (Fluka, #85680), warfarin (
  • ITC Isothermal titration calorimetry
  • ITC experiments were carried out on VP-ITC (MicroCal, GE Healthcare) using the siRNA and albumin described previously, diluted in PBS (pH 7.4) or water. Samples were degased at 37°C without magnetic stirring for 10 min. The VP-ITC was thermostated and run at 37°C. Contrad-70 5% (Decon Labs Inc., #1002), methanol and double distilled water were used between experiments for cleaning, according to manufacturer specification. For settings see Table 12, below, regarding "ITC”. Cell culture
  • Caco-2 DMEM (Gibco, #41965), 10 % fetal bovine serum (FBS), lx non-essential amino acids (Gibco #11140), penicillin,
  • HepG2 DMEM (Gibco, #41965), 10 % FBS, penicillin, 100 pg/ml; streptomycin 100 pg/mL.
  • SFM serum free media
  • TEER Trans Epithelial Electrical Resistance
  • H1299 cells were seeded at a density of 5*10 4 cells per well in 24 well plates (Nunc) 24 hours before transfection, and supplied with lmL growth media.
  • Mirus transfectant was prepared by mixing 50 ⁇ _ SFM + 3 ⁇ _ TransIT-TKO® reagent (Mirus, #MIR2154), incubating 15 min, adding 2.5 ⁇ _ 5 ⁇ siRNA, incubating 15 min. Transfectant added to cells with 200 ⁇ _ SFM for total transfection volume of 250 ⁇ _ per well and siRNA concentration of 50 nM.
  • Lipofectamine Mix 50 ⁇ _ SFM + 1 ⁇ _ LipofectamineTM 2000 reagent (Invitrogen, 11668-019), incubate for 5 min, and mix 50 ⁇ SFM + 2.5 ⁇ 5 ⁇ siRNA, incubate 5 min. Combine the two dilutions, incubate for 20 min, add to cells with 150 ⁇ _ SFM for total transfection volume of 250 ⁇ _ pr. well and siRNA concentration of 50 nM.
  • Albumin transfectants were prepared by mixing the albumin and siRNA to a total volume of 20 ⁇ _ in Nuclease-Free water, incubating for 4 hours and adding to cells with 230 ⁇ _ SFM. Transfections were carried out for 4 (TransIT-TKO, Lipofectamine) or 16 hours (Albumin). Transfectant was removed and 1 ml_ growth media was added.
  • Membrane was blocked in 5 % milk with PBS-Tween 0.005 % (Sigma, #P1379) for 1 hour at RT (Room Temperature), followed by Rabbit-anti-albumin antibody (1 : 1000; 1 pg/mL) (Abeam, #ab2406) incubation in 5 % milk with PBS-Tween 0.005 % for 1 hour at RT. This was followed by incubation in anti-rabbit antibodies with either HRP (1 :2000; 0.5 pg/mL) or Alexa 488 1 : 1000; 1 pg/mL) in 5 % milk with PBS-Tween 0.005 % for 1 hour at RT (1 : 2000). The blots were scanned for chemiluminescence or fluorescence, respectively, using Typhoon Trio+ scanner (GE Healthcare).
  • Cells were cultured in Lab-Tek 4-Chamber Slides (Nunc, #177399) or 24-well plates (Nunc) with glass coverslips (Menzel Glaser). Labeled albumin was added for 4 hours in SFM. Recombinant albumin uptake was carried out for 4 hours in SFM or 24 hours in serum containing media. Cells were fixed with 4 % paraformaldehyde, permeabilized with 1 % Triton X-100 in PBS.
  • Cells were cultured as in above "cellular uptake studies”. Albumin uptake was carried out for 4 hours in SFM or 24 hours in serum containing media. Cells were fixed with 4 % paraformaldehyde, permeabilized with 1 % Triton X-100 in PBS. Cells were stained with primary AB 1 : 1000 (ab2406), secondary AB 1 : 300 (Alexa 488), rhodamine- phalloidin 1 : 50 in PBS (Invitrogen, R415) and Hoechst 33342 1 : 10000 (Invitrogen, H35701). Cells were preserved as in 4.8. For detailed protocol see below regarding "ICC”.
  • siRNA concentrations were generally designed to have siRNA concentrations within this range. Each sample was prepared in a total volume of 12.5 ⁇ _ (10 ⁇ _ sample in nuclease free water (Ambion) +2.5 ⁇ _ loading buffer (5X)). siRNA and albumin were incubated at RT for 30 min and gels were run according to this protocol :
  • ITC Isothermal titration calorimetry
  • Binding experiments were carried out with albumin concentrations of 5 ⁇ for siRNA experiments and 10 ⁇ for octanoate experiments. Between experiments the cell was flushed manually with 3x2 ml_ Contrad-70, 5 %, 3x2 ml_ methanol and 3x2 ml_ water.
  • the emptying can be done by tilting the slide upside-down and carefully tapping it to release most fluid
  • Step 10-12 can be carried out on Para-film, where 100 ⁇ _ AB solution will be enough to cover sample
  • Rhodamine-Phalloidin 1 50 in PBS, enough to cover the samples 11.2. Add Rhodamine-Phalloidin, covering the sample, and incubate for 30 min
  • the siRNA amount was kept constant (3.75pmol) in each well and the albumin amount was varied from 86.6 to 1.6pmol, using a dilution series of 15 points, where each sample was diluted 1 : 1.33 compared to the previous.
  • the fluorescence labeled Albumins were synthesized by conventional conjugation chemistry, in which the Cys34 of rAlbumin is coupled to a maleimide group using Alexa
  • Alexa Fluor ® 488 C5 Maleimide (Molecular Probes ® , Invitrogen, Life Technologies Europe
  • Multivalent labeling of rAlb was conducted using Fluorescein isothiocyanate (FITC), which couples to thiol and amine groups.
  • FITC Fluorescein isothiocyanate
  • a reference library was created for the spectral unmixing was recorded by injecting the fluorophores (0.2nmol) subcutaneously in a NMRI mouse and scanning at the same wavelength settings as used for the experiment.
  • organ quantification spectral unmixing was used.
  • serum quantification of Alexa 680 labeled albumin the excitation/emission pair 645/740nm was used, which has the highest signal intensity.
  • the images were quantified using Living Image 4.3.1 software (PerkinElmen).
  • the human peripheral blood mononuclear cells PBMCs was isolated from the human blood buffy coats using Ficoll-PaqueTM PLUS density gradient centrifugation medium (GE Healthcare) according to the following protocol :
  • PBMCs were seeded in 96-well round-bottom microtiter plates at 200.000 cells/well. For the experiment, each well was incubated with siRNA or siRNA:albumin complexes containing 20pmol ( ⁇ in 200 ⁇ _ total volume) siRNA and 200pmol albumin ( ⁇ ). The cells were incubated with the complexes for 18 hours.
  • ELISA assay was performed according to the manufacturer's protocol, as follows. Each incubation step was followed by sealing and shaking the microtiter plate on the rotating table at 150- 200 rpm, except the overnight incubation with the Capture Antibody, where plates were not shaken. One day prior to carrying out ELISA the 96-well assay plates were coated with the Capture Antibody, diluted 1 : 200 in lx Coating Buffer (5x Coating Buffer diluted in ddH 2 0).
  • 1 ml_ of the top standard 250 pg/mL was prepared in lx Assay Diluent A (lx AD) from the TNF-a stock solution (55 ng/ ml_).
  • the six two-fold serial dilutions of the 250 pg/mL top standard were performed, with the human TNF-a standard concentration : 250 pg/mL, 125 pg/mL, 62.5 pg/mL, 31.2 pg/mL, 15.6 pg/mL, 7.8 pg/mL and 3.9 pg/mL, respectively, lx Assay Diluent A serves as the zero standard (0 pg/mL).
  • Serum was frozen and stored at -80°C for later scanning.
  • the serum samples were scanned in quartz capillaries of different sizes:
  • the terminal blood samples were analysed using the larger, 20 ⁇ _ capillary tubes and the signals were obtained (see Figure 36).
  • Table 18 Half-life calculated on the basis of Figure 2, and initial loss of signal over 4 hours.
  • the WT and HB can be fitted to an exponential function with R 2 >0.99, whereas the LB behaves differently with R 2 ⁇ 0.9.
  • Half-lives for the first 48 hours are calculated to be 12.6 hours, 14.1 hours and 15.8 hours, for the LB, WT and HB respectively.
  • the trend is no longer observable, with very small variations between the variants.
  • 650pg of either LB or HB was injected into the tail vein of female Wistar rats.
  • the animals were split into two groups of 5 animals: 1 PBS, 2 low-binder (LB), 2 high bind (HB).
  • Blood samples were taken according to the following time schedule (table 19) :
  • Peripheral tissues Abdominal fat, external fat (from hip), skin (from back), stomach lining and muscle (hind leg).
  • Table 20 Half-lives calculated from the blood fluorescence at time intervals 0-24 hours and 32-72 hours respectively.
  • the biodistribution data from the IVIS has been supplemented with
  • Tissue sections from control, low-binder (LB) and high-binder (HB) treated rats were fixed in ice cold acetone and kept at -20°C for 10 minutes.
  • DAPI dark gray
  • ATT0488-phalloidine light gray
  • co-staining was performed for nucleus and actin cytoskeleton, respectively.
  • the sections were analyzed for Alexa Fluorophore 680- labeled albumin presence (gray).
  • Intravenous injected albumin can migrate to intestinal epithelium
  • HB may accumulate more than LB - Albumin can potentially be utilised as a payload carrier for treatment of intestinal diseases such as Inflammatory Bowel Disease (IBD) due to IBD.
  • IBD Inflammatory Bowel Disease
  • siRNA was labeled on the AS strand with Cy7, and contains for increased stability LNA nucleotides in the sense strand.
  • the siRNA was pre-formulated 1 : 10 with HB. 1 nmol of siRNA and 10 nmol of albumin were injected in each animal in 400 ⁇ _ PBS via tail vein injection. After injection, blood samples of 5 ⁇ _ were taken from the tail vein at 1, 15, 30, 60, 120 and 240 min, and analyzed in the IVIS scanner. At 360 mins the animals were terminated and the organs and bodies scanned for presence of fluorescent signal.
  • siRNA 56 was only detected in trace amounts in the kidney, this suggests rapid excretion.
  • siRNA 57 was observed in the liver and kidney and was strongly present in the gallbladder.
  • siRNA 58 was observed in larger amounts than siRNA 57 in kidney, liver, gallbladder. Trace amounts were also seen in spleen, lungs and stomach.
  • Modifications can change the biodistribution of the siRNA.
  • mice were kept on a special, non-fluorescent diet ("phytoestrogen-free" mouse chow D10001 AIN-76A, Research Diets, Inc., NJ, USA) for 10 days prior to
  • the organs of the animals were scanned using the IVIS live imager, and analysed by spectral unmixing using a library previously created by subcutaneous injection of Alexa 680 albumin (See Figure 53).
  • LNA locked nucleic acid
  • siRNA can be incorporated into siRNA to improve the efficiency of annealing and to reduce degradation and deannealing in vitro and in vivo.
  • LNA modified siRNA allow the characterization of the cholesteryhalbumin binding in serum and in vivo where non-modified siRNA would be rapidly degraded.
  • the modified siRNA 54 binds with similar affinity as non-modified. Very little dimer formation is observed, however, possible due to increased steric hindrance because of the more rigid oligo.
  • Cy7 modified siRNA was included (See Figure 59).
  • the Cy7 was combined with cholesteryl and LNA, to enable studies on the effect of cholesteryl modifications in vitro.
  • albumin complexes which correspond on the gel to albumin dimers and trimmers (See Figure 60).
  • the formation is likely caused by one siRNA molecule being able to interact with several albumin molecules, and can potentially influence significantly the serum half-life of such complex.
  • the dimer formation is also observed for the single stranded RNA with 2 cholesteryl modifications (see Figure 61).
  • siRNA 33 and 34 which are only modified on the SS are still highly active (>80% KD), whereas modification of both strands somewhat reduces the efficiency ( ⁇ 40% KD). This data also indicates that the current system does not induce knockdown with albumin as the only transfectant.
  • Aim was to determine the effect of mutations on the FcRn binding region of albumin on the albumin :siRNA interaction of cholesteryl modified siRNA 34.
  • three variants were included : Low binder (LB), wild type (WT) and high binder (HB). Further, a rat serum albumin was included.
  • the aim is to determine whether the preformulation of cholesteryl siRNA with albumin can influence the rate of serum degradation of the siRNA.
  • the serum degradation was repeated using the LNA modified siRNA 52, 53 and 54, to determine whether the siRNA: albumin complex stays together over time when incubated in serum, See Figure 67.
  • the aim of this experiment was to determine the TNF-a response of Primary Blood Monocytic Cells (PBMC) towards the siRNA, to assess the therapeutic potential of the system.
  • PBMC Primary Blood Monocytic Cells
  • Annealing at lower temperature 80 Annealing OK, still precipitated below 70 degrees degrees
  • siRNA 56 no cholesteryl, Cy7
  • 57 and 58 were annealed with WT albumin
  • siRNA 58 was annealed with LB, WT and HB albumin.
  • the oligonucleotides were run on a gel and scanned for both Cy7 and SYBR Gold. See Figure 70.
  • siRNA 58 forms some dimer, as expected, whereas siRNA 56 shows no binding.
  • albumin can help efficiently anneal and solubilize siRNA which is very hydrophobic.
  • siRNA annealing with albumin To further characterize the siRNA annealing with albumin, a serum degradation experiment was done on siRNA 58 annealed with albumin. A non-albumin control could not be included because this siRNA cannot readily be solubilized, and therefore cannot be annealed, without albumin.
  • siRNA is Cy7 labeled
  • SYBR-Gold SYBR-Gold
  • Cy7 was detected directly. See Figure 74. This data again indicates that some the siRNA:albumin complex is still present after 72 hours serum incubation.
  • the aim was to determine the effect of Alexa 680 labeling of albumin on the Cys34 on cholesteryl interactions.
  • the LB, WT and HB previously described have been assessed for this, all labeled with Alexa 680 using standard maleimide chemistry and protocols supplied by the manufacturer. See Figure 75.
  • oligonucleotides As control, an unmodified oligonucleotide in the same buffer and DMSO amount should be used.
  • HPLC should reveal >90% SMCC modified material.
  • the oligonucleotide is purified by precipitation to get rid of DMSO:

Abstract

Cette invention concerne une composition comprenant une molécule cargo, par exemple un petit acide nucléique interférent (siNA), et de l'albumine ou ses fragments, ou des polypeptides hybrides comprenant un variant d'une albumine mère ou ses fragments. L'invention concerne aussi une molécule cargo, par exemple un siNA conjugué avec un ou plusieurs cholestéryles et/ou un ou plusieurs acides gras. L'invention concerne également une molécule cargo, par exemple un siNA, la composition selon l'invention étant utilisée comme médicament. L'invention concerne par ailleurs une molécule cargo, par exemple un siNA, ou la composition de l'invention utilisée pour réguler l'expression génétique d'un produit de transcription ou d'une protéine associé(e) à une maladie.
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US10696732B2 (en) 2009-10-30 2020-06-30 Albumedix, Ltd Albumin variants
US10711050B2 (en) 2011-11-18 2020-07-14 Albumedix Ltd Variant serum albumin with improved half-life and other properties
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EP3865576A1 (fr) * 2014-12-15 2021-08-18 Dicerna Pharmaceuticals, Inc. Acides nucléiques double brin modifiés par un ligand
WO2021206917A1 (fr) * 2020-04-07 2021-10-14 Alnylam Pharmaceuticals, Inc. Compositions arni d'enzyme de conversion de l'angiotensine 2 (eca2) et procédés d'utilisation associés
WO2021206922A1 (fr) * 2020-04-07 2021-10-14 Alnylam Pharmaceuticals, Inc. Compositions d'arni de la serine protease 2 transmembranaire (tmprss2) et leurs procédés d'utilisation
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US10233228B2 (en) 2010-04-09 2019-03-19 Albumedix Ltd Albumin derivatives and variants
US10711050B2 (en) 2011-11-18 2020-07-14 Albumedix Ltd Variant serum albumin with improved half-life and other properties
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US10633428B2 (en) 2015-08-20 2020-04-28 Albumedix Ltd Albumin variants and conjugates
US10894962B2 (en) 2016-08-16 2021-01-19 Biontech Delivery Technologies Gmbh Carboxylated 2′-amino-LNA nucleotides and oligonucleotides comprising the same
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WO2020257194A1 (fr) * 2019-06-17 2020-12-24 Alnylam Pharmaceuticals, Inc. Administration d'oligonucléotides au striatum
WO2021092145A1 (fr) * 2019-11-06 2021-05-14 Alnylam Pharmaceuticals, Inc. Composition d'arni de la transthyrétine (ttr) et ses procédés d'utilisation pour le traitement ou la prévention de maladies oculaires associées à ttr
WO2021206917A1 (fr) * 2020-04-07 2021-10-14 Alnylam Pharmaceuticals, Inc. Compositions arni d'enzyme de conversion de l'angiotensine 2 (eca2) et procédés d'utilisation associés
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