WO2014166500A2 - Peptides ayant des domaines immunosuppresseurs pour la transfection - Google Patents

Peptides ayant des domaines immunosuppresseurs pour la transfection Download PDF

Info

Publication number
WO2014166500A2
WO2014166500A2 PCT/DK2014/050089 DK2014050089W WO2014166500A2 WO 2014166500 A2 WO2014166500 A2 WO 2014166500A2 DK 2014050089 W DK2014050089 W DK 2014050089W WO 2014166500 A2 WO2014166500 A2 WO 2014166500A2
Authority
WO
WIPO (PCT)
Prior art keywords
peptide
kit
parts
virus
composition according
Prior art date
Application number
PCT/DK2014/050089
Other languages
English (en)
Other versions
WO2014166500A3 (fr
Inventor
Shervin Bahrami
Christian KANSTRUP HOLM
Søren RIIS PALUDAN
Original Assignee
Skau Aps
Aarhus Universitet (University Of Aarhus)
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Skau Aps, Aarhus Universitet (University Of Aarhus) filed Critical Skau Aps
Priority to EP14722536.1A priority Critical patent/EP2984171A2/fr
Publication of WO2014166500A2 publication Critical patent/WO2014166500A2/fr
Publication of WO2014166500A3 publication Critical patent/WO2014166500A3/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/11—Orthomyxoviridae, e.g. influenza virus
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to a use of a peptide, comprising an immune suppressive domain, for introducing entities, such as foreign entities, to cells.
  • the invention further relates to a use of a peptide, comprising an immune suppressive domain, for transfection.
  • the present invention relates to the use of the peptide for immune suppression in a transfection mix.
  • Transfection is the process of deliberately introducing nucleic acids into cells.
  • the term is used notably for non-viral methods in eukaryotic cells.
  • Transfection is used for the introduction of DNA plasmids into live cells with the purpose of expressing either proteins or silencing / interfering RNA encoded in the plasmid.
  • Transfection is also used to introduce immune-stimulatory DNA or RNA into cells with the purpose of studying aspects of immune reactions to DNA or RNA. Further, transfection can be used to introduce non-DNA non-RNA molecules into cells, which directly interfere with cell function.
  • the International patent application WO 98/40502 discloses transfection compositions in which a peptide is covalently linked to a transfection agent. The application purports that enhanced transfection efficiency is obtained.
  • the International patent application WO 2009/79635 discloses a delivery system for delivering interfering RNA molecules into a cell and methods for using the delivery system.
  • Membrane fusion and STING pathway Membrane disturbance as induced by fusion induces type I interferon responses with expression of interferon-stimu!ated genes, in vivo recruitment of leukocytes and potentiation of signaling via Toil- like receptor 7 (TLR7) and TLR9.
  • TLR7 Toil- like receptor 7
  • the fusion-dependent response is dependent on the stimulator of interferon genes STING.
  • Membrane disturbance can be induced by fusion of cellular membranes with viral envelopes, cellular membranes of other cells, and fusogenic liposomes.
  • STING stimulation of interferon genes
  • M ITA/MPYS/ERIS is also essential for cytosolic DNA-mediated type I IFNs induction.
  • STING contains multi-putative transmembrane regions in the amino terminal region, and is found to associate with membranes.
  • immune suppressive domains in the viral fusion proteins is expected to insert the immune suppressive activity partly through interference with this pathway either through direct or indirect interaction with STING.
  • an antagonist of this putative interaction will enhance the immune responses to proteins containing such immune suppressive domains and can be used as adjuvants.
  • fusion proteins typically undergo a conformational transition triggered by receptor recognition or low pH, leading to the insertion of a fusion peptide into the plasma membrane or the membrane of an endocytic vesicle.
  • fusion proteins typically include members of the paramyxovirus family, separate envelope proteins mediate attachment and fusion.
  • Membrane fusion can occur either at the plasma membrane or at an intracellular location following internalization of virus by receptor-mediated endocytosis. Fusion is mediated by viral
  • transmembrane proteins known as fusion proteins.
  • the fusion protein interacts with the target membrane through a hydrophobic fusion peptide and undergoes a conformational change that drives the membrane fusion reaction.
  • fusion triggers including various combinations of receptor binding, receptor/coreceptor binding, and exposure to the mildly acidic pH within the endocytic pathway. Fusion proteins from different viruses have different names in spite of the common functionality.
  • virus membrane fusion proteins are currently annotated to either the "class I" membrane fusion proteins exemplified by the influenza
  • hemagglutinin (HA) or HIV-1 gp41 or the "class II" proteins of the alphaviruses and flaviviruses.
  • the alphaviruses and flaviviruses are members of the Togaviridae and Flaviviridae families, respectively. These small enveloped positive-sense NAviruses are composed of a capsid protein that assembles with the RNA into the nucleocapsid, and a lipid bilayer containing the viral transmembrane (TM) proteins.
  • Class I fusion proteins are synthesized as single chain precursors, which then assemble into trimers.
  • the polypeptides are then cleaved by host proteases, which is an essential step in rendering the proteins fusion competent.
  • This proteolytic event occurs late in the biosynthetic process because the fusion proteins, once cleaved are metastable and readily activated. Once activated, the protein refolds into a highly stable conformation. The timing of this latter event is of crucial importance in the fusion process. Maintenance of the intact precursor polypeptide during folding and assembly of the oligomeric structure is essential if the free energy that is released during the refolding event is to be available to overcome the inherent barriers to membrane fusion.
  • the new amino-terminal region that is created by the cleavage event contains a hydrophobic sequence, which is known as the fusion peptide.
  • the authentic carboxy-terminal region of the precursor polypeptide contains the
  • transmembrane anchor In the carboxy-terminal polypeptide, there are sequences known as the heptad repeat that are predicted to have an alpha helical structure and to form a coiled coil structure. These sequences participate in the formation of highly stable structure that characterizes the post- fusion conformation of the fusion protein.
  • the class II fusion proteins are elongated finger-like molecules with three globular domains composed almost entirely of ⁇ -sheets. Domain I is a ⁇ -barrel that contains the N-terminus and two long insertions that connect adjacent ⁇ -strands and together form the elongated domain II.
  • the first of these insertions contains the highly conserved fusion peptide loop at its tip, connecting the c and d ⁇ -strands of domain II (termed the cd loop) and containing 4 conserved disulfide bonds including several that are located at the base of the fusion loop.
  • the second insertion contains the ij loop at its tip, adjacent to the fusion loop, and one conserved disulfide bond at its base.
  • a hinge region is located between domains I and II.
  • a short linker region connects domain I to domain III, a ⁇ -barrel with an immunoglobulin-like fold stabilized by three conserved disulfide bonds.
  • domain III is followed by a stem region that connects the protein to the virus TM anchor.
  • Fusion peptides are moderately hydrophobic segments of viral and non-viral membrane fusion proteins that enable these proteins to disrupt and connect two closely apposed biological membranes. This process, which results in membrane fusion occurs in a well-controlled manner with a surprisingly small amount of leakage of the contents of the encapsulated volumes to the outside world.
  • the sequences of fusion peptides are highly conserved within different groups of fusion proteins, for example within different virus families, but not between them. Most fusion peptides are located at the extreme N-termini of the transmembrane subunits of the fusion proteins.
  • Fusion proteins of a subset of enveloped Type I viruses have previously been shown to feature an immune suppressive activity. Inactivated retroviruses are able to inhibit proliferation of immune cells upon stimulation. Expression of these proteins is enough to enable allogenic cells to grow to a tumor in immune competent mice. In one study, introduction of ENV expressing construct into MCA205 murine tumor cells, which do not proliferate upon s.c.
  • immunosuppressive domains have been found in a variety of different viruses with type 1 fusion mechanism such as gamma-retroviruses like Mason pfeizer monkey virus (MPMV) and murine leukemia virus (MLV), lentiviruses such as HIV and in filoviruses such as Ebola and Marburg viruses.
  • MPMV Mason pfeizer monkey virus
  • MMV murine leukemia virus
  • lentiviruses such as HIV
  • filoviruses such as Ebola and Marburg viruses.
  • This immune suppressive activity was in all cases located to a very well-defined structure within the class I fusion proteins, more precisely at the bend in the heptad repeat just N-terminale of the transmembrane structure in the fusion protein.
  • the immunosuppressive effects range from significant inhibition of lymphocyte proliferation, cytokine skewing (up regulating IL-10; down regulating TNF-a, IL-12, IFN- ⁇ ) and inhibition of monocytic burst to cytotoxic T cell killing.
  • peptides spanning ISD in these assays must either be linked as dimers or coupled to a carrier (i.e. >monomeric) to be active.
  • Such peptides derived from immune-suppressive domains are able to reduce or abolish immune responses such as cytokine secretion or proliferation of T-cells upon stimulation.
  • the protection mediated by the immunosuppressive properties of the fusion protein from the immune system of the host is not limited to the fusion protein but covers all the viral envelope proteins displayed at viral or cellular membranes in particular also the protein mediating attachment of the virus to the cell.
  • immunosuppressive domains of viruses like but not limited to retro-, lenti-, Orthomyxo-, flavi- and filoviruses overlap structurally important parts of the fusion subunits of the surface
  • glycoproteins In several cases the primary structure (sequence) of the ISD can vary greatly from virus to virus, but the secondary structure, which is very well preserved among different virus families, is that of an alpha helix that bends in different ways during the fusion process This structure plays a crucial role during events that result in fusion of viral and cellular membranes. It is evident that the immunosuppressive domains of these (retroviral, lentiviral and filoviral) class I fusion proteins overlap with a very important protein structure needed for the fusion mechanistic function.
  • the energy needed for mediating the fusion of viral and cellular membranes is stored in the fusion proteins, which are thus found in a meta-stable conformation on the viral surface. Once the energy is released to drive the fusion event, the protein will find its most energetically stable conformation. In this regard fusion proteins can be compared with loaded springs that are ready to be sprung. This high energy conformation makes the viral fusion proteins very susceptible to modifications; Small changes in the primary structure of the protein often result in the protein to be folded in its stable post fusion conformation. The two conformations present very different tertiary structures of the same protein. It has been shown in the case of simple retroviruses that small structural changes in the envelope protein are sufficient to remove the immune suppressive effect without changing structure and hence the antigenic profile.
  • the mutated non-immune suppressive envelope proteins are much better antigens for vaccination.
  • the proteins can induce a 30-fold enhancement of anti-env antibody titers when used for vaccination and are much better at launching an effective CTL response.
  • viruses that contain the non-immunosuppressive form of the friend murine leukemia virus envelope protein although fully infectious in irradiated immunocompromised mice cannot establish an infection in
  • Immunosuppressive domains in the fusion proteins have been known since 1985 for retrovirus, since 1988 for lentivirus and since 1992 for filoviruses. These viruses, as mentioned above, all belong to enveloped RNA viruses with a type I fusion mechanism.
  • the immunosuppressive domains of lentivirus, retroviruses and filoviruses show large structural similarity. Furthermore the immunosuppressive domain of these viruses are all located at the same position in the structure of the fusion protein, more precisely in the linker between the two heptad repeat structures just N-terminal of the transmembrane domain in the fusion protein.
  • the immune suppressive domains can be located in relation to two well conserved cystein residues that are found in these structures. These cystein residues are between 4 and 6 amino acid residues from one another and in many cases are believed to form disulfide bridges that stabilize the fusion proteins.
  • the immune suppressive domains in all three cases include at least some of the first 22 amino acids that are located N-terminal to the first cysteine residue.
  • Immunosuppressive domains are found in type II fusion proteins. Immunosuppressive domains have been identified at different positions in different groups of viruses. For example an immune suppressive domain might co-localize with the fusion peptide exemplified by the identification of an common immunosuppressive domain in the fusion peptide of Flavirius (Dengue virus, west Nile virus etc), or with the hydrophobic alpha helix N-terminal of the transmembrane domain in the fusion protein exemplified by the finding of an immunosuppressive domain in said helixes of all flaviridae e.g. Hepatitis C virus, Dengue, west nile etc.
  • flaviridae e.g. Hepatitis C virus, Dengue, west nile etc.
  • the immune suppressive domains can also be located in the fusion peptide of the fusion protein among enveloped RNA viruses with type I fusion mechanism.
  • HIV or influenza A and B types have an immune suppressive domain that co-localized with their fusion peptide.
  • immunosuppressive domains are identified among enveloped RNA viruses with type II fusion mechanism at different positions in different groups of viruses: i. Co-localizing with the fusion peptide exemplified by the identification of an common
  • Immunosuppressive domains have been identified in the fusion protein among enveloped RNA viruses with type I fusion mechanism. This position co-localizes with the fusion peptide of said fusion protein as demonstrated by the identification of a common immunosuppressive domain in the fusion peptide of all Influenza A and B types as well as HIV.
  • Functional homolog refers to homologues of the molecules according to the present invention and is meant to comprise any molecule which is capable of mimicking the function of molecules as described herein. Thus, the terms refer to functional similarity or, interchangeably, functional identity, between two or more molecular entities.
  • functional homology is further used herein to describe that one molecular entity are able to mimic the function of one or more molecular entities.
  • Functional homologues according to the present invention may comprise any molecule that can function as an antagonist of the immune suppressive activity exerted by an immune suppressive domains.
  • a molecule when added to the composition containing said immune suppressive domains reduces the immune suppressive activity exerted by the latter in either an in vitro test system (e.g. CTLL-2 or PBMC proliferation assays) or in vivo seen as an enhanced T- and/or B-cell responses.
  • Functional homologues according to the present invention may comprise polypeptides with an amino acid sequence, which are sharing at least some homology with the predetermined polypeptide sequences as outlined herein.
  • polypeptides are at least about 40 percent, such as at least about 50 percent homologous, for example at least about 60 percent homologous, such as at least about 70 percent homologous, for example at least about 75 percent homologous, such as at least about 80 percent homologous, for example at least about 85 percent homologous, such as at least about 90 percent homologous, for example at least 92 percent homologous, such as at least 94 percent homologous, for example at least 95 percent homologous, such as at least 96 percent homologous, for example at least 97 percent homologous, such as at least 98 percent homologous, for example at least 99 percent homologous with the predetermined polypeptide sequences as outlined herein above.
  • the homology between amino acid sequences may be calculated using well known algorithms such as for example any one of BLOSUM 30, BLOSUM 40, BLOSUM 45, BLOSUM 50, BLOSUM 55, BLOSUM 60, BLOSUM 62, BLOSUM 65, BLOSUM 70, BLOSUM 75, BLOSUM 80, BLOSUM 85, and BLOSUM 90.
  • Functional homologues may comprise an amino acid sequence that comprises at least one substitution of one amino acid for any other amino acid.
  • a substitution may be a conservative amino acid substitution or it may be a non-conservative substitution.
  • a conservative amino acid substitution is a substitution of one amino acid within a predetermined group of amino acids for another amino acid within the same group, wherein the amino acids within predetermined groups exhibit similar or substantially similar characteristics.
  • one amino acid may be substituted for another within groups of amino acids characterized by having i) hydrophilic (polar) side chains (Asp, Glu, Lys, Arg, His, Asn, Gin, Ser, Thr, Tyr, and Cys,) ⁇ ) hydrophobic (non-polar) side chains (Gly, Ala, Val, Leu, lie, Phe, Trp, Pro, and Met) iii) aliphatic side chains (Gly, Ala Val, Leu, lie)
  • amino acids being monoamino-dicarboxylic acids or monoamino-monocarboxylic- monoamidocarboxylic acids (Asp, Glu, Asn, Gin).
  • Non-conservative substitutions are any other substitutions.
  • a non-conservative substitution leading to the formation of a functional homologue would for example i) differ substantially in hydrophobicity, for example a hydrophobic residue (Val, lie, Leu, Phe or Met) substituted for a hydrophilic residue such as Arg, Lys, Trp or Asn, or a hydrophilic residue such as Thr, Ser, His, Gin, Asn, Lys, Asp, Glu or Trp substituted for a hydrophobic residue; and/or ii) differ substantially in its effect on polypeptide backbone orientation such as substitution of or for Pro or Gly by another residue; and/or iii) differ substantially in electric charge, for example substitution of a negatively charged residue such as Glu or Asp for a positively charged residue such as Lys, His or Arg (and vice versa); and/or iv) differ substantially in steric bulk, for example substitution of a bulky residue such as His, Trp, Phe or Tyr for one having
  • Functional homologues according to the present invention may comprise more than one such substitution, such as e.g. two amino acid substitutions, for example three or four amino acid substitutions, such as five or six amino acid substitutions, for example seven or eight amino acid substitutions, such as from 10 to 15 amino acid substitutions, for example from 15 to 25 amino acid substitution, such as from 25 to 30 amino acid substitutions, for example from 30 to 40 amino acid substitution, such as from 40 to 50 amino acid substitutions, for example from 50 to 75 amino acid substitution, such as from 75 to 100 amino acid substitutions, for example more than 100 amino acid substitutions.
  • substitutions such as e.g. two amino acid substitutions, for example three or four amino acid substitutions, such as five or six amino acid substitutions, for example seven or eight amino acid substitutions, such as from 10 to 15 amino acid substitutions, for example from 15 to 25 amino acid substitution, such as from 25 to 30 amino acid substitutions, for example from 30 to 40 amino acid substitution, such as from 40 to 50 amino acid substitutions, for example from 50 to 75 amino acid substitution,
  • the addition or deletion of an amino acid may be an addition or deletion of from 2 to 5 amino acids, such as from 5 to 10 amino acids, for example from 10 to 20 amino acids, such as from 20 to 50 amino acids.
  • additions or deletions of more than 50 amino acids, such as additions from 50 to 200 amino acids are also comprised within the present invention.
  • polypeptides according to the present invention may in one embodiment comprise more than 5 amino acid residues, such as more than 10 amino acid residues, for example more than 20 amino acid residues, such as more than 25 amino acid residues, for example more than 50 amino acid residues, such as more than 75 amino acid residues, for example more than 100 amino acid residues, such as more than 150 amino acid residues, for example more than 200 amino acid residues.
  • the genetic code is the set of rules by which information encoded within genetic material (DNA or m NA sequences) is translated into proteins (amino acid sequences) by living cells. Biological decoding is accomplished by the ribosome, which links amino acids in an order specified by mRNA, using transfer RNA (tRNA) molecules to carry amino acids and to read the mRNA three nucleotides at a time.
  • tRNA transfer RNA
  • the code defines how sequences of these nucleotide triplets, called codons, specify which amino acid will be added next during protein synthesis. With some exceptions a three-nucleotide codon in a nucleic acid sequence specifies a single amino acid. Because the vast majority of genes are encoded with exactly the same code (see the RNA codon table), this particular code is often referred to as the canonical or standard genetic code, or simply the genetic code, though in fact some variant codes have evolved. For example, protein synthesis in human mitochondria relies on a genetic code that differs from the standard genetic code. Not all genetic information is stored using the genetic code.
  • All organisms' DNA contains regulatory sequences, intergenic segments, chromosomal structural areas, and other non-coding DNA that can contribute greatly to phenotype. Those elements operate under sets of rules that are distinct from the codon-to-amino acid paradigm underlying the genetic code.
  • Codons Compressed Codons Compressed acid acid
  • GCU GCC, GCA, UUA, UUG, CUU,
  • ACU ACC, ACA,
  • L-amino acids represent all of the amino acids found in proteins during translation in the ribosome
  • D-amino acids are found in some proteins produced by enzyme posttranslational modifications after translation and translocation to the endoplasmic reticulum, as in exotic sea-dwelling organisms such as cone snails. They are also abundant components of the peptidoglycan cell walls of bacteria, and D-serine may act as a neurotransmitter in the brain.
  • L and D convention for amino acid configuration refers not to the optical activity of the amino acid itself, but rather to the optical activity of the isomer of
  • glyceraldehyde from which that amino acid can, in theory, be synthesized (D-glyceraldehyde is dextrorotary; L-glyceraldehyde is levorotatory).
  • Fatty acids, or fatty acid residues when they form part of a lipid are a diverse group of molecules synthesized by chain-elongation of an acetyl-CoA primer with malonyl-CoA or methylmalonyl-CoA groups in a process called fatty acid synthesis. They are made of a hydrocarbon chain that terminates with a carboxylic acid group; this arrangement confers the molecule with a polar, hydrophilic end, and a nonpolar, hydrophobic end that is insoluble in water.
  • the fatty acid structure is one of the most fundamental categories of biological lipids, and is commonly used as a building-block of more structurally complex lipids.
  • the carbon chain typically between four and 24 carbons long, may be saturated or unsaturated, and may be attached to functional groups containing oxygen, halogens, nitrogen, and sulfur.
  • a double bond exists, there is the possibility of either a cis or trans geometric isomerism, which significantly affects the molecule's configuration.
  • Cis-double bonds cause the fatty acid chain to bend, an effect that is compounded with more double bonds in the chain. This in turn plays an important role in the structure and function of cell membranes.
  • Most naturally occurring fatty acids are of the cis configuration, although the trans form does exist in some natural and partially hydrogenated fats and oils.
  • biologically important fatty acids are the eicosanoids, derived primarily from arachidonic acid and eicosapentaenoic acid, that include prostaglandins, leukotrienes, and thromboxanes.
  • Docosahexaenoic acid is also important in biological systems, particularly with respect to sight.
  • Other major lipid classes in the fatty acid category are the fatty esters and fatty amides.
  • Fatty esters include important biochemical intermediates such as wax esters, fatty acid thioester coenzyme A derivatives, fatty acid thioester ACP derivatives and fatty acid carnitines.
  • the fatty amides include N-acyl ethanolamines, such as the cannabinoid neurotransmitter anandamide.
  • Glycerolipids are composed mainly of mono-, di-, and tri-substituted glycerols, the most well-known being the fatty acid triesters of glycerol, called triglycerides.
  • the word "triacylglycerol” is sometimes used synonymously with "triglyceride", though the latter lipid contains no hydroxyl group.
  • the three hydroxyl groups of glycerol are each esterified, typically by different fatty acids. Because they function as an energy store, these lipids comprise the bulk of storage fat in animal tissues.
  • the hydrolysis of the ester bonds of triglycerides and the release of glycerol and fatty acids from adipose tissue are the initial steps in metabolising fat.
  • glycosylglycerols are characterized by the presence of one or more sugar residues attached to glycerol via a glycosidic linkage.
  • structures in this category are the digalactosyldiacylglycerols found in plant membranes and seminolipid from mammalian sperm cells.
  • Glycerophospholipids usually referred to as phospholipids, are ubiquitous in nature and are key components of the lipid bilayer of cells, as well as being involved in metabolism and cell signaling.
  • Neural tissue including the brain contains relatively high amounts of glycerophospholipids, and alterations in their composition has been implicated in various neurological disorders.
  • Glycerophospholipids may be subdivided into distinct classes, based on the nature of the polar headgroup at the sn-3 position of the glycerol backbone in eukaryotes and eubacteria, or the sn-1 position in the case of archaebacteria.
  • Examples of glycerophospholipids found in biological membranes are phosphatidylcholine (also known as PC, GPCho or lecithin), phosphatidylethanolamine (PE or GPEtn) and phosphatidylserine (PS or GPSer).
  • glycerophospholipids in eukaryotic cells such as phosphatidylinositols and phosphatidic acids are either precursors of or, themselves, membrane- derived second messengers.
  • phosphatidylinositols and phosphatidic acids are either precursors of or, themselves, membrane- derived second messengers.
  • one or both of these hydroxyl groups are acylated with long- chain fatty acids, but there are also alkyl-linked and lZ-alkenyl-linked (plasmalogen)
  • glycerophospholipids as well as dialkylether variants in archaebacteria.
  • Sphingolipids are a complicated family of compounds that share a common structural feature, a sphingoid base backbone that is synthesized de novo from the amino acid serine and a long-chain fatty acyl CoA, then converted into ceramides, phosphosphingolipids, glycosphingolipids and other compounds.
  • the major sphingoid base of mammals is commonly referred to as sphingosine.
  • Ceramides are a major subclass of sphingoid base derivatives with an amide-linked fatty acid.
  • the fatty acids are typically saturated or mono-unsaturated with chain lengths from 16 to 26 carbon atoms.
  • the major phosphosphingolipids of mammals are sphingomyelins (ceramide phosphocholines), whereas insects contain mainly ceramide phosphoethanolamines and fungi have phytoceramide phosphoinositols and mannose-containing headgroups.
  • the glycosphingolipids are a diverse family of molecules composed of one or more sugar residues linked via a glycosidic bond to the sphingoid base. Examples of these are the simple and complex glycosphingolipids such as cerebrosides and gangliosides.
  • Sterol lipids such as cholesterol and its derivatives, are an important component of membrane lipids, along with the glycerophospholipids and sphingomyelins.
  • the steroids all derived from the same fused four-ring core structure, have different biological roles as hormones and signaling molecules.
  • the eighteen-carbon (C18) steroids include the estrogen family whereas the C19 steroids comprise the androgens such as testosterone and androsterone.
  • the C21 subclass includes the progestogens as well as the glucocorticoids and mineralocorticoids.
  • the secosteroids comprising various forms of vitamin D, are characterized by cleavage of the B ring of the core structure.
  • sterols are the bile acids and their conjugates, which in mammals are oxidized derivatives of cholesterol and are synthesized in the liver.
  • the plant equivalents are the phytosterols, such as ⁇ -sitosterol, stigmasterol, and brassicasterol; the latter compound is also used as a biomarker for algal growth.
  • the predominant sterol in fungal cell membranes is ergosterol.
  • Prenol lipids are synthesized from the five-carbon-unit precursors isopentenyl diphosphate and dimethylallyl diphosphate that are produced mainly via the mevalonic acid (MVA) pathway.
  • the simple isoprenoids (linear alcohols, diphosphates, etc.) are formed by the successive addition of C5 units, and are classified according to number of these terpene units. Structures containing greater than 40 carbons are known as polyterpenes.
  • Carotenoids are important simple isoprenoids that function as antioxidants and as precursors of vitamin A.
  • quinones and hydroquinones which contain an isoprenoid tail attached to a quinonoid core of non-isoprenoid origin.
  • Vitamin E and vitamin K as well as the ubiquinones, are examples of this class.
  • Prokaryotes synthesize polyprenols (called bactoprenols) in which the terminal isoprenoid unit attached to oxygen remains unsaturated, whereas in animal polyprenols (dolichols) the terminal isoprenoid is reduced.
  • Saccharolipids describe compounds in which fatty acids are linked directly to a sugar backbone, forming structures that are compatible with membrane bilayers.
  • a saccharolipid In the saccharolipids, a
  • glycerophospholipids The most familiar saccharolipids are the acylated glucosamine precursors of the Lipid A component of the lipopolysaccharides in Gram-negative bacteria. Typical lipid A molecules are disaccharides of glucosamine, which are derivatized with as many as seven fatty-acyl chains.
  • the minimal lipopolysaccharide required for growth in E. coli is Kdo2-Lipid A, a hexa-acylated disaccharide of glucosamine that is glycosylated with two 3-deoxy-D-manno-octulosonic acid (Kdo) residues.
  • Polyketides are synthesized by polymerization of acetyl and propionyl subunits by classic enzymes as well as iterative and multimodular enzymes that share mechanistic features with the fatty acid synthases. They comprise a large number of secondary metabolites and natural products from animal, plant, bacterial, fungal and marine sources, and have great structural diversity. Many polyketides are cyclic molecules whose backbones are often further modified by glycosylation, methylation, hydroxylation, oxidation, and/or other processes.
  • anti-microbial, anti-parasitic, and anti-cancer agents are polyketides or polyketide derivatives, such as erythromycins, tetracyclines, avermectins, and antitumor epothilones.
  • the glycerophospholipids are the main structural component of biological membranes, such as the cellular plasma membrane and the intracellular membranes of organelles; in animal cells the plasma membrane physically separates the intracellular components from the extracellular environment.
  • the glycerophospholipids are amphipathic molecules (containing both hydrophobic and hydrophilic regions) that contain a glycerol core linked to two fatty acid-derived "tails" by ester linkages and to one "head” group by a phosphate ester linkage. While
  • glycerophospholipids are the major component of biological membranes, other non-glyceride lipid components such as sphingomyelin and sterols (mainly cholesterol in animal cell membranes) are also found in biological membranes.
  • sphingomyelin and sterols mainly cholesterol in animal cell membranes
  • galactosyldiacylglycerols, and sulfoquinovosyldiacylglycerol which lack a phosphate group, are important components of membranes of chloroplasts and related organelles and are the most abundant lipids in
  • Bilayers have been found to exhibit high levels of birefringence, which can be used to probe the degree of order (or disruption) within the bilayer using techniques such as dual polarization interferometry and Circular dichroism.
  • a biological membrane is a form of lipid bilayer.
  • the formation of lipid bilayers is an energetically preferred process when the glycerophospholipids described above are in an aqueous environment. This is known as the hydrophobic effect.
  • the hydrophobic tails minimize their contact with water and tend to cluster together, forming a vesicle; depending on the concentration of the lipid, this biophysical interaction may result in the formation of micelles, liposomes, or lipid bilayers.
  • Other aggregations are also observed and form part of the polymorphism of amphiphile (lipid) behavior.
  • Phase behavior is an area of study within biophysics and is the subject of current academic research.
  • Micelles and bilayers form in the polar medium by a process known as the hydrophobic effect.
  • the polar molecules i.e., water in an aqueous solution
  • the polar molecules become more ordered around the dissolved lipophilic substance, since the polar molecules cannot form hydrogen bonds to the lipophilic areas of the amphiphile. So in an aqueous environment, the water molecules form an ordered "clathrate" cage around the dissolved lipophilic molecule.
  • the present invention concerns a use of a peptide, comprising an immune suppressive domain, for transfection.
  • the invention concerns a use of a peptide comprising an immune suppressive domain for transfection, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, subject to the proviso that a monomeric peptide should comprise at least two immune suppressive domain sequences.
  • the invention concerns a use of a peptide comprising an immune suppressive domain in a transfection mix, subject to the proviso that said peptide is a dimer or multimer or comprises at least two immune suppressive domain motifs.
  • a dimer or multimer may comprise more than one immune suppressive domain motif.
  • the invention concerns a use of a peptide comprising an immune suppressive domain for transfection of a cell, said peptide providing immune suppression of the transfected cell.
  • the invention concerns a use of a molecule, which comprises at least two parts, each part comprising an immune suppressive domain, for transfection.
  • the invention concerns a kit-of-parts or composition comprising: a. a transfection agent; and b. a peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences.
  • the invention concerns a transfection reagent comprising a lipid based molecule coupled covalentely to a peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences.
  • the invention concerns a nano particle coupled covalentely to a peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences.
  • the invention concerns a hydrophobic vehicle for delivery of hydrophobic molecules coupled covalently to a peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences.
  • the invention concerns a drug coupled covalently to a peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences.
  • the invention concerns a use of a peptide, composition, a kit-of-parts, a transfection agent, a nano particle, a hydrophobic vehicle or a drug according to the invention for inhibiting immune response.
  • the invention concerns a method for transfecting a cell, said method comprising: a. Providing a peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences; b. Incubating the cell with said peptide; c. Providing a transfection agent; d. Providing a material to be transfected into the cell; and e. Further incubating the cell with said transfection agent and said material.
  • the invention concerns a cell obtainable with said method for transfecting a cell.
  • the invention concerns a cell obtainable by incubating a cell with a peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences.
  • the invention concerns a cell comprising a peptide, said peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences.
  • the invention concerns a use of a cell according to the invention, for diagnostics, prophylaxis or therapy.
  • the present invention concerns a use of a peptide, comprising an immune suppressive domain, for transfection.
  • Transfection is the process of deliberately introducing nucleic acids into cells.
  • the term "for transfection” is meant to encompass that the peptide is used as part of the transfection process, for example together with at least one transfection agent, and/or for preparing a cell for additional steps of the transfection process.
  • immune suppressive activity means that it can inhibit proliferation of CTLL-2 or PBMCs in assays as described in the examples, i.e. by more than 20% or interfere with type I IFN production in response to lipid based formulations.
  • the invention concerns a use of a peptide comprising an immune suppressive domain for transfection, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, subject to the proviso that a monomeric peptide should comprise at least two immune suppressive domain sequences.
  • peptide as used here, comprises shorter amino sequences as well as longer sequences such as proteins, and monomers as well as dimers and multimers.
  • the immune suppressive peptides do not need to be covalently bound to form a dimer or multimer. If they contain multimerization domains, the monomeric peptides may associate together through non-covalent interactions and form dimers or multimers that will also be functional.
  • the invention concerns the use of a peptide of the invention, wherein said peptide is a dimer.
  • the invention concerns a use of a peptide comprising an immune suppressive domain in a transfection mix, subject to the proviso that said peptide is a dimer or multimer or comprises at least two immune suppressive domain motifs.
  • the word "or” is not necessarily an exclusive expression.
  • a dimer or multimer may comprise more than one immune suppressive domain motif, and still be within the scope of the present invention.
  • the invention concerns the use of the invention, wherein said at least two immune suppressive domain motifs are two identical or different motifs.
  • the invention concerns a use of a peptide comprising an immune suppressive domain for transfection of a cell, said peptide providing immune suppression of the transfected cell.
  • the invention concerns the use of a peptide according to the invention, which is soluble in water.
  • the peptide is preferably sufficiently soluble to provide immune suppressive activity in an aqueous solution.
  • the invention concerns the use of a peptide according to the invention, wherein said immune suppressive domain is part of a virus.
  • the invention concerns the use of a peptide of the invention, wherein said virus is an influenza virus.
  • the invention concerns the use of a peptide according to the invention, wherein said peptide forms part of a protein.
  • the invention concerns a use of a molecule, which comprises at least two parts, each comprising an immune suppressive domain, for transfection.
  • the invention concerns a kit-of-parts or composition comprising: a. a transfection agent; and b. a peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences.
  • the invention concerns the kit-of-parts or composition of the invention, wherein said transfection agent is coupled covalentely to said peptide.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said transfection agent is a lipid based transfection agent.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said lipid based transfection agent is selected among an anionic and a cationic transfection agent.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said transfection agent is a lipid based liposome or virosome or viral vector.
  • the invention concerns the kit-of-parts or composition according to the invention, comprising a nano particle.
  • a nanoparticle may e.g. be used for drug delivery, it may have an inherent therapeutic effect, or it may be fluorescent and used for tests.
  • the invention concerns the kit-of-parts or composition according to the invention, further comprising a hydrophobic vehicle for delivery of hydrophobic molecules.
  • the invention concerns the kit-of-parts or composition according to the invention, comprising a drug.
  • the immune suppressive domain may suppress an unwanted immune response induced by the drug, which e.g. may comprise a carrier oil, known to induce an immune response.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide is an immune suppressive peptide.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide forms part of a protein of a pathogen.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said pathogen is a virus.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide forms part of a protein on the surface of a pathogen.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide forms part of a virus surface glycoprotein.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said immune suppressive peptide forms part of an enveloped virus surface glycoprotein.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said immune suppressive peptide has a length of at least 6, preferably 7, more preferred 8, preferably 9, more preferred 10, preferably 11, more preferred 12, preferably 13, more preferred 14, preferably 15, more preferred 16, preferably 17, more preferred 18, preferably 19, more preferred 20, preferably 21 more preferred 22, preferably 23, more preferred 24, preferably 25 amino acids.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide has a length selected among 5 - 200, preferably 10 - 100, more preferred 20 - 50, preferably 30 - 40 amino acids.
  • the invention concerns the kit-of-parts or composition according to the invention, further comprising a fusion peptide from a fusion protein.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said fusion peptide is from the fusion protein of an enveloped virus.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said fusion peptide is from a type I fusion protein.
  • the invention concerns the kit-of-parts or composition according to the invention, comprising a fusion peptide from a type II fusion protein.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said fusion peptide has 1, 2, 3 or 4 mutations, deletions or insertions with respect to the wild type.
  • mutation is used with a number about this number of point mutation(s), i.e. 3 mutations mean 3 point mutations.
  • deletion is used with a number about the deletion of this number of amino acid(s), i.e. 2 deletions means the deletion of 2 amino acids.
  • insertion is used with a number about insertion of this number of amino acid(s), i.e. 1 insertion means the insertion of 1 amino acid.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide, or a functional homologue thereof, binds either directly or indirectly to a cellular protein complex containing the protein STING encoded by the gene Tmeml73.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide, or a functional homologue thereof, affects type I interferon responses.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide, or a functional homologue thereof, affects type I interferon responses induced by membrane fusion.
  • the invention concerns the kit-of-parts or composition according to the invention, comprising a peptide from selected among the group consisting of the lists of Table 1 and among the sequences with Seq. Id. 1 - 287.
  • the invention concerns the kit-of-parts or composition according to the invention, comprising a peptide from an influenza virus or a Flu peptide.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide has immune suppressive activity as dimer or mulitimer or when coupled to carrier proteins.
  • the coupling of peptides to larger proteins is one of the known methods for producing mulitimeric peptides or antigens.
  • Proteins such as Bovine serum albumin (BSA) or Keyhole limpet hemocyanin (KLH) can be used as carrier proteins.
  • BSA Bovine serum albumin
  • KLH Keyhole limpet hemocyanin
  • KLH is used extensively as a carrier protein in the production of antibodies for research
  • KLH is an effective carrier protein for several reasons. Its large size and numerous epitopes generate a substantial immune response, and abundance of lysine residues used as coupling sites.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide comprises at least one non-genetically encoded amino acid residue.
  • the non-genetically encoded amino acid residues are amino acid residues, which are not genetically encoded.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide comprises at least one D-amino acid. According to an embodiment, the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide comprises at least one D-amino acid residue.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide is coupled to any other molecule.
  • Two peptides may be joined via another molecule, and each of the two peptides may comprise an immune suppressive domain sequence.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide is attached to lipids.
  • Lipids are here defined as cationic, anionic or neutrally charged fatty acids and their derivatives (including tri-, di-, monoglycerides, and phospholipids), as well as sterol-containing metabolites such as cholesterol).
  • Liposomes may be used as a delivery system, and vesicles may be at least partly covered by immune suppressive domains, thereby suppressing a potential immune system response to the delivery system.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide is coupled to a molecule through a peptide bond.
  • the molecule may e.g. be a ligand of a receptor, thereby targeting the peptide, or it may e.g. be a molecule providing different solubility characteristics of the combination of the peptide and the molecule as compared to the peptide alone, or the molecule may be a nanoparticle.
  • the peptide may further form part of a protein, which may provide advantages such as easy production, as the protein may be derived from natural sources.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide is coupled to a protein.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide is a circular peptide.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide is attached to at least one biological membrane.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide is modified in a way in which one of the peptide bonds is replaced by a non-peptide bond.
  • the invention concerns the kit-of-parts or composition according to the invention, wherein said peptide interferes with an interferon response induced by the transfection agent upon addition to a cell.
  • the invention concerns the kit-of-parts or composition comprising a functional homologue of a peptide according to the invention.
  • a functional homologue may be replaced by an altered peptide, obtained from a peptide of the invention, by making 1, 2, 3, or 4 mutations, deletions or insertions of the immune suppressive domain.
  • An alternated peptide may have a %identity vis-a-vis the unaltered form of at least 60%, preferably at least 70%, more preferred at least 80%, preferably at least 90%, more preferred at least 95%, preferably at least 98%, more preferred at least 99%.
  • the invention concerns a transfection reagent comprising a lipid based molecule coupled covalentely to a peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences.
  • the invention concerns a nano particle coupled covalentely to a peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences.
  • the invention concerns a hydrophobic vehicle for delivery of hydrophobic molecules coupled covalently to a peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences.
  • the invention concerns a drug coupled covalently to a peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences.
  • the invention concerns a use of a peptide, composition, a kit-of-parts, a transfection agent, a nano particle, a hydrophobic vehicle or a drug according to the invention for inhibiting immune response.
  • the invention concerns the use of a peptide, composition, or a kit of parts according to the invention for introducing a molecule into a cell.
  • the invention concerns the use of a peptide, composition, or a kit of parts according to the invention, for introducing a DNA or NA molecule into a cell.
  • the invention concerns the use of a peptide, composition, or a kit of parts according to the invention, for introducing a pharmaceutical molecule into a cell.
  • the invention concerns the use of a peptide, composition, or a kit of parts according to the invention, for introducing a pharmaceutical molecule into a tissue.
  • the invention concerns the use of a peptide, composition, or a kit of parts according to the invention, for introducing a gene-therapeutic pharmaceutical molecule into a cell.
  • the invention concerns the use of a peptide, composition, or a kit of parts according to the invention for initiating expression of proteins via transfected plasmids. According to an embodiment, the invention concerns the use of a peptide, composition, or a kit of parts according to the invention for targeting active genes by micro NA silencing.
  • the invention concerns the use of a peptide, composition, or a kit of parts according to the invention for examining the immune response to DNA or RNA. According to an embodiment, the invention concerns the use of a peptide, composition, or a kit of parts according to the invention for gene therapy.
  • the invention concerns the use of a peptide, composition, or a kit of parts according to the invention for delivery of any molecule using liposomes.
  • the invention concerns a method for transfecting a cell, said method comprising: a. Providing a peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences; b. Incubating the cell with said peptide; c. Providing a transfection agent; d. Providing a material to be transfected into the cell; and e. Further incubating the cell with said transfection agent and said material.
  • the invention concerns a cell obtainable with said method for transfecting a cell.
  • the cells obtainable by this process produce less or no cytokines, in particular interferons, compared to cells obtained without the use of the peptides of the invention. I.e. when compared to untreated cells, cells treated with the INF-F#2 peptide produce less or no cytokines, in particular interferons, but appear to retain the ability to react to other and stronger stimuli.
  • the invention concerns a cell obtainable by incubating a cell with a peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences. Such a cell may be used e.g.
  • the invention concerns a cell comprising a peptide, said peptide comprising an immune suppressive domain, wherein said peptide is selected among a dimer, a multimer, and a monomeric peptide, which comprises at least two immune suppressive domain sequences.
  • the peptide may e.g. be bound to the surface of the cell, internally or externally, or may be present inside of the cell.
  • the invention concerns a use of a cell according to the invention, for diagnostics, prophylaxis or therapy.
  • the invention concerns the use according to the invention, for cell therapy and/or gene therapy.
  • Gene therapy and cell therapy are overlapping fields of biomedical research with similar therapeutic goals.
  • Gene therapy can be defined as the use of genetic material (usually deoxyribonucleic acid - DNA) to manipulate a patient's cells for the treatment of an inherited or acquired disease.
  • Cell Therapy can be defined as the infusion or transplantation of whole cells into a patient for the treatment of an inherited or acquired disease.
  • the invention concerns the use according to the invention, for stem cell transplantation.
  • Table 1 is provided below. Table 1 likewise provides a number of immunosuppressive domains and motifs.
  • Flavivirus sp Flavivirus sp .
  • Hepa Hepatitis C Hepatitis C virus genotype 1 a seqid3
  • Border disease virus 2 SYFQQYMLKGQYQYWFDLE
  • Rubella virus strain TO-336 MSVFALASYVQHPHKTVRVKFHT vaccine .
  • Phlebovirus JS24 Phlebovirus JS24
  • Phlebovir sp Phlebovir sp .
  • Bovine parainfluenza virus 3 Porcine paramyxovirus strain Frost Porcine paramyxovirus strain Texas Human parainfluenza virus 1 Human parainfluenza virus 3 Simian Agent 10
  • Tuhoko virus 3 unclassified Atlantic salmon paramyxovirus Paramyxovirinae Beilong virus
  • the I N F-F#2 peptide has the sequence GLFGAIAGFI ENGWEGCGGEKEKEK [Seq id 287] and is dimerized through a disulfide bridge between the cystein residues.
  • Fig. 1 shows type I IFN production in cells either untreated (UT) or treated with cationic liposomes.
  • Cells were either pre-treated with IN F-F#2 or not pre-treated with any substance.
  • treatment with cationic liposomes induce the production of type I I FN and that this production was inhibited when cells were pre-treated with IN F-F#2.
  • the cells used were bonemarrow derived dendritic cells (BM DCs) from mice of the laboratory strain C57BL.
  • the peptide IN F-F#2 was used at a concentration of ⁇ and initiated 15minutes before treatment with cationic liposomes.
  • Cells were then left to incubate 16 hours at 5% C0 2 and 37.5 degrees Celcius. After 16 hours of incubation the supernatants were harvested. Type I I FN activity was then measured using a type I I FN bioassay.
  • Cationic liposomes were prepared using a mix of the lipids DOTAP/DOPE/L (Lissamine Rhodamine)- DOPE in a w/w/w of 1/1/0.1 dissolved in chloroform.
  • the mix was purchased from the company Avanti Polar Lipids, inc. Chloroform was evaporated using rotation evaporation using a dry nitrogen stream. The lipids film was then dried in vaccum for 4 hours. To created liposomes, the lipid film was hydrated in phosphate buffered saline solution at pH 7.4 at ambient temperature. Liposomes were then size restricted using a 0.2 ⁇ filter and an Avanti Mini-extruder purchased form Avanti Polar Lipids. Fig.
  • FIG. 2 shows type I I FN production in cells either untreated (UT) or treated with the transfection reagent Lipofectamine2000 (Lipofect) according to the manufactures instructions.
  • Lipofectamine2000 was used alone and not with the purpose of transfecting DNA or RNA into the cells.
  • the type I IFN produces therefore represent the cellular response to the transfection reagent Lipofectamine2000 itself.
  • Cells were either pre-treated with IN F-F#2 or not pre-treated with any substance.
  • treatment with Lipofectamine2000 induced the production of type I IFN and that this production was inhibited when cells were pre-treated with IN F-F#2.
  • Cells used were BM DCs as described in the Figure 1 text.
  • Fig. 3 shows type I IFN production in cells either untreated (UT) or transfected with double stranded DNA (dsDNA) using the reagent Lipofectamine2000 according to the manufactures instructions.
  • Cells were either pre-treated with IN F-F#2 or not pre-treated with any substance.
  • transfection with dsDNA induces the production of type I IFN and that this production was NOT inhibited when cells were pre-treated with IN F-F#2. Therefore, pretreatment does not interfere with type I I FN levels induced by the transfected dsDNA but only with the type I I FN induced by the transfection reagent itself.
  • Cells used were BM DCs as described in the Figure 1 text.
  • Beads were then collected by centrifugation at 2000xg for 2min. Beads were then washed x3 in PBS at pH 7.4. Beads were then headed to 95 deg. Celcius in reducing denaturating loading buffer for 5 min. yielding the pull-down lysate. This was then loaded and run on a SDS-PAGE for 45 min at 120V. Proteins were then transferred to protein PVDF membrane and blotted for STING and Giantin. The protein Giantin is a golgi-resident transmembrane protein unrelated to STING function. As depicted in figure 4, the INF-F#2 but not beads alone or the peptide DI6 was able to precipitate STING. Further, INF-F#2 was unable to precipitate other transmembrane proteins such as Giantin.
  • the peptide INF-F#2 therefore blocks STING dependent type I IFN production in response to membrane disturbance as introduced by liposome based transfection agents. This is possibly through direct or indirect interaction with STING itself as INF-F#2 binds either directly to STING or to a complex which contains STING. This occurs without significant interference with type I IFN production with response to transfected immuno-stimulatory DNA itself (Fig. 3)
  • Fig. 5 shows the effect of INF-F#2 (INF ISD) on STING dimerization in response to cationic liposomes.
  • Cells of the human moncytic cell line THP-1 were either culture in the presence or absence of INF-F#2 (INF ISD) as depicted. Cells were then either treated with or left untreated with cationic liposomes (as in Figure 1) for 3.5 hours. Cells were then lysed using IPA lysis buffer with
  • Pretreatment of cells with INF-F#2 resultet in an inhibition of the formation of STING dimers.
  • Light panel Lysates from cells treated with cationic liposomes either treated with the reducing agent DTT or left untreated for 5min.
  • INF-F#2 binds either directly to STING or to a STING containing complex ( Figure 4), and interferes with STING dimerization in response to cellular treatment with cationic liposomes.
  • Figure 6 shows inflammation-related enzyme and transcription factor gene expression kinetics of THP-1 monocytes stimulated with ⁇ g/ml LPS. Gene expression was expressed as relative gene expression towards RPL13a-expression and non-stimulated cells at time zero (AACt). Data shown are means + standard deviation from two independent biological replications.
  • FIG. 7 shows effects of INF-F#2 peptide on expression of NF-kappaB mRNA in LPS-stimulated THP-1 cells.
  • THP-1 cells were incubated with either medium alone, 30 ⁇ , 60 ⁇ INF-F#2 peptide or 30 ⁇ , 60 ⁇ control peptide, and stimulated with ⁇ g/ml LPS. Data shown are the medians ⁇ standard deviation from two independent biological replications.
  • FIG. 8 shows effects of INF-F#2 peptide on expression of SP-1 mRNA in LPS-stimulated THP-1 cells.
  • THP-1 cells were incubated with either medium alone, 30 ⁇ , 60 ⁇ INF-F#2 peptide or 30 ⁇ , 60 ⁇ control peptide, and stimulated with ⁇ g/ml LPS. Data shown are the medians ⁇ standard deviation from two independent biological replications.
  • FIG 9 shows effects of INF-F#2 peptide on protein secretion of IL-8 in LPS-stimulated THP-1 cells.
  • THP-1 cells were incubated with either medium alone, 30 ⁇ or 60 ⁇ INF-F#2 peptide or 30 ⁇ , 60 ⁇ control peptide, and stimulated with ⁇ g/ml LPS. Data shown are the median ⁇ standard deviation from three independent experiments performed in duplicates.
  • Figure 10 shows effects of INF-F#2 peptide on protein secretion of IL-10 in LPS-stimulated THP-1 cells. THP-1 cells were incubated with either medium alone, 30 ⁇ or 60 ⁇ INF-F#2 peptide or 30 ⁇ , 60 ⁇ control peptide, and stimulated with ⁇ g/ml LPS.
  • Figure 12 shows expression kinetics of IFN gamma expression in response to PMA/ionomycin treatment. Gene expression was expressed as relative gene expression towards RPL13a expression and non-stimulated cells at time zero ( ⁇ Ct). Data shown are the medians ⁇ standard deviation from three independent technical replicates.
  • Figure 13 shows effect of INF-F#2 peptide on secretion of protein of IFN-gamma in PMA/ionomycin stimulated PBMCs.
  • PBMCs were incubated with either medium alone, 30 ⁇ or 60 ⁇ INF-F#2 peptide or 30 ⁇ or 60 ⁇ control peptide, and stimulated with 50ng/ml PMA and ⁇ g/ml ionomycin.
  • Data shown are the medians ⁇ standard deviation from three independent experiments performed in duplicates.
  • Figure 14 shows effects of SARS ([Seq id 285] AEVQIDRLITGRLQSLQTYVCGGEKEKEK) or Filo ISD ([Seq id 286] GAAIGLAWIPYFGPAAECGGEKEKEK) on expression of TNF-alpha mRNA in LPS-stimulated THP-1 cells.
  • THP-1 cells were incubated with either medium alone, 30 ⁇ , 60 ⁇ SARS or Filo ISD peptide or 30 ⁇ , 60 ⁇ control peptide, and stimulated with ⁇ g/ml LPS. Data shown are the medians ⁇ standard deviation from two independent biological replications.
  • FIG. 15 shows effects of SARS or Filo ISD on expression of IL-1 ⁇ mRNA in LPS-stimulated THP-1 cells.
  • THP-1 cells were incubated with either medium alone, 30 ⁇ , 60 ⁇ SARS or Filo ISD peptide or 30 ⁇ , 60 ⁇ control peptide, and stimulated with ⁇ g/ml LPS. Data shown are the medians ⁇ standard deviation from two independent biological replications.
  • FIG. 16 shows effects of SARS or Filo ISD on expression of IL-1 ⁇ mRNA in LPS-stimulated THP-1 cells.
  • THP-1 cells were incubated with either medium alone, 30 ⁇ , 60 ⁇ SARS or Filo ISD peptide or 30 ⁇ , 60 ⁇ control peptide, and stimulated with ⁇ g/ml LPS. Data shown are the medians ⁇ standard deviation from two independent biological replications.
  • Figure 17 shows interactions between INF-F#2 peptide and STING depends on distinct STING domains.
  • STING was either in a wt form or with deletions. Lysates from tansfected cells were used for pulldown using biotinylated INF-F#2 peptide and streptavidin coated beads. The bead eluate was then immunoblotted using antibodies against HA-tag.
  • FIG. 18 Bonemarrow derived dendritic cells (BM DCs) were pretreated with indicated peptides at 5 ⁇ . After 30min cells were then treated with cationic liposomes (Lipo) or iwht lmw(low molecular weight) ds NA (Poly l:C). After 18 hours supernatants were analyzed for type I IFN using a bioassay based on vesicular stomatitis virus (VSV) and L929 cells.
  • VSV vesicular stomatitis virus
  • INF wt is the INF-F#2 peptide.
  • INF D4-6 and INF DI6 are deletion mutants of the INF ISD (negative controls) INF mono is the monomeric form of the peptide that does not have any effect.
  • the data show that the INF-F#2 peptide inhibits interferon production induced by liposom fusion with the cells.
  • Figure 19 a+b) BMDCs were pretreated with indicated peptides at 5 ⁇ . After 30min cells were then treated with dsDNA ⁇ g/mL) by transfection using lipofectamine 2000. After 18hours supernatants were analyzed for type I IFN using a bioassay based on vesicular stomatitis virus (VSV) and L929 cells, c-e) Human monocyte derived macrophages were treated with virus like particles (Vlp) or cationic liposomes. Cells were either not pretreated or pretreated with INF-F#2 peptide (pFlu). After 4 hours cells were fixed, stained for STING (green) and with DAPI (blue).
  • VSV vesicular stomatitis virus
  • Vlp virus like particles
  • pFlu cationic liposomes
  • Influenza peptide or pFlu are identical to INF ISD peptide which is identical to INF-F#2 peptide.
  • Figure 20 BMDCs were treated with Iipofectamine2000 according to manufactures directions for usage of Iipofectamine2000 for transfection with DNA (but in this instance without DNA). After 18 hours supernatants were harvested and analyzed for type I IFN by bioassay. Before Iipofectamin2000 treatment cells received either no pretreatment or pretreatment for 30 minutes with INF-F#2 peptide (product). The data shows that the INF-F#2 peptide prevents production of IFN caused by lipofectamine transfection.
  • INF-F#2 a peptide from Influenza HA2, blocks type I IFN production induced by membrane fusion.
  • transfection reagents such as Lipofectamine2000 from Invitrogen
  • Lipofectamine2000 is based on cationic liposomes and function because such liposomes can transport charged molecules such as DNA into living cells.
  • the reagent itself is harmful to the treated cells and induce these to produce cytokines such as type I IFN.
  • INF-F#2 could also inhibit the response to Lipofectamine2000 we pretreated BMDCs with INF-F#2 for 30min and then treated the cells with Lipofectamine2000 according to instructions by Invitrogen. As seen in Figure 2, INF-F#2 completely blocked the type I IFN response to Lipofectamine.
  • INF-F#2 blocks STING signalling in response to cationic liposomes

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Virology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Communicable Diseases (AREA)
  • Pulmonology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne une utilisation d'un peptide, comprenant un domaine immunosuppresseur, pour la transfection. En particulier, la présente invention concerne l'utilisation du peptide pour l'immunosuppression dans un mélange de transfection, le peptide étant choisi parmi un dimère, une multimère et un peptide monomérique, qui comprend au moins deux séquences de domaine immunosuppresseur.
PCT/DK2014/050089 2013-04-10 2014-04-10 Peptides ayant des domaines immunosuppresseurs pour la transfection WO2014166500A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP14722536.1A EP2984171A2 (fr) 2013-04-10 2014-04-10 Peptides ayant des domaines immunosuppresseurs pour la transfection

Applications Claiming Priority (10)

Application Number Priority Date Filing Date Title
DKPA201370201 2013-04-10
DKPA201370202 2013-04-10
DKPA201370201 2013-04-10
DKPA201370200 2013-04-10
DKPA201370202 2013-04-10
DKPA201370200 2013-04-10
DKPA201370203 2013-04-11
DKPA201370203 2013-04-11
DKPA201370204 2013-04-11
DKPA201370204 2013-04-11

Publications (2)

Publication Number Publication Date
WO2014166500A2 true WO2014166500A2 (fr) 2014-10-16
WO2014166500A3 WO2014166500A3 (fr) 2014-12-24

Family

ID=50685717

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/DK2014/050089 WO2014166500A2 (fr) 2013-04-10 2014-04-10 Peptides ayant des domaines immunosuppresseurs pour la transfection

Country Status (2)

Country Link
EP (1) EP2984171A2 (fr)
WO (1) WO2014166500A2 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11136353B2 (en) 2019-04-15 2021-10-05 Qwixel Therapeutics Llc Fusion protein composition(s) comprising masked type I interferons (IFNA and IFNB) for use in the treatment of cancer and methods thereof
EP4056582A4 (fr) * 2019-11-07 2024-02-07 Inst Microbiology Cas Vaccin contre le zika/la dengue et son application

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998040502A1 (fr) 1997-03-14 1998-09-17 Life Technologies, Inc. Transfections activees par des peptides
WO2009079635A1 (fr) 2007-12-18 2009-06-25 Alcon Research, Ltd. Système de délivrance d'arn d'interférence et ses utilisations

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2860004A1 (fr) * 2003-09-18 2005-03-25 Roussy Inst Gustave Nouveau vecteur adenoviral pour l'infection de cellules deficientes ou depourvues en recepteurs car
WO2009065618A2 (fr) * 2007-11-22 2009-05-28 Biontex Laboratories Gmbh Amélioration de résultats de transfection de systèmes de livraison de gènes non viraux par action sur le système immunitaire inné

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998040502A1 (fr) 1997-03-14 1998-09-17 Life Technologies, Inc. Transfections activees par des peptides
WO2009079635A1 (fr) 2007-12-18 2009-06-25 Alcon Research, Ltd. Système de délivrance d'arn d'interférence et ses utilisations

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
TAMM; HAN, BIOSCIENCE REPORTS, vol. 20, no. 6, 2000

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11136353B2 (en) 2019-04-15 2021-10-05 Qwixel Therapeutics Llc Fusion protein composition(s) comprising masked type I interferons (IFNA and IFNB) for use in the treatment of cancer and methods thereof
US11795198B2 (en) 2019-04-15 2023-10-24 Qwixel Therapeutics Llc Fusion protein composition(s) comprising masked type I interferons (IFNA and IFNB) for use in the treatment of cancer and methods thereof
EP4056582A4 (fr) * 2019-11-07 2024-02-07 Inst Microbiology Cas Vaccin contre le zika/la dengue et son application

Also Published As

Publication number Publication date
WO2014166500A3 (fr) 2014-12-24
EP2984171A2 (fr) 2016-02-17

Similar Documents

Publication Publication Date Title
JP2021035979A (ja) Ｒｎａの免疫原性を低減するための方法
ES2633470T3 (es) Métodos y composiciones de generación de una respuesta inmune mediante la inducción de CD40 y adaptadores de receptores de reconocimiento de patrones
CN113286603A (zh) 工程化的细胞外囊泡及其用途
CN110869507A (zh) 促进膜融合的组合物和其用途
ES2623147T3 (es) Secuencias polipeptídicas inductoras de cuerpos proteicos
NZ740946A (en) 3’ utr sequences for stabilization of rna
EP4226938A2 (fr) Vaccin contre le coronavirus
WO2006101201A1 (fr) Liposome capable de liberer efficacement une substance donnee dans le noyau
WO2014166500A2 (fr) Peptides ayant des domaines immunosuppresseurs pour la transfection
WO2023147092A2 (fr) Vaccin anti-coronavirus
AU2022260466A1 (en) Virus vaccine
KR20230082033A (ko) 융합 연관된 작은 막횡단 단백질로 제제화된 단백지질 소포
Park et al. Enhanced delivery of adenovirus, using proteoliposomes containing wildtype or V156K apolipoprotein AI and dimyristoylphosphatidylcholine
WO2019103151A1 (fr) Structure membranaire lipidique pour distribuer un acide nucléique dans une cellule
EP2983705A2 (fr) Utilisation de peptides immunosuppresseurs en tant qu'adjuvants
US20230392169A1 (en) Nucleic acid nanostructures for delivery of nucleic acid sequences to cells
JP2024514364A (ja) 緩衝物質を含むｒｎａ組成物ならびにその製造、保存および使用のための方法
WO2023057935A1 (fr) Nanoparticule lipidique comprenant une protéine de liaison à un acide nucléique
JP2021193893A (ja) 改変型ｐｉｇｇｙＢａｃトランスポゼースのポリペプチド、それをコードするポリヌクレオチド、導入キャリア、キット、細胞のゲノムに目的配列を組込む方法及び細胞製造方法
AU2022336160B2 (en) Potency assay for therapeutic potential of coding nucleic acid
KR102515464B1 (ko) 핵산 또는 유전자 형질감염 효율을 개선하는 세포독성 완화 막 투과 펩타이드 및 이의 용도
US20240042011A1 (en) Coronavirus vaccine
WO2024026257A2 (fr) Polynucléotides modifiés pour l'expression sélective de cellules
WO2024038407A1 (fr) Nanoparticule lipidique comprenant une protéine de liaison à l'adn
TW202345870A (zh) 具有延長半衰期之信使核糖核酸

Legal Events

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

Ref document number: 14722536

Country of ref document: EP

Kind code of ref document: A2

WWE Wipo information: entry into national phase

Ref document number: 2014722536

Country of ref document: EP