WO2023146791A1 - Administration à médiation peptidique d'agents actifs - Google Patents

Administration à médiation peptidique d'agents actifs Download PDF

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Publication number
WO2023146791A1
WO2023146791A1 PCT/US2023/011156 US2023011156W WO2023146791A1 WO 2023146791 A1 WO2023146791 A1 WO 2023146791A1 US 2023011156 W US2023011156 W US 2023011156W WO 2023146791 A1 WO2023146791 A1 WO 2023146791A1
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peptide
nucleic acid
complex
cells
rna
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PCT/US2023/011156
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English (en)
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Han-Chung Wu
Ming-Hsiang HONG
James C. Liao
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Academia Sinica
Chou, Mei-Yin
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Publication of WO2023146791A1 publication Critical patent/WO2023146791A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • 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
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
    • C12N2810/60—Vectors comprising as targeting moiety peptide derived from defined protein from viruses
    • C12N2810/609—Vectors comprising as targeting moiety peptide derived from defined protein from viruses positive strand RNA viruses

Definitions

  • the present disclosure in general relates to the field of carrier peptides, particularly, relates to novel synthetic peptides capable of delivering active agents including nucleic acids into cells; and uses thereof in the treatment and/or prophylaxis of diseases.
  • the present disclosure provides novel synthetic peptides for the delivery of active agents such as nucleic acids into host cells and uses thereof in the treatment and/or prophylaxis of diseases (e.g., an infection caused by a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)).
  • active agents such as nucleic acids into host cells and uses thereof in the treatment and/or prophylaxis of diseases (e.g., an infection caused by a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)).
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • a synthetic peptide for the delivery of nucleic acids of a target protein.
  • the synthetic peptide has the structure of formula (I), wherein,
  • X is a stabilizing residue of cysteine (C);
  • EED is an endosomal escaping domain consisting of 5 consecutive repeats of histidine (H) residues;
  • CPD is a cationic peptide domain having an amino acid sequence at least 90% identical to SEQ ID NOs: 1 or 2.
  • the synthetic peptide has the amino acid sequence of SEQ ID NO: 3.
  • the synthetic peptide has the amino acid sequence of SEQ ID NO: 4.
  • the synthetic peptide is acetylated at its N-terminus and amidated at its C-terminus.
  • step (b) aerating the mixture of step (a) with oxygen to form a complex
  • step (b) incubating the complex of step (b) with the host cell thereby allowing the host cell to express the exogeneous target protein therein.
  • the synthetic peptide in the step (a), is mixed with the nucleic acid at the weight ratio of 20: 1 ; and in the step (b), the mixture is aerated with oxygen at room temperature for 16 hrs.
  • the nucleic acid is a double strand DNA (dsDNA), a single strand DNA (ssDNA), a small interference RNA (siRNA), a short hairpin RNA (shRNA), a messenger RNA (mRNA), a micro RNA (miRNA), a transfer RNA (tRNA) or a combination thereof.
  • the nucleic acid is a dsDNA encoding a receptor binding domain (RBD) of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein.
  • the nucleic acid is a mRNA encoding RBD of a SARS-CoV-2 spike protein.
  • the host cell is an antigen presenting cell.
  • the antigen presenting cell including, but are not limited to, an alveolar macrophage, a peritoneal macrophage, a splenic macrophage, a monocyte, and a dendritic cell.
  • the host cell is a dendritic cell that is any one of a L angerhans cell, an immature dendritic cell, or a mature dendritic cell.
  • a further aspect of the present disclosure is directed to a complex of the present synthetic peptide and a nucleic acid of a target protein useful for transforming a cell to express the target protein therein.
  • the complex of the present synthetic peptide and a nucleic acid of a target protein is produced by a method comprising, (i) mixing the synthetic peptide with the nucleic acid of the target protein at a weight ratio ranging from 50: 1 to 1 : 1 to form a mixture; and (ii) aerating the mixture of step (i) with oxygen to form the complex.
  • the nucleic acid is a double strand DNA (dsDNA), a single strand DNA (ssDNA), a small interference RNA (siRNA), a short hairpin RNA (shRNA), a messenger RNA (mRNA), a micro RNA (miRNA), a transfer RNA (tRNA) or a combination thereof.
  • the nucleic acid is a dsDNA encoding RBD of a SARS-CoV-2 spike protein.
  • the nucleic acid is a mRNA encoding RBD of SARS-CoV-2 spike protein.
  • step (i) the synthetic peptide of claim 1 is mixed with the nucleic acid at the weight ratio of 20: 1; while in step (ii), the mixture is aerated with oxygen for 16 hrs.
  • the complex formed by the present method described above is stable at 25 °C , 4°C or -20°C for at least 14 days.
  • the term “express” or “expression” refers to the processes by which polynucleotides are transcribed into mRNA and mRNA is translated into peptides, polypeptides, or proteins. If tire polynucleotide is derived from genomic DNA of an appropriate eukaryotic host expression may include splicing of the mRNA. Regulatory elements required for expression include promoter sequences to bind RNA polymerase and transcription initiation sequences for ribosome binding.
  • a bacterial expression vector includes a promoter such as the lac promoter and for transcription initiation the Shine- Dalgamo sequence and the start codon AUG.
  • a eukaryotic expression vector includes a heterologous or homologous promoter for RNA polymerase II, a downstream polyadenylation signal, the start codon AUG, and a termination codon for detachment of the ribosome.
  • a heterologous or homologous promoter for RNA polymerase II for RNA polymerase II
  • a downstream polyadenylation signal for RNA polymerase II
  • the start codon AUG a downstream polyadenylation signal
  • a termination codon for detachment of the ribosome.
  • peptide denotes a polymer of amino acid residues.
  • synthetic peptide as used herein, it is meant a peptide which does not comprise an entire naturally occurring protein molecule.
  • the peptide is “synthetic” in that it may be produced by human intervention using such techniques as chemical synthesis, recombinant genetic techniques, or fragmentation of whole antigen or the like.
  • the synthetic peptide of the present disclosure refers to a polymer of 8 to 40, preferably 10 to 35, more preferably 12 to 30, most preferably 13 to 26 amino acid residues, amino acid analogues, peptidomimetics, or a combination thereof.
  • the amino acid residues in the synthetic peptide may be linked by peptide bonds, or by other bonds (e.g., ester, ether, and etc). Throughout the present disclosure, the positions of any specified amino acid residues within a peptide are numbered starting from the N terminus of the peptide.
  • amino acids are not designated as either D- or L-amino acids, the amino acid is either L-amino acid or could be either D- or L- amino acid, unless the context requires a particular isomer.
  • the terms “D-amino acid” and “L-amino acid” are used to refer to absolute configuration of the amino acid, rather than a particular direction of rotation of plane-polarized light.
  • amino acid sequences of proteins/peptides are contemplated as being encompassed by the presently disclosed and claimed inventive concept(s), providing that the variations in the amino acid sequence maintain at least 70%, such as at least 70%, 71%, 72%, 73%, 75%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99%.
  • the present synthetic peptide may be modified specifically to alter a feature of the peptide unrelated to its physiological activity.
  • amino acids can be changed and/or deleted without affecting the activity of the peptide in this study (i.e. , its ability in mediating the delivery of an active agent (e.g., nucleic acids) into a host cell).
  • conservative amino acid replacements are contemplated. Conservative replacements are those that take place within a family of amino acids that are related in their side chains.
  • More preferred families are: serine and threonine are aliphatic-hydroxy family; asparagine and glutamine are an amide-containing family; alanine, valine, leucine and isoleucine are an aliphatic family; and phenylalanine, tryptophan, and tyrosine are an aromatic family.
  • serine and threonine are aliphatic-hydroxy family
  • asparagine and glutamine are an amide-containing family
  • alanine, valine, leucine and isoleucine are an aliphatic family
  • phenylalanine, tryptophan, and tyrosine are an aromatic family.
  • an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the binding or properties of the resulting molecule, especially
  • identity refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues that are the same, when compared and aligned for maximum correspondence. To determine the percent identity, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid sequence for optimal alignment with a second amino acid sequence). The amino acid residues at corresponding amino acid positions are then compared. When a position in the first sequence is occupied by the same amino acid residue as the corresponding position in the second sequence, then the molecules are identical at that position.
  • EED endosomal escaping domain
  • macromolecule e.g. , a nucleic acid, a peptide/DNA complex and etc
  • EED of the present synthetic peptide helps a nucleic acid translocated into cytoplasm of a host cell from being transported into endosomes, thereby preventing the translocated nucleic acid from endolytic digestion.
  • cationic peptide domain refers to an amino acid sequence having one or more basic amino acid residues (e.g., lysin (K) and arginine (R)) thereby results in a net positive charge at selected condition (e.g., pH ⁇ 7.0).
  • selected condition e.g., pH ⁇ 7.0
  • the CPD of the present synthetic peptide helps binding of a macromolecule (e.g., a nucleic acid).
  • APC antigen presenting cells
  • APC antigen presenting cells
  • APCs include, but are not limited to, macrophages, B-cells and dendritic cells, such as immature dendritic cells, mature dendritic cells and Langerhans cells.
  • the term “host cell” is intended to include any individual cell, multiple cells, cell culture or cell in an organism that can be or has been the recipient for transfection with a peptide/nucleic acid complex of this invention. It also is intended to include progeny of a single cell, and the progeny may not necessarily be completely identical (in morphology or in genomic or total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation.
  • the cells may be prokaryotic or eukaryotic, in vivo or in vitro, and include, but are not limited to. bacterial cells, yeast ceils, animal cells, and mammalian ceils, including human cells.
  • the host cell is an antigen presenting cell, most preferably a dendritic cell.
  • dendritic cell refers to a diverse population of morphologically similar cell types found in a variety of lymphoid and non-iymphoid tissues, Dendritic cells constitute die most potent and preferred APCs in the organism. While the dendritic cells can be differentiated from monocytes, they possess distinct phenotypes. For example, a particular differentiating marker, CD-14 antigen, is not found in dendritic cells but is possessed by monocytes. Also, mature dendritic cells are not phagocytic, whereas the monocytes are strongly phagocytosing cells. It has been shown that mature DCs can provide all the signals necessary for T cell activation and proliferation.
  • the present disclosure is based, at least in part, on the discovery of novel synthetic peptides capable of transfecting cells with nucleic acids, thereby allowing the nucleic acids to be expressed in the cells.
  • the present synthetic peptide has the structure of formula (I), wherein,
  • X is a stabilizing residue of cysteine (C);
  • EED is an endosomal escaping domain consisting of 5 consecutive repeats of histidine (H) residues;
  • CPD is a cationic peptide domain having an amino acid sequence at least 70% identical to NGERSGARSKQRRP (SEQ ID NO: 1) or SKKPRQKRTATKA (SEQ ID NO: 2).
  • the synthetic peptide has the amino acid sequence of CHHHHHNGERSGARSKQRRPHHHHHC (SEQ ID NO: 3). [0039] According to other embodiments, the synthetic peptide has the amino acid sequence of CHHHHHSKKPRQKRTATKAHHHHHC (SEQ ID NO: 4).
  • the present synthetic peptide may be synthesized in accordance with any standard peptide synthesis protocol in the art.
  • the present synthetic peptides were synthesized by use of a solid-phase peptide synthesizer in accordance with the manufacturer’s protocols.
  • the present synthetic peptides may be prepared using recombinant technology.
  • the expressed recombinant polypeptide can be purified from the host cell by methods such as ammonium sulfate precipitation and fractionation column chromatography.
  • a peptide thus prepared can be tested for its activity according to the method described in the examples below.
  • nucleic acids or polynucleotide can be delivered by the use of polymeric, biodegradable microparticle or microcapsule delivery devices known in the art. Another way to achieve uptake of the nucleic acid in a host is using liposomes, prepared by standard methods.
  • the polynucleotide can be incorporated alone into these delivery vehicles or co-incorporated with tissue-specific antibodies. Alternatively, one can prepare a molecular conjugate composed of a plasmid or other vector attached to poly-L-lysine by electrostatic or covalent forces. Alternatively, tissue specific targeting can be achieved by the use of tissue- specific transcriptional regulatory elements that are known in the art. Delivery of “naked DNA” (i.e., without a delivery vehicle) to an intramuscular, intradermal, or subcutaneous site is another means to achieve in vivo expression.
  • the present synthetic peptide may be modified at its N-terminus or C-terminus.
  • N-terminal modifications include, but are not limited to, N-glycated, N-alkylated, and N-acetylated amino acid.
  • a terminal modification can include a pegylation.
  • An example of C-terminal modification is a C-terminal amidated amino acid.
  • one or more peptide bond may be replaced by a non-peptidyl linkage, the individual amino acid moieties may be modified through treatment with agents capable of reacting with selected side chains or terminal residues.
  • Various functional groups may also be added at various points of the synthetic peptide that are susceptible to chemical modification. Functional groups may be added to the termini of the peptide. In some embodiments, the function groups improve the activity of the peptide with regard to one or more characteristics, such as improving the stability, efficacy, or selectivity of the synthetic peptide; improving the penetration of the synthetic peptide across cellular membranes and/or tissue barrier; improving tissue localization; reducing toxicity or clearance; and improving resistance to expulsion by cellular pump and the like.
  • Non-limited examples of suitable functional groups are those that facilitate transport of a peptide attached thereto into a cell, for example, by reducing the hydrophilicity and increasing the lipophilicity of the peptide, these functional groups may optionally and preferably be cleaved in vivo, either by hydrolysis or enzymatically, inside the cell.
  • Hydroxy protecting groups include esters, carbonates and carbamate protecting groups.
  • Amine protecting groups include alkoxy and aryloxy carbonyl groups.
  • Carboxylic acid protecting groups include aliphatic, benzylic and aryl esters.
  • a “peptidomimetic organic moiety” can optionally be substituted for amino acid residues in the present synthetic peptide both as conservative and as non-conservative substitutions.
  • the peptidomimetic organic moieties optionally and preferably have steric, electronic or configuration properties similar to the replaced amino acid and such peptidomimetics are used to replace amino acids in the essential positions, and are considered conservative substitutions.
  • Peptidomimetics may optionally be used to inhibit degradation of peptides by enzymatic or other degradative processes.
  • the peptidomimetics can optionally and preferably be produced by organic synthetic techniques.
  • Non-limiting examples of suitable petidomimetics include isosteres of amide bonds, 3-amino-2-propenidone-6- carboxylic acid, hydroxyl-1,2,3,4-tetrahydro-isoquinoline-3-carboxylate, 1,2,3,4-tetrahydro- isoquinoline-3 -carboxylate, and histidine isoquinolone carboxylic acid.
  • Any part of the synthetic peptide may optionally be chemically modified, such as by the addition of functional groups.
  • the modification may optionally be performed during the synthesis of the present peptide.
  • Non-limiting exemplary types of the modification include carboxymethylation, acylation, phosphorylation, glycosylation or fatty acylation.
  • the present synthetic peptides may be used to deliver nucleic acids of interest into the cytoplasm or a specific organelle of a cell (e.g., the nucleus) in vitro or in vivo. Accordingly, there is provided a method of expressing an exogeneous target protein in a host cell. The method comprises,
  • step (b) aerating the mixture of step (a) with oxygen to form a complex of the present synthetic peptide and the nucleic acid;
  • step (b) incubating the complex of step (b) with the host cell thereby allowing the host cell to express the exogeneous target protein therein.
  • the nucleic acid may be a double strand DNA (dsDNA), a single strand DNA (ssDNA), a small interference RNA (siRNA), a short hairpin RNA (shRNA), a messenger RNA (mRNA), a micro RNA (miRNA), a transfer RNA (tRNA) or a combination thereof.
  • dsDNA double strand DNA
  • ssDNA single strand DNA
  • siRNA small interference RNA
  • shRNA short hairpin RNA
  • mRNA messenger RNA
  • miRNA micro RNA
  • tRNA transfer RNA
  • Nucleic acids may be extracted from one or more cells or pathogens or produced in vitro using conventional molecular techniques. Nucleic acids may be amplified by polymerase chain reaction (PCR) and in vitro transcription. Such methods are known to those skilled in the art.
  • the nucleic acid is a dsDNA isolated from a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and encodes a receptor binding domain (RBD) of a SARS-CoV-2 spike protein.
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • RBD receptor binding domain
  • the nucleic acid is a mRNA isolated from SARS-CoV-2 and encodes RBD of a SARS-CoV-2 spike protein.
  • the present method starts by mixing the present synthetic peptide with a nucleic acid (e.g., dsDNA or mRNA encoding RBD of SARS-CoV-2 spike protein) at a weight ratio ranging from 50: 1 to 1 : 1 to form a mixture (step (a)), such as at the weight ratio of 50: 1 , 40: 1 , 30:1, 20:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, and 1:1; preferably, at the weight ratio of 20:1.
  • a nucleic acid e.g., dsDNA or mRNA encoding RBD of SARS-CoV-2 spike protein
  • step (b) the mixture of step (a) is aerated with oxygen until the present synthetic peptide and the nucleic acid are covalently connected to each other thereby forming a covalent peptide/nucleic acid complex (step (b)).
  • the aeration may be performed at room temperature for a period of 10 to 24 hrs, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, and 24 hrs; preferably for 16 hrs.
  • the covalent peptide/nucleic acid complex is allowed to contact with a host cell for a sufficient period of time until the nucleic acid is translocating into cytoplasm or nucleus of the host cell and the exogeneous target protein encoded by the nucleic acid is expressed therein (step (c)).
  • the peptide/nucleic acid complex of this disclosure may be used to transform any type of cells, including prokaryotic and eukaryotic cells, cells in culture, cells in tissue slices, or cells in an animal, including humans.
  • the cell is a eukaryotic cell.
  • cells suitable for use in the present method include, but are not limited to, hepatocytes, epithelial cells, hematopoietic cells, endothelial cells, macrophages, dendritic cells, lung cells, bone cells, stem cells, mesenchymal stem cells, neural cells, cardiac cells, adipocytes, vascular smooth muscle cells, skeletal muscle cells, B cells, T cells, leukocytes, granulocytes, fibroblasts, reticulocytes, and etc.
  • the host cell is an antigen presenting cell.
  • the antigen presenting cell including, but are not limited to, an alveolar macrophage, a peritoneal macrophage, a splenic macrophage, a monocyte, and a dendritic cell.
  • the host cell is a dendritic cell.
  • Dendritic cells are derived from bone marrow progenitor cells, circulating in small numbers in the peripheral blood and appear either as immature Langerhans’ cells or terminally differentiated mature cells. Dendritic cells can also differentiate from monocytes. Methods for the isolation of antigen presenting cells, dendritic precursor cells and/or mature dendritic cells are well known to skilled persons in the related art.
  • the exogeneous target protein expressed in the host cell may be detected by any method known in the related art including but not limiting to radioimmunoassays, enzyme linked immunosorbent assay (ELISA), sandwich immunoassays, immunoradiometric assays, in situ immunoassays (e.g., by using colloidal gold, enzyme or radioisotope labels), western blot analysis, immunoprecipitation assays, immunofluorescent assays and PAGE-SDS.
  • the expressed target protein e.g., RBD of a SARS-CoV-2 spike protein
  • ELISA enzyme linked immunosorbent assay
  • the present synthetic peptide described in section 2 of this paper may also be used to construct a peptide-modified lipid nanoparticle for the delivery of nucleic acids in vitro or in vivo.
  • the lipid nanoparticle will include in its structure, a hydrophilic core; a lipid bilayer shell formed by a cholesterol and one or more a phospholipid optionally modified with a polyethylene glycol (PEG); and the present synthetic peptide (e.g., SEQ ID NO: 3) linked to the PEG-modified phospholipid; wherein, the synthetic peptide linked PEG-modified phospholipid is arranged in the manner that the synthetic peptide and the PEG are both disposed within the hydrophilic core and/or outside the lipid bilayer shell.
  • the lipid nanoparticle further includes nucleic acids encapsulated in the hydrophilic core of the present lipid nanoparticle.
  • Examples of the phospholipid suitable for forming the lipid bilayer shell of the present peptide-modified lipid nanoparticle include, but are not limited to, phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), distearoylphosphatidylethanolamine (DSPE), dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylcholine (DOPC), distearoylphosphatidylcholine (DSPC), 1,2-dioleoyl- 3 -trimethylammonium propane (DOTAP), l,2-distearoyl-3-trimethylammonium propane (DSTAP), 1,2-dimyristoyl glycerol (DMG), ceramide phosphoryl choline, ceramide phosphorylethanolamine, and ceramide phosphoryllipid.
  • PE phosphatidylethanolamine
  • PC phosphatidylcholine
  • PS
  • the phospholipid may be optionally modified with a PEG molecule.
  • PEG polyethylene glycol
  • the term polyethylene glycol (PEG) as used herein refers to oligomers and/or polymers of ethylene oxide, and in general refers to those with a molecular mass below 20,000 g/mol (e.g., PEG 400, PEG 2000, and etc).
  • PEG molecule in the present preparation is to prevent aggregation, as well as to prolong the half-life of the present synthetic peptide, and the nanoparticles thus formed.
  • the lipid bilayer shell of the present peptide-modified lipid nanoparticle is formed by a phospholipid (e.g., DSPC), a PEG-modified phospholipid (e.g., DMG-PEG 2000) and cholesterol in a molar ratio of 118: 3: 77.
  • a phospholipid e.g., DSPC
  • PEG-modified phospholipid e.g., DMG-PEG 2000
  • cholesterol in a molar ratio of 118: 3: 77.
  • the synthetic peptide (e.g., SEQ ID NO: 3) is first conjugated to a PEG-modified phospholipid (e.g., DSPE-PEG 2000) to form a pegylated peptide; then the pegylated peptide is mixed with a cholesterol, a phospholipid (e.g., DSPC) and the PEG- modified phospholipid (e.g., DMG-PEG 2000) in an alcohol solution (e.g., ethanol); DNA or RNA is reconstituted in a sodium acetate solution (pH 4.5); the mixture is then subjected to a nano mixer (e.g., Precision NanoSystems Ignite microfluidic system, San Jose, CA, USA) to produce the desired peptide-modified lipid nanoparticles.
  • a nano mixer e.g., Precision NanoSystems Ignite microfluidic system, San Jose, CA, USA
  • the peptide-modified lipid nanoparticles may also be produced
  • the present peptide-modified lipid nanoparticles are capable of attracting, capturing or withholding nucleic acids that are negatively charged, thereby turning the peptide-modified lipid nanoparticles into a vehicle capable of packaging and loading nucleic acids at selected pH.
  • the peptide-modified lipid nanoparticles may take up or internalize negatively charged nucleic acids, such as by holding the negatively charged nucleic acid on its outer surface, or by encapsulating the entire negatively charged nucleic acid in its interior space.
  • nucleic acid suitable for taken up by the present peptide-modified lipid nanoparticle include, but are not limited to, double strand DNA (dsDNA), single strand DNA (ssDNA), RNA, small interfering RNA (siRNA), short hairpin RNA (shRNA), mRNA, double strand RNA (dsRNA), transfer RNA (tRNA), microRNA (miRNA) and a mixture thereof.
  • the nucleic acids are mRNA of proteins of interest (e.g., the mRNA of viral proteins).
  • the peptide- modified lipid nanoparticle of the present disclosure is loaded with mRNA of one or more viral protein (e.g., RBD of SARS-CoV-2 spike protein).
  • the present disclosure may also provide compositions and methods for intracellularly delivery of nucleic acids (e.g., dsDNA or mRNA of SARS-CoV-2) to a target cell for the production of therapeutic levels of target proteins (e.g., RBD of SARS-CoV-2 spike protein).
  • nucleic acids e.g., dsDNA or mRNA of SARS-CoV-2
  • target proteins e.g., RBD of SARS-CoV-2 spike protein
  • the term “subject” refers to any animal including, but not limited to, humans, non-human primates, rodents, and the like, to which the compositions and methods of the present disclosure are administered. Typically, the term “subject” as used herein refers to a human subject.
  • the composition of the present disclosure comprises the present peptide-modified lipid nanoparticle preloaded with nucleic acids of interest (e.g., dsDNA or mRNA of RBD of SARS-CoV-2 spike protein) intended to be delivered to a target cell, and one or more agents to facilitate contact with, and subsequent transfection of the target cell.
  • nucleic acids of interest e.g., dsDNA or mRNA of RBD of SARS-CoV-2 spike protein
  • the present peptide-modified lipid nanoparticle allows the encapsulated nucleic acids to reach the target cells, and then transfect the target cells.
  • the nucleic acids will encode one or more of target proteins in the target cells.
  • compositions and methods may be used to target a vast number types of cells that include, but are not limited to, hepatocytes, epithelial cells, hematopoietic cells, endothelial cells, lung cells, bone cells, stem cells, macrophages, mesenchymal stem cells, neural cells, cardiac cells, adipocytes, dendritic cells, vascular smooth muscle cells, skeletal muscle cells, B cells, T cells, leukocytes, granulocytes, fibroblasts, reticulocytes, and etc.
  • the peptide-modified lipid nanoparticle pre-loaded with nucleic acids of interest are successfully taken up by dendritic cells, and subsequently transfect the dendritic cells to express the protein of interest (e.g. , the spike protein of SARS-CoV-2) encoded by the nucleic acid (e.g., mRNA of the spike protein of SARS-CoV-2) delivered thereto.
  • the protein of interest e.g., the spike protein of SARS-CoV-2
  • the nucleic acid e.g., mRNA of the spike protein of SARS-CoV-2
  • the protein of interest is produced at levels higher than that of a control (i.e., the baseline level of cells not being treated with the present peptide-modified lipid nanoparticle).
  • the protein of interest is expressed by the target cells (e.g., dendritic cells) at least 1 to 100,000-folds greater than that of the control, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 55, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, and 100,000- folds greater than that of the control; preferably, at least 5 to 50,000-folds greater than that of the control, such as 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 55, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000,
  • compositions and methods for the delivery of nucleic acids to treat or prevent a number of diseases in a subject are for the delivery of nucleic acids of a virus to a subject, in which the composition comprises the present peptide-modified lipid nanoparticle pre-loaded with the viral nucleic acids of interest, and the method includes the step of administering an effective amount of the composition to a target tissue of the subject, so that the encapsulated viral nucleic acids of interest are delivered to the target tissue (e.g., lung, liver, and etc).
  • target tissue e.g., lung, liver, and etc.
  • viral proteins encoded by said viral nucleic acids are expressed in the target tissue and act as antigens to elicit a controlled level of an immune response in the subject to immune the subject, thereby preventing the subject from being subsequently infected by said virus and/or from developing diseases caused by said viral infection (e.g., severe acute respiratory syndrome, SARS).
  • SARS severe acute respiratory syndrome
  • compositions of the present disclosure is formulated in combination with one or more additional carriers, excipients, or stabilizing agents.
  • the composition of the present disclosure may be administered and dosed in accordance with current medical practice, considering the clinical condition of the subject, the site and method of administration, the scheduling of administration, the subject’s age, sex, body weight and other factors relevant to the clinical condition.
  • the effective amount for the purpose herein may be determined by such relevant factors known to those of ordinary skill in clinical research, pharmacological, clinical, and medical arts.
  • the amount administered is effective to achieve at least some level of stabilizing, improving, or eliminating symptoms of the disease, or prevents the disease from progressing.
  • a suitable amount and dosing regimen is one that causes at least transient protein production.
  • Suitable routes of administration include, for example, oral, rectal, vaginal, transmucosal, pulmonary (e.g., intratracheal, inhaled, and etc), or intestinal administration; parenteral delivery including intramuscular, subcutaneous, intramedullary injection, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal or intraocular injection.
  • the composition of the present disclosure may be administered in a local rather systemic manner, for example, via injection of the composition directly into a target tissue.
  • Local delivery may be affected in various ways, depending on the tissue to be targeted.
  • aerosols containing the present composition can be inhaled (for nasal, tracheal or bronchial delivery); compositions of the present disclosure can be injected into the site where the disease manifests, or where pain occurs.
  • the compositions may be provided in lozenges for oral, tracheal, or esophageal application; or can be supplied in liquid, tablet or capsule form for administration to the stomach or intestines; or can be delivered to the eye by use of creams, drops, or even injection.
  • the composition of the present disclosure is formulated such that it is suitable for extended-release of nucleic acids contained therein.
  • extended- release composition may be administered to the subject at extended dosing intervals.
  • the composition of the present disclosure may be administered to a subject daily, or twice per day, or every other day.
  • the composition is administered to the subject once a week, twice a week, every 10 days, every two weeks, every three weeks, or every four weeks, once a month, every six weeks, every eight weeks, every other month, every other month, every three months, every four months, every six months, every eight months, every nine months or annually.
  • compositions which are formulated for depot administration e.g., by intramuscularly, subcutaneously, and etc to either deliver or release nucleic acids over an extended period.
  • lyophilized compositions comprising one or more of the peptide-modified lipid nanoparticles disclosed herein.
  • the lyophilized composition of the present disclosure may be reconstituted prior to administration or can be reconstituted in vivo.
  • a lyophilized composition can be formulated in an appropriate dosage form (e.g., an intradermal dosage such as a disk, rod, or membrane) and administered such that the dosage form is rehydrated over time in vivo by the individual’s body fluid.
  • the peptides Pl, P2 and their respective mutants P1-1 to P1-6 and P2-1 were independently prepared by Fmoc SPPS using a CEM Liberty automated microwave peptide synthesizer, in which each peptide was modified by acetylation at the NH2 termini and amidation at the COOH termini to improve its stability, and was subsequently characterized using mass spectrometry (>95% purity).
  • the amino acid sequences of the synthetic peptides are summarized in Table 1.
  • Plasmid DNA was mixed with peptides at a ratio of 1:20 (w/w) at 25 °C for 10 min. The mixtures were further reacted with oxygen in a shaking incubator at 250 rpm for 16 h at 25°C to generate stable complexes.
  • Example 1 The present synthetic peptide mediated protein expression in 293T cells
  • the synthetic peptides prepared in accordance with procedures described in the “Materials and Methods” section were used to transfect nucleic acid encoding green fluorescent protein (GFP) or the receptor binding domain of SARS-CoV-2 Spike protein into 293T cells.
  • GFP green fluorescent protein
  • GFP-encoding DNA was mixed with each of the synthetic peptide listed in Table 1 at a ratio of 1 :20 (w/w) at 25°C for 10 min. The mixtures were further reacted with oxygen in a shaking incubator at 250 rpm for 16 h at 25°C to generate stable peptide/DNA complexes. Then, the thus formed peptide/DNA complex was added to adherent 293T cells (3 x 10 4 cells) and incubated at 37°C for 48 hr. Images of the cells were captured by a fluorescent microscope, and the quantified results are summarized in Table 2.
  • DNA encoding the RBD of SARS-CoV-2 spike protein was mixed with P1 (SEQ ID NO: 3) or P2 (SEQ ID NO: 4) peptides to form corresponding peptide/DNA complex, which was then used to transfect adherent 293T cells in accordance with procedures described in Example 1.1. The supernatant of transfected 293T cells were then harvested and the amount of RBD of SARS-CoV-2 spike protein expressed therein was determined by ELISA.
  • Example 2 Storage stability of the present peptide-DNA complex

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Abstract

<i />L'invention concerne de nouveaux peptides synthétiques et leurs utilisations dans l'administration d'agents actifs (par exemple, des acides nucléiques) in vitro ou in vivo. La présente invention concerne ainsi un complexe peptide covalent/acide nucléique utile dans de telles utilisations. Le complexe peptide covalent/acide nucléique est formé par le présent peptide synthétique et un acide nucléique codant pour une protéine cible liée l'un à l'autre par l'intermédiaire de liaisons covalentes formées entre eux. Un tel complexe peptide covalent/acide nucléique est particulièrement utile pour la transformation de cellules présentatrices d'antigène, telles que des cellules dendritiques.
PCT/US2023/011156 2022-01-27 2023-01-19 Administration à médiation peptidique d'agents actifs WO2023146791A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060110777A1 (en) * 1999-06-14 2006-05-25 Genentech, Inc. Structured peptide scaffold for displaying turn libraries on phage
US20170275650A1 (en) * 2014-07-22 2017-09-28 The Regents Of The University Of California Endosomal escape domains for delivery of macromolecules into cells
WO2019213562A1 (fr) * 2018-05-03 2019-11-07 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Hydrogels peptidiques pour l'administration de médicaments immunosuppresseurs et utilisations associées
WO2021236854A1 (fr) * 2020-05-19 2021-11-25 Gritstone Bio, Inc. Vaccins contre le sars-cov-2

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060110777A1 (en) * 1999-06-14 2006-05-25 Genentech, Inc. Structured peptide scaffold for displaying turn libraries on phage
US20170275650A1 (en) * 2014-07-22 2017-09-28 The Regents Of The University Of California Endosomal escape domains for delivery of macromolecules into cells
WO2019213562A1 (fr) * 2018-05-03 2019-11-07 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Hydrogels peptidiques pour l'administration de médicaments immunosuppresseurs et utilisations associées
WO2021236854A1 (fr) * 2020-05-19 2021-11-25 Gritstone Bio, Inc. Vaccins contre le sars-cov-2

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LÓPEZ-LAGUNA HÈCTOR, CUBARSI RAFAEL, UNZUETA UGUTZ, MANGUES RAMÓN, VÁZQUEZ ESTHER, VILLAVERDE ANTONIO: "Endosomal escape of protein nanoparticles engineered through humanized histidine-rich peptides", SCIENCE CHINA MATERIALS, vol. 63, no. 4, 1 April 2020 (2020-04-01), pages 644 - 653, XP093083501, ISSN: 2095-8226, DOI: 10.1007/s40843-019-1231-y *

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