Cell-penetrating peptides and uses thereof

Abstract

The present invention relates to novel membrane-permeable constructs and membrane-permeable constructs for use in transporting cargo across lipid membranes and subsequently delivering the cargo to cells. The cargo may be mRNA, and the cell may be in vivo, e.g., a spleen cell. Pharmaceutical compositions comprising the membrane-permeable constructs and their use in methods of eliciting an immune response in a subject are also provided.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to novel membrane-permeable constructs and membrane-permeable constructs for use in transporting cargo across lipid membranes and subsequently delivering the cargo to cells. The cargo may be mRNA, and the cell may be in vivo, e.g., a spleen cell. Pharmaceutical compositions comprising the membrane-permeable constructs and their use in methods of eliciting an immune response in a subject are also provided. [Background technology]

[0002] (background) Cell-penetrating amino acid sequences (also called cell-penetrating peptides or "CPPs") are relatively short peptides that have the ability to gain access into cells and mediate the delivery of cargo covalently or non-covalently bound to them.

[0003] Cell-penetrating amino acids have been used to deliver many different molecules, including, but not limited to, siRNA. Cell-penetrating amino acid sequences typically contain several positively charged amino acids (arginine, lysine, histidine, and the non-proteinogenic amino acid ornithine), which can therefore associate with the negatively charged backbone of nucleic acids. These primarily form non-covalent complexes through electrostatic interactions, although hydrophobic interactions may also play a role in the stability of the complexes formed. While non-covalent cell-penetrating amino acid sequence / cargo complexation strategies allow for simple complex formation with high mixing versatility, limitations of this approach include complex heterogeneity and low stability, as well as premature or limited dissociation of the cargo from the complex.

[0004] When associated with a cargo, the cell-penetrating amino acid sequence / cargo complex primarily enters via the endosomal pathway and ends up in endosomal compartments or lysosomes, and internalization must be quickly followed by release of the complex from these organelles to produce its intended effect.

[0005] Cell membranes have amphipathic properties. To enhance membrane interaction of cell-penetrating amino acids, some cell-penetrating amino acid sequences have been designed to contain both hydrophilic and hydrophobic regions and / or portions within their sequences. NickFect55 (NF55) is a cell-penetrating peptide useful as a plasmid DNA (pDNA) delivery vector for in vitro and in vivo nucleic acid delivery applications. It contains an N-terminal fatty acid and the non-proteinogenic amino acid ornithine at aa7. It was originally designed for the delivery of pDNA, which has optimal CPP / pDNA complex stability and positive charges distributed along the theoretical α-helix. Several amphipathic CPPs have been used to efficiently deliver siRNA into mammalian cells.

[0006] mRNA therapeutics are a class of drugs that enable two therapeutic approaches: mRNA-based vaccination and mRNA as protein replacement therapy. The COVID-19 pandemic (caused by the SARS-CoV2 virus) has brought unprecedented attention to mRNA vaccines (Dolgin E. (2021) Nature, 589(7841):189-191, doi: https: / / doi.org / 10.1038 / d41586-021-00019-w). Although frequently presented as a "new technology," it is actually the result and combination of previous technological breakthroughs and discoveries in various disciplines (Pardi et al. (2018) Nat Rev Drug Discov, 17(4):261-279, doi: https: / / doi.org / 10.1038 / nrd.2017.243). Chief among these technological components is the mRNA delivery technology (Le et al. (2020) Trends Biochem Sci, 46(5):351-365, doi: https: / / doi.org / 10.1016 / j.tibs.2020.11.010), which is currently the bottleneck that defines the potential of the entire platform (Sahin et al. (2014) Nat Rev Drug Discov, 13(10):759-80, doi: https: / / doi.org / 10.1038 / nrd4278).Current mRNA vaccine technology is based on liposomes (LNPs; Corbett et al. (2020) Nature, 586(7830):567-571, doi: https: / / doi.org / 10.1038 / s41586-020-2622-0), and significant developments over the past decade have advanced these to systemic use in vivo (Semple et al. (2010) Nature Biotechnology, 28(2):172-176, doi: https: / / doi.org / 10.1038 / nbt.1602; and Dong et al. (2014) Proc Natl Acad Sci USA, 111(11):3955-60, doi: https: / / doi.org / 10.1073 / pnas.1322937111). Nevertheless, the biggest controversies and challenges with LNPs are: (i) their side effects; and (ii) the fact that nucleic acid delivery, despite its effectiveness, is limited to liver tissue. To fully address the potential of mRNA therapeutics, delivery technologies must be improved. For example, the current SARS-CoV2 vaccination regime requires the local administration of LNPs and low doses of mRNA, sufficient to activate the immune system (Servick, K. (2020) Science News, https: / / www.science.org / content / article / mysterious-2-billion-biotech-revealing-secrets-behind-its-new-drugs-and-vaccines). However, to treat life-threatening chronic diseases, delivery technologies must be: (i) significantly more efficient; and (ii) less toxic.

[0007] To date, CPPs from the NickFect (NF) and PepFect (PF) families have been developed for plasmid (pDNA), siRNA, and miRNA delivery. Within the PepFect family, PF14 has been successfully used both in vitro and in vivo for pDNA (Veimann et al. (2013) Mol. Pharmaceutics, 10(1):199-210, doi: https: / / doi.org / 10.1021 / mp3003557) and siRNA delivery (Sirmane et al. (2018) Peptides, 104(6):62-69, doi: https: / / doi.org / 10.1016 / j.peptides.2018.04.015). We have also previously shown that PF1452 (Kurrikoff et al. (2017) Sci. Rep., 7(1):17056, doi: https: / / doi.org / 10.1038 / s41598-017-17316-y) is more efficient than PF14 for pDNA delivery in vivo. NF55 (Freiman et al. (2016) J of Control. Release, 241(10):135-143, doi: https: / / doi.org / 10.1016 / j.jconrel.2016.09.022) is an excellent vector for DNA delivery in vivo. In Porosk et al. (2019, Biomater. Sci., 7:4363-4374, doi: https: / / doi.org / 10.1039 / c9bm00688e), the inventors designed and tested a series of histidine-containing peptides for siRNA delivery and found that NF70 and NF71 were the most efficient, even more so than NF55. In Carreras-Badosa et al. (2020, Biomaterials 262:120316, doi: https: / / doi.org / 10.1016 / j.biomaterials.2020.120316), different CPPs from the NF and PF families (PF14, NF55, NF70, and NF71) were tested for miRNA delivery.Of all the tested peptides, NF71 was the most efficient in vivo. NF70 has also been used to transfect therapeutic siRNA in vitro and in vivo (Kiisholts et al. (2021) Pharmaceutics, 13(10):1618, doi: https: / / doi.org / 10.3390 / pharmaceutics13101618). Therefore, although several NFs and PFs have been developed and shown to deliver pDNA, siRNA, and miRNA, no studies to date have demonstrated successful delivery of mRNA. Additionally, van den Brand et al. (2019, EJ of Pharmaceutics and Biopharmaceutics, 141:180-190, doi: https: / / doi.org / 10.1016 / j.ejpb.2019.05.014) used PF14 to deliver mRNA, but with limited success.

[0008] Therefore, there is a great need to develop CPPs and membrane-permeable constructs that allow improved delivery of cargo, particularly delivery of mRNA in vivo, for example, to the spleen. Such CPPs and membrane-permeable constructs can then be used, for example, in therapy to elicit an immune response when used as part of an mRNA vaccine. Summary of the Invention

[0009] (Summary of the Invention) According to a first aspect of the present invention there is provided a membrane-permeable construct for use in transporting a cargo across a lipid membrane and subsequently delivering said cargo to a cell in vivo, said construct comprising a cell-permeable amino acid sequence and a fatty acid chain attached to the N-terminus of said amino acid sequence, wherein said cargo is mRNA, and wherein said cell-permeable amino acid sequence is [ka] (wherein X1 represents Lys(K), Orn(O), Dab, or Dap; where: * indicates that the peptide is optionally continued from a side chain amino group rather than from the α-amino group, wherein X2 represents KA or AK, and wherein the cell-penetrating amino acid sequence is optionally chemically modified at the C-terminus. A membrane-permeable construct is provided, comprising the sequence:

[0010] According to a second aspect of the present invention, there is provided a membrane-permeable construct for transport of a cargo across a lipid membrane and subsequent delivery of said cargo to a cell, said construct comprising a cell-permeable amino acid sequence and a fatty acid chain attached to the N-terminus of said amino acid sequence, wherein said cell-permeable amino acid sequence is [ka] (wherein X1 represents Lys(K), Orn(O), Dab, or Dap; where: * indicates that the peptide continues from a side chain amino group rather than from the α-amino group, and where X2 represents KA or AK. The membrane permeable construct is provided, comprising the sequence:

[0011] In certain embodiments, the cargo is mRNA. In further particular embodiments, the cell is in vivo and / or the cell is a splenic cell, e.g., a dendritic cell (DC).

[0012] In another aspect, there is provided a pharmaceutical composition comprising a membrane permeable construct described herein and a pharmaceutically acceptable carrier, and optionally further comprising one or more adjuvants.

[0013] In a still further aspect, there is provided a membrane permeable construct for use as described herein or a membrane permeable construct or a pharmaceutical composition as described herein for use in a method of raising an immune response in a subject, wherein such an immune response is therapeutically or prophylactically useful, for example in a vaccine. [Brief explanation of the drawings]

[0014] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] Figure 1: Delivery of reporter mRNA by CPPs in cell culture - Assessment of reporter levels after transfection (relative light units (RLU / mg) normalized to total protein in cell lysates). Cells treated with naked mRNA were used as a control to compare transfection efficiency using a liposome-based transfection reagent, Lipofectamine3000 (LF3000). [Figure 2] Figure 2: Comparison of reporter levels in cells after transfection of pDNA or mRNA delivered by CPPs (P14, NF55, and NF71; relative light units (RLU / mg) normalized to total protein in cell lysates). Naked mRNA and untreated cells (UT) were used as controls. [Figure 3] Figure 3: Biodistribution of reporter levels after transfection of mRNA or pDNA with NF or PF CPPs in vivo. Reporter gene product levels were assessed from whole tissue homogenates collected postmortem. Results are shown as fold change relative to mice treated with mRNA or pDNA. [Figure 4] Figure 4: Live animal imaging 1–48 hours after administration of NF424 / mRNA or NF424 / pDNA complexes. Reporter gene product levels were assessed with a whole-body imager at 1, 6, 12, 24, and 48 hours after injection. [Figure 5]Figure 5: CPP-mediated mRNA delivery in vivo - reporter levels in the spleen. Spleens were harvested post-mortem 16 hours after injection. Reporter levels (RLU) were assessed from whole tissue homogenates. [Figure 6] Figure 6: Reporter levels detected in splenocyte subtypes recovered from mice treated with NF55 / mRNA. Reporter protein levels were assessed from sorted splenocytes: T cells, macrophages, dendritic cells, and remaining lymphocytes. Results are shown as fold change compared to untreated mice. DETAILED DESCRIPTION OF THE INVENTION

[0015] (Brief explanation of the array) SEQ ID NO: 1 represents the consensus amino acid sequence of the cell-permeable amino acid sequence included in the membrane-permeable constructs described herein for use in transporting cargo across lipid membranes and subsequently delivering the cargo to cells in vivo (where the cargo is mRNA).

[0016] SEQ ID NO: 2 represents the consensus amino acid sequence of the cell-penetrating amino acid sequence contained in the novel membrane-penetrating constructs described herein.

[0017] SEQ ID NO: 3 represents the amino acid sequence contained in the membrane-permeable constructs NF419, NF55, NF410, and NF411 of the first aspect of the present invention.

[0018] SEQ ID NO: 4 represents the amino acid sequence contained in the membrane-permeable constructs NF420, NF554, NF412, and NF413 of the first aspect of the present invention.

[0019] SEQ ID NO: 5 represents the amino acid sequence contained in the membrane permeable constructs NF422, NF553, and NF423 of the first aspect of the present invention.

[0020] SEQ ID NO: 6 represents the amino acid sequence contained in the membrane permeable construct NF559 of the first aspect of the present invention.

[0021] SEQ ID NO: 7 represents the amino acid sequence contained in the membrane permeable construct NF550 of the first aspect of the present invention.

[0022] SEQ ID NO: 8 represents the amino acid sequence contained in the membrane permeable construct NF54 of the first aspect of the present invention.

[0023] SEQ ID NO: 9 represents the amino acid sequence contained in the membrane-permeable constructs NF430 and NF437 of the second aspect of the present invention.

[0024] SEQ ID NO: 10 represents the amino acid sequence contained in the membrane permeable constructs NF425, NF424, and NF426 of the second aspect of the present invention.

[0025] SEQ ID NO: 11 shows the amino acid sequence contained in the membrane-permeable constructs NF436 and NF438 according to the second aspect of the present invention.

[0026] SEQ ID NOs: 12 to 17 represent the amino acid sequences contained in the control / comparison standard membrane-permeable constructs (NF1, NF70, NF71, NF72, PF14, PF1452) used herein.

[0027] (Detailed Description of the Invention) According to a first aspect of the present invention there is provided a membrane-permeable construct for use in transporting a cargo across a lipid membrane and subsequently delivering said cargo to a cell in vivo, said construct comprising a cell-permeable amino acid sequence and a fatty acid chain attached to the N-terminus of said amino acid sequence, wherein said cargo is mRNA, and wherein said cell-permeable amino acid sequence is [ka] (wherein X1 represents Lys(K), Orn(O), Dab, or Dap; where: * indicates that the peptide is optionally continued from a side chain amino group rather than from the α-amino group, wherein X2 represents KA or AK, and wherein the cell-penetrating amino acid sequence is optionally chemically modified at the C-terminus. A membrane-permeable construct is provided, comprising the sequence:

[0028] The inventors have found that membrane-permeable constructs according to this first aspect of the invention have a surprisingly good ability to deliver mRNA cargo to intracellular compartments, in particular to cells of the spleen in vivo. Such delivery is predicted to be efficient in eliciting an immune response in a subject, such as a priming or vaccination response, because professional antigen-presenting cells (APCs), such as dendritic cells (DCs), migrate to secondary lymphoid organs such as the spleen after exposure to antigen, and the spleen contains the largest number of APCs (including DCs) in mammals.

[0029] Therefore, all of the membrane-permeable constructs described herein and their use are likely to be a significant improvement over commonly used liposome (LNP) delivery systems, which target cargo to liver tissue and contain polyethylene glycol (PEG), thus having side effects and toxicity after systemic administration (Semple et al., 2010; and Dong et al., 2014). By effectively delivering cargo (e.g., mRNA) to the spleen, the present invention provides higher efficiency and lower toxicity than liposomes / LNPs. Furthermore, due to their reduced toxicity, all of the membrane-permeable constructs described herein can be used to deliver higher doses of cargo, particularly mRNA, than liposomes / LNPs, which have traditionally used lower doses due to their toxicity, side effects, and liver-restricted delivery. Thus, the membrane-permeable constructs described herein may also be useful in methods for treating chronic diseases.

[0030] Furthermore, all of the membrane-permeable constructs of the present invention are easier to formulate with nucleic acids (e.g., mRNA) than liposomes / LNPs due to their ability to spontaneously complex (e.g., form nanoparticles). This is due, in part, to the presence of only one amino acid component compared to the four components that make up liposome / LNP capsules, and also due to the presence of positively charged amino acids such as ornithine (Orn, O) and lysine (K), which neutralize the nucleic acid (e.g., mRNA) with the peptide backbone. All of the membrane-permeable constructs described herein also provide a fully biodegradable delivery platform without intracellular accumulation or adverse effects, even long after delivery.

[0031] Thus, in certain embodiments, all of the membrane-permeable constructs described herein are particularly useful for delivering large, linear nucleic acid constructs, such as messenger RNA (mRNA), that encode antigenic proteins or peptides. It is therefore believed that the membrane-permeable constructs described herein can be effectively used to deliver mRNA encoding an antigenic protein or peptide in a method for eliciting an immune response against the antigenic protein / peptide in a subject, or can be administered to a subject as part of a method for eliciting such an immune response in the subject.

[0032] (construct) A "construct" as referred to herein includes an amino acid peptide sequence of any length with a fatty acid chain attached to the N-terminus of the amino acid sequence. The constructs described herein may also further include cargo, particularly mRNA cargo.

[0033] The construct is membrane-permeable. Thus, it can cross or pass through a membrane (e.g., a lipid membrane) from the extracellular environment to enter a cell or intracellular environment. The membrane can be a monolayer or multilayer structure (e.g., a lipid bilayer). The construct can transport a cargo across the membrane and deliver it into the cytoplasm of a cell. The construct can also transport a cargo across the membrane and deliver it to an organelle of a cell. Alternatively or additionally, the construct can transport a cargo across the membrane and deliver it to the intracellular surface. The construct can enter the cell via the endosomal pathway. The construct can be packaged within an organelle, preferably an endosomal compartment or lysosome, and then released into an intracellular compartment or environment.

[0034] The membrane may be an artificial membrane, such as, for example, a lipid, a phospholipid, or an artificially constructed composite membrane formed from molecules having both hydrophilic and hydrophobic compounds or structures. The membrane may also be a biological membrane, including, but not limited to, a eukaryotic cell membrane and a prokaryotic cell membrane. The membrane may be a lipid bilayer or a phospholipid bilayer. The membrane may be a lipid membrane of a phospholipid membrane. The membrane may also be a plasma membrane, for example, the plasma membrane of a cell. Thus, in one embodiment, the membrane-permeable construct disclosed herein is for transporting cargo across a lipid membrane and subsequently delivering the cargo to a cell. In another embodiment, the membrane-permeable construct disclosed herein is for use in such transport. Eukaryotic cell membranes include, but are not limited to, membranes of immune cells such as leukocytes, erythrocytes, monocytes, macrophages, neutrophils, T cells, B cells, or dendritic cells; epithelial cells; endothelial cells; keratinocytes; muscle cells; skin cells; nerve cells; and adipocytes.

[0035] (cell-permeable amino acid sequence) Cell-penetrating amino acid sequences, also known as cell-penetrating peptides or "CPPs," are short amino acid sequences that transport different types of cargo molecules across lipid membranes and facilitate cellular uptake of the cargo molecules. Cell-penetrating amino acid sequences can contain 19 to 25 or 25 to 30 amino acids. Cell-penetrating amino acid sequences can contain 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, or 30 or more amino acids. Cell-penetrating amino acid sequences can contain 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. Thus, in some embodiments, cell-penetrating amino acid sequences contain 19 or more, 20 or more, or 21 or more amino acids. In further embodiments, cell-penetrating amino acid sequences contain 19, 20, or 21 amino acids.

[0036] The characteristic of the cell-permeable amino acid described herein is its ability to translocate lipid membranes and facilitate the delivery of various molecular cargoes to the cytoplasm, or to cellular organelles, or to the intracellular cell surface.In some embodiments, the cell-permeable amino acid contained in the membrane-permeable construct described herein delivers cargo to the cytoplasm of cells, so that when the cargo is mRNA, the translational machinery (for example, ribosomes) can produce proteins or peptides encoded by the mRNA, such as antigenic proteins or peptides.

[0037] In one embodiment, the cell-penetrating amino acid sequence is [ka] :Containing or consisting of:

[0038] According to this embodiment: X1 represents Lys(K), Orn(O), Dab, or Dap; * indicates that the peptide is optionally continued from a side chain amino group rather than from the α-amino group, and X2 represents KA or AK.

[0039] Dab refers to 2,4-diaminobutyric acid. Dap refers to 2,3-diaminopropionic acid.

[0040] The naturally occurring proteinogenic and non-proteinogenic (also known as unnatural) amino acids of the cell-penetrating peptides may be of the natural L-enantiomeric configuration (which may be represented by capital letters).

[0041] In further embodiments, X1 is K, Orn(O), or Dab. In certain embodiments, X1 is Dab. In still further embodiments, the peptide is * In yet a further embodiment, X2 is KA.

[0042] In another embodiment, the cell-penetrating amino acid sequence is [ka] :Containing or consisting of:

[0043] According to this embodiment: X1 represents K, Orn(O), Dab, or Dap; * indicates that the peptide continues from the side chain amino group rather than from the α-amino group, and X2 represents KA or AK.

[0044] Accordingly, in a second aspect of the present invention there is provided a membrane-permeable construct for the transport of a cargo across a lipid membrane and subsequent delivery of said cargo to a cell, said construct comprising a cell-permeable amino acid sequence and a fatty acid chain attached to the N-terminus of said amino acid sequence, wherein said cell-permeable amino acid sequence is [ka] (wherein X1 represents Lys(K), Orn(O), Dab, or Dap; where: * indicates that the peptide continues from a side chain amino group rather than from the α-amino group, and where X2 represents KA or AK. A membrane-permeable construct is provided, comprising the sequence:

[0045] In further embodiments, X1 is Orn(O) or Dab. In certain embodiments, X1 is Dab. In certain embodiments, X2 is KA. In another particular embodiment, X2 is AK.

[0046] The inventors have found that the novel membrane-permeable construct of this second aspect of the present invention has excellent ability to deliver cargo, e.g., cargo selected from: mRNA, plasmid DNA (pDNA), small interfering RNA (siRNA), microRNA (miRNA), or any other coding or non-coding nucleic acid, as well as peptides, proteins, non-peptide pharmaceuticals, polysaccharides, lipids (including lipoproteins and glycolipids), small molecule drugs, and imaging agents, to intracellular compartments. Similar to the membrane-permeable construct of the first aspect herein, such delivery is expected to be efficient in eliciting an immune response, e.g., a priming or vaccination response, in a subject, particularly when the cargo is mRNA encoding an antigenic protein or peptide, because professional antigen-presenting cells (APCs), e.g., dendritic cells (DCs), migrate to secondary lymphoid organs such as the spleen after exposure to antigen, and the spleen contains the largest number of APCs (including DCs) in mammals. However, as will be appreciated from the disclosure herein, the membrane permeable construct according to the second aspect of the present invention is not limited to use in the transport of mRNA across lipid membranes and subsequent delivery of said mRNA into cells, and may therefore be for the transport of any suitable cargo.

[0047] In some embodiments, the membrane-permeable construct is more effective at delivering cargo (e.g., mRNA) than existing cell-penetrating peptides, such as NF55 or NF54. In certain embodiments, the membrane-permeable construct according to the second aspect of the present invention is more effective at delivering cargo (e.g., mRNA) than existing cell-penetrating peptides, such as NF55 or NF54. The construct also, at least in some embodiments, advantageously has low toxicity and is expected to be cheaper to synthesize compared to existing cell-penetrating peptides, such as NF55 or NF54.

[0048] Throughout this disclosure, residue and / or position numbers in cell-penetrating amino acid sequences refer to positions in the sequence, with reference to the numbering of SEQ ID NO: 1 or SEQ ID NO: 2, as appropriate, and it is understood that the sequence may have any or none of the modifications described herein. Thus, by way of example only, the numbering of residues / positions within the cell-penetrating peptide sequences described herein can be represented as follows: [ka] .

[0049] Furthermore, the "N-terminal portion" referred to herein includes the amino acid residue at the "end" of the cell-penetrating peptide amino acid sequence, which is the N-terminus. Thus, in one embodiment, the N-terminal portion includes positions 1 to 6 of SEQ ID NO: 1 or SEQ ID NO: 2. In another embodiment, the N-terminal portion includes positions 1 to 5 of SEQ ID NO: 1 or SEQ ID NO: 2. The N-terminal portion of the cell-penetrating amino acid sequence may also include amino acids at positions 1 to 4, 1 to 3, 1 to 2, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 6, 3 to 5, and 3 to 4 of the cell-penetrating amino acid sequence. Substitution refers to the exchange of one amino acid for another, different amino acid.

[0050] In some embodiments, the cell-penetrating amino acid sequence comprises one or more additional amino acid substitutions at positions 9-21 of SEQ ID NO:1 or positions 9-19 of SEQ ID NO:2.

[0051] Thus, in some embodiments, the cell-penetrating amino acid sequence is [ka] (where, * indicates that the peptide continues from the side chain amino group rather than from the α-amino group. : It comprises or consists of the sequence:

[0052] In certain embodiments, the cell-penetrating amino acid sequence is [ka] : It comprises or consists of the sequence:

[0053] As demonstrated by the data presented herein, membrane-permeable constructs comprising cell-permeable amino acid sequences according to these embodiments provide better or equivalent delivery of mRNA cargo than existing cell-permeable peptides NF55 and NF54. In particular, certain cell-permeable peptides comprising the sequences of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:8 provide significantly better delivery of mRNA cargo, resulting in significantly increased activity of the expressed protein (i.e., increased expression of the protein) compared to NF55 (see Figure 1).

[0054] In one embodiment, a membrane permeable construct for use in transporting mRNA across a lipid membrane and subsequently delivering said mRNA to a cell comprises: [ka] especially: [ka] (where, * indicates that the peptide continues from the side chain amino group rather than from the α-amino group. The present invention further comprises a cell-permeable amino acid sequence comprising or consisting of the sequence:

[0055] In another embodiment, a membrane permeable construct for transport of a cargo across a lipid membrane and subsequent delivery of said cargo to a cell comprises: [ka] especially: [ka] (where, * indicates that the peptide continues from the side chain amino group rather than from the α-amino group. The present invention further comprises a cell-permeable amino acid sequence comprising or consisting of the sequence:

[0056] In another embodiment, the cell-penetrating amino acid sequence is optionally chemically modified at C-terminus.Such C-terminus modification includes amidation, for example, where C-terminus is CONH2.Therefore, in certain embodiments, the C-terminus of the cell-penetrating amino acid sequence is CONH2.

[0057] The cell-permeable amino acid sequence may be further modified to add specific targeting elements, for example, to target the membrane-permeable constructs described herein to specific cells or organs. In certain embodiments, the targeting element may be directed to a surface receptor or ligand present on cells of the spleen. In certain embodiments, the targeting element may be directed to a surface receptor or ligand on dendritic cells. The targeting element may include, and therefore may be selected from, any one or more of the following: peptides and / or proteins (including antibodies and fragments thereof, e.g., antigen-binding fragments (Fab) and heavy chain-only / VHH fragments), and aptamers.

[0058] (fatty acid) Fatty acids are carboxylic acids with long aliphatic chains that are either saturated or unsaturated. Short-chain fatty acids are fatty acids with an aliphatic tail of 5 or fewer carbon atoms. Medium-chain fatty acids are fatty acids with an aliphatic tail of 6-12 carbon atoms. Long-chain fatty acids are fatty acids with an aliphatic tail of 13-21 carbon atoms. Very-long-chain fatty acids are fatty acids with an aliphatic tail of 22 or more carbon atoms.

[0059] The fatty acid chain of the present invention may be a saturated or unsaturated carbon chain, but is preferably a saturated carbon chain. A saturated fatty acid does not have a carbon-carbon double bond. An unsaturated fatty acid has one or more (e.g., 1 to 4, e.g., 1 or 2) carbon-carbon double bonds. The carbon-carbon double bonds can give rise to either cis or trans isomers.

[0060] The cis configuration means that the two hydrogen atoms adjacent to the double bond protrude from the same side of the chain. The rigidity of the double bond fixes the conformation, causing the chain to bend in the cis isomer, limiting the conformational freedom of the fatty acid. The more double bonds a chain has in the cis configuration, the less flexibility it has.

[0061] The trans configuration means that two adjacent hydrogen atoms are located on opposite sides of the chain, which results in the chain being less curved and more similar in shape to a straight-chain saturated fatty acid.

[0062] A saturated fatty acid chain with 16 carbon atoms is also called palmitic acid. A saturated fatty acid chain with 18 carbon atoms is also called stearic acid. A saturated fatty acid chain with 20 carbon atoms is also called arachidic acid. A saturated fatty acid chain with 22 carbon atoms is also called behenic acid.

[0063] Examples of unsaturated fatty acids with 16 carbon atoms are palmitoleic acid or sapienic acid. Examples of unsaturated fatty acids with 18 carbon atoms are oleic acid or elaidic acid. Examples of unsaturated fatty acids with 20 carbon atoms are arachidonic acid or eicosapentaenoic acid. Examples of unsaturated fatty acids with 22 carbon atoms are erucic acid or docosahexaenoic acid.

[0064] The cell-penetrating amino acid sequences described herein have a fatty acid chain attached thereto. The fatty acid chain is sometimes referred to as a fatty acid moiety. The fatty acid chain is attached to the amino acid sequence or peptide by a covalent bond. The fatty acid chain may be attached to the peptide via a linker molecule. In a preferred embodiment of the present invention, the fatty acid is attached to the N-terminus of the cell-penetrating amino acid sequence. In a further preferred embodiment, the fatty acid is attached to the N-terminal amine of the amino acid at position 1, e.g., position 1 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0065] A linker can separate the peptide and the fatty acid chain. The linker is a chemical moiety containing two reactive groups / functional groups, one of which can react with the peptide and the other of which can react with the fatty acid chain. The two reactive groups / functional groups of the linker are connected via a linking moiety or spacer, where the linking moiety or spacer does not interfere with the coupling of the linker to the peptide and the fatty acid chain. The linker can be composed of amino acids linked by peptide bonds.

[0066] The fatty acid chain may have 16 to 22 carbon atoms, 16 to 20 carbon atoms, 16 to 18 carbon atoms, 16 carbon atoms, 18 to 22 carbon atoms, 18 to 20 carbon atoms, 18 carbon atoms, 20 to 22 carbon atoms, 20 carbon atoms, or 22 carbon atoms. The fatty acid chain preferably has 18 to 22 carbon atoms. Alternatively, or in addition, the fatty acid chain has 16, 18, 20, or 22 carbon atoms. In certain embodiments, the fatty acid chain has 16, 18, or 20 carbon atoms. In particularly preferred embodiments, the fatty acid chain has 18 carbon atoms.

[0067] Thus, in some embodiments, the membrane permeable constructs described herein for use in transporting mRNA across lipid membranes and subsequently delivering said mRNA to cells comprise: C16-SEQ ID NO:3) (referred to herein as NF419); C18-(SEQ ID NO:3) (referred to herein as NF55); C20-(SEQ ID NO:3) (referred to herein as NF410); C22-(SEQ ID NO:3) (referred to herein as NF411); C16-SEQ ID NO:4) (referred to herein as NF420); C18-(SEQ ID NO:4) (referred to herein as NF554); C20-(SEQ ID NO:4) (referred to herein as NF412); C22-(SEQ ID NO:4) (referred to herein as NF413); C16-SEQ ID NO:5) (referred to herein as NF422); C18-(SEQ ID NO:5) (referred to herein as NF553); C22-(SEQ ID NO:5) (referred to herein as NF423); C18-(SEQ ID NO:6) (referred to herein as NF559); C18-(SEQ ID NO:7) (referred to herein as NF550); C18-(SEQ ID NO:8) (referred to herein as NF54); C18-(SEQ ID NO:9) (referred to herein as NF430); C20-(SEQ ID NO:9) (referred to herein as NF437); C16-(SEQ ID NO: 10) (referred to herein as NF425); C18-(SEQ ID NO: 10) (referred to herein as NF424); C20-(SEQ ID NO: 10) (referred to herein as NF426); C18-(SEQ ID NO: 11) (referred to herein as NF436); or C20-(SEQ ID NO: 11) (referred to herein as NF438) :Contains or consists of these.

[0068] In certain embodiments, the membrane permeable constructs described herein for use in transporting mRNA across lipid membranes and subsequently delivering said mRNA to cells comprise: C22-(SEQ ID NO:3) (referred to herein as NF411); C18-(SEQ ID NO:4) (referred to herein as NF554); C18-(SEQ ID NO:5) (referred to herein as NF553); C18-(SEQ ID NO:8) (referred to herein as NF54); C18-(SEQ ID NO: 10) (referred to herein as NF424); C20-(SEQ ID NO: 10) (referred to herein as NF426); or C18-(SEQ ID NO: 11) (referred to herein as NF436) :Contains or consists of these.

[0069] In one embodiment, the membrane-permeable construct for use comprises or consists of a cell-permeable amino acid sequence of SEQ ID NO: 3 and a fatty acid chain having 22 carbon atoms, for example, where the fatty acid is behenic acid. This membrane-permeable construct is referred to herein as NF411. As shown by the data presented herein, NF411 shows mRNA delivery comparable to NF54 and better than NF55.

[0070] In another embodiment, the membrane-permeable construct for use comprises or consists of a cell-permeable amino acid sequence of SEQ ID NO: 4 and a fatty acid chain having 18 carbon atoms, for example, where the fatty acid is stearic acid. This membrane-permeable construct is referred to herein as NF554. As shown by the data presented herein, NF554 exhibits mRNA delivery similar to NF54 and improved mRNA delivery over NF55.

[0071] In another embodiment, the membrane-permeable construct for use comprises or consists of a cell-permeable amino acid sequence of SEQ ID NO: 5 and a fatty acid chain having 18 carbon atoms, for example, where the fatty acid is stearic acid. This membrane-permeable construct is referred to herein as NF553. As shown by the data presented herein, NF553 exhibits mRNA delivery similar to NF54 and improved mRNA delivery over NF55.

[0072] In another embodiment, the membrane-permeable construct for use comprises or consists of a cell-permeable amino acid sequence of SEQ ID NO: 8 and a fatty acid chain having 18 carbon atoms, for example, where the fatty acid is stearic acid. This membrane-permeable construct is referred to herein as NF54. As shown by the data presented herein, NF54 has significantly improved mRNA delivery over NF55 and better in vivo mRNA delivery improvement than NF55.

[0073] In another embodiment, the membrane-permeable construct for use comprises or consists of a cell-permeable amino acid sequence of SEQ ID NO: 10 and a fatty acid chain having 18 carbon atoms, for example, where the fatty acid is stearic acid. This membrane-permeable construct is referred to herein as NF424. As shown by the data presented herein, NF424 exhibits better mRNA delivery improvement than NF54 and significantly improved mRNA delivery than NF55. Also shown herein, NF424 exhibits significantly improved in vivo mRNA delivery compared to both NF54 and NF55.

[0074] In another embodiment, the membrane-permeable construct for use comprises or consists of a cell-permeable amino acid sequence of SEQ ID NO: 10 and a fatty acid chain having 20 carbon atoms, for example, wherein the fatty acid is arachidic acid. This membrane-permeable construct is referred to herein as NF426. As shown by the data presented herein, NF426 exhibits mRNA delivery comparable to NF54 and better than NF55 in improved mRNA delivery. Also shown herein, NF426 exhibits better in vivo mRNA delivery than NF55.

[0075] In another embodiment, the membrane-permeable construct for use comprises or consists of a cell-permeable amino acid sequence of SEQ ID NO: 11 and a fatty acid chain having 18 carbon atoms, for example, where the fatty acid is stearic acid. This membrane-permeable construct is referred to herein as NF436. As demonstrated by the data presented herein, NF436 has significantly improved in vivo mRNA delivery over both NF54 and NF55.

[0076] In other embodiments, the membrane permeable constructs described herein for transport of cargo across lipid membranes and subsequent delivery of said cargo to cells comprise: C18-(SEQ ID NO:9) (referred to herein as NF430); C20-(SEQ ID NO:9) (referred to herein as NF437); C16-(SEQ ID NO: 10) (referred to herein as NF425); C18-(SEQ ID NO: 10) (referred to herein as NF424); C20-(SEQ ID NO: 10) (referred to herein as NF426); C18-(SEQ ID NO: 11) (referred to herein as NF436); or C20-(SEQ ID NO: 11) (referred to herein as NF438) :Contains or consists of these.

[0077] In certain further embodiments, the membrane permeable constructs for transport of cargo across lipid membranes and subsequent delivery of said cargo to cells described herein comprise: C18-(SEQ ID NO: 10) (referred to herein as NF424); C20-(SEQ ID NO: 10) (referred to herein as NF426); or C18-(SEQ ID NO: 11) (referred to herein as NF436) :Contains or consists of these.

[0078] In one embodiment, the membrane-permeable construct comprises or consists of a cell-permeable amino acid sequence of SEQ ID NO: 10 and a fatty acid chain having 18 carbon atoms, for example, wherein the fatty acid is stearyl. This membrane-permeable construct is referred to herein as NF424. As shown by the data presented herein, NF424 exhibits improved delivery of cargo such as mRNA over NF54 and significantly improved delivery over NF55. Also shown herein, NF424 exhibits significantly improved in vivo mRNA delivery over both NF54 and NF55.

[0079] In another embodiment, the membrane-permeable construct comprises or consists of a cell-permeable amino acid sequence of SEQ ID NO: 10 and a fatty acid chain having 20 carbon atoms, for example, wherein the fatty acid is arachidic acid. This membrane-permeable construct is referred to herein as NF426. As shown by the data presented herein, NF426 exhibits comparable delivery of cargo such as mRNA to NF54 and better delivery improvement than NF55. Also shown herein, NF426 exhibits better in vivo mRNA delivery improvement than NF55.

[0080] In another embodiment, the membrane-permeable construct comprises or consists of a cell-permeable amino acid sequence of SEQ ID NO: 11 and a fatty acid chain having 18 carbon atoms, for example, where the fatty acid is stearic acid. This membrane-permeable construct is referred to herein as NF436. As demonstrated by the data presented herein, NF436 has significantly improved in vivo mRNA delivery over both NF54 and NF55.

[0081] (cargo) The cargo molecule may be a substance associated with a cell-penetrating peptide intended to be delivered to a cell. The cargo molecule may be associated with the cell-penetrating peptide either by chemical linkage via a covalent bond or by non-covalent or ionic bonds or interactions. The attached cargo associated with the cell-penetrating peptide can be transported from the outside of the cell across the cell's membrane and enter the cell. The cargo can then be released into the cell's cytoplasm and targeted to an intracellular organelle or presented on the intracellular or extracellular cell surface. The cargo associated with the cell-penetrating peptide (forming a construct, complex, or nanoparticle) can enter the cell via the endosomal pathway. The construct, complex, or nanoparticle can then be packaged within an organelle, preferably an endosomal compartment or lysosome, and then released into an intracellular compartment or the environment.

[0082] Cargos include peptides, proteins, non-peptide pharmaceuticals, polysaccharides, lipids (including combinations thereof, including lipoproteins and glycolipids), nucleic acids (e.g., DNA, small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), antisense oligonucleotides, decoy DNA, plasmid DNA), small molecule drugs, and imaging agents (e.g., fluorophores, radioactive tracers, and metal chelates). When the cargo molecule is a peptide, polypeptide, or protein, it can include one or more linked peptides, polypeptides, or proteins. Peptides may be selected from the group consisting of, but not limited to, cell or tumor-targeting peptides, aptamers, receptor ligands, peptide ligands, cytotoxic peptides, bioactive peptides, antibodies, and diagnostic agents. When the cargo molecule is a nucleic acid, the nucleic acid can include one or more nucleic acids, each one encoding a peptide or polypeptide. The cargo molecule may be a combination of proteins, lipids, and / or polysaccharides, including lipoproteins and glycolipids. The cargo may be selected from the group consisting of oligonucleotides, including, but not limited to, single-stranded oligonucleotides (e.g., DNA, RNA, PNA, LNA, and analogs thereof), double-stranded oligonucleotides (e.g., siRNA, shRNA, microRNA, and decoy DNA), and circular DNA (e.g., plasmids, e.g., pDNA). In certain embodiments of the present invention, the cargo is messenger RNA (mRNA). Particularly, where the membrane-permeable constructs described herein are intended for use in transporting said mRNA cargo across a lipid membrane and subsequently delivering the mRNA cargo to a cell, it is preferred that the cargo is mRNA.

[0083] In one embodiment, the membrane-permeable construct or membrane-permeable construct for use as described herein comprises a cargo covalently bound thereto. In the case of a covalent bond, a linker moiety may optionally be present between the cell-permeable peptide and the cargo molecule, i.e., such a linker may link or connect the cell-permeable peptide and the cargo molecule together. The covalent bond, e.g., the linker moiety, may be biodegradable to facilitate release of the cargo into an intracellular compartment or the environment.

[0084] In further embodiments, the complex comprises a membrane-permeable construct for use as described herein or a membrane-permeable construct and a cargo that interacts therewith non-covalently, e.g., via ionic interactions. Such cargo is preferably ionic and negatively charged. In certain embodiments, the complex forms a nanoparticle. The complex self-assembles into nanoparticles. The nanoparticles of the present invention comprise an amphiphilic, cell-permeable amino acid sequence and associated cargo, and the nanoparticles remain intact upon entry into cells. The nanoparticles of the present invention may have a diameter of, for example, 20 to 60 nm, preferably 30 to 40 nm. Once inside the cell, the nanoparticles undergo a phase transition, e.g., within an endosome / lysosome providing a sufficiently low pH environment, thereby releasing the associated cargo into the cell.

[0085] In a preferred embodiment, the cargo interacts with the construct non-covalently (e.g., via ionic interaction) to form a complex. In one embodiment, the siRNA cargo interacts with the construct non-covalently (e.g., via ionic interaction) to form a complex. In a further embodiment, the complex formed of the siRNA that interacts with the construct non-covalently (e.g., via ionic interaction) forms a nanoparticle. In a particular embodiment, the mRNA cargo interacts with the construct non-covalently (e.g., via ionic interaction) to form a complex. In a further embodiment, the complex formed of the mRNA that interacts with the construct non-covalently (e.g., via ionic interaction) forms a nanoparticle.

[0086] In certain embodiments, the mRNA cargo encodes an antigenic protein or peptide. It is anticipated that mRNA encoding an antigenic protein or peptide delivered as described herein will be particularly useful for eliciting an immune response in a subject, e.g., a mammalian subject (e.g., a human). Thus, the cargo can form part of an mRNA vaccine, such that the membrane-permeable construct described herein carrying the mRNA cargo is an mRNA vaccine. In further embodiments, the mRNA cargo encodes an antigenic protein or peptide for eliciting an immune response in a subject. In certain embodiments, the antigenic protein or peptide is a surface protein of a pathogen, e.g., a viral surface protein. Thus, in certain embodiments, the antigenic protein or peptide is a viral surface protein. Viral surface proteins include those constituting the capsid or viral envelope, e.g., capsid protein and / or viral envelope protein. In certain embodiments, the antigenic protein or peptide is a viral glycoprotein. Such glycoproteins include spike and hemagglutinin proteins, e.g., the spike protein of coronaviruses, particularly CARS-CoV2. Thus, in one embodiment, the antigenic protein or peptide is a spike protein of a coronavirus, particularly CARS-CoV2. In other embodiments, the antigenic protein or peptide is a surface protein, e.g., a spike protein, of influenza, respiratory syncytial virus (RSV), or tick-borne encephalitis (TBE). In yet other embodiments, the antigenic protein or peptide is a bacterial surface protein. In yet other embodiments, the antigenic protein or peptide is a surface protein of a parasite.

[0087] In an alternative embodiment, the antigenic protein or peptide may be an antigen associated with cancer, such as a cancer marker protein. Such cancer-associated antigens may be suitably identified and selected depending on the particular cancer to be targeted. Thus, in a further embodiment, the present invention may be for use in a method of eliciting an anti-cancer immune response in a subject. Thus, the subject may be suffering from cancer.

[0088] Pharmaceutical Compositions, Uses, and Therapeutic Applications / Methods In a further aspect of the present invention, there is provided a membrane-permeable construct as described herein for use in transporting a cargo across a lipid membrane and subsequently delivering the cargo to a cell. As previously described herein, in certain embodiments, the cargo is mRNA, which may be either covalently bound to the membrane-permeable construct or may interact non-covalently with it.

[0089] The membrane-permeable construct of the present invention can be formulated for delivery in a pharmaceutical composition. Thus, in another aspect, a pharmaceutical composition is provided that includes the membrane-permeable construct described herein. In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents, and / or excipients. In a further embodiment, the pharmaceutical composition optionally further comprises one or more adjuvants.

[0090] As used herein, "pharmaceutically acceptable carriers" include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Carriers may be suitable for parenteral (e.g., topical), oral, nasal, intravenous, intramuscular, intradermal, intracranial, intraocular, intraperitoneal, subcutaneous, spinal, or other parenteral administration routes, such as by injection or infusion. For parenteral administration, the carrier preferably comprises water and may contain buffers for pH control, stabilizers (e.g., surfactants and amino acids), and osmolality modifiers (e.g., salts and sugars). If the composition is intended to be provided in lyophilized form for dilution at the time of use, the formulation may contain a cryoprotectant, such as a sugar, e.g., trehalose. For oral administration, any of the above carriers or solid carriers, such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be utilized. Alternatively, the carrier may be suitable for parenteral administration, e.g., topical, epidermal, or mucosal routes of administration. The carrier may be suitable for oral administration. Depending on the route of administration, the modulator may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0091] Thus, compositions of the invention can include buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose, or dextran), mannitol, proteins, polypeptides, or amino acids such as glycine, antioxidants, bacteriostats, chelating agents such as EDTA or glutathione, solutes that render the formulation isotonic, hypotonic, or weakly hypertonic with the blood of the recipient, suspending agents, viscosity increasing agents, and / or preservatives. Alternatively, compositions of the invention can be formulated as a lyophilizate.

[0092] The pharmaceutical compositions of the present invention may contain one or more pharmaceutically acceptable salts. A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart undesired toxicological effects. Examples of such salts include acid addition salts and base addition salts. Such salts can be prepared from pharmaceutically acceptable non-toxic bases, including organic bases (e.g., salts of primary, secondary, and tertiary amines, and basic amino acids) and inorganic bases (e.g., sodium, potassium, lithium, ammonium, calcium, and magnesium salts).

[0093] Pharmaceutically acceptable carriers include aqueous carriers or diluents.Suitable aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, buffered water, and physiological saline.Other carrier examples include ethanol, polyols (for example, glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.In many cases, it is desirable to include isotonic agents, for example, sugars, polyhydric alcohols such as mannitol, sorbitol, or sodium chloride in the composition.

[0094] Therapeutic compositions must usually be sterile and stable under the conditions of manufacture and storage. The compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentration. The pharmaceutical compositions of the present invention may contain additional active ingredients. In therapeutic applications, compounds are administered to a subject already suffering from the above-mentioned disorders or diseases in an amount sufficient to cure, alleviate, or partially arrest the disease or one or more of its symptoms. Such therapeutic treatment may result in a decrease in the severity of disease symptoms or an increase in the frequency or duration of symptom-free periods. An amount adequate to accomplish this is defined as a "therapeutically effective amount."

[0095] In prophylactic applications, formulations are administered to a subject at risk of a disorder or disease described herein in an amount sufficient to prevent or reduce the subsequent effects of the disease or one or more of its symptoms. An amount adequate to accomplish this is defined as a "prophylactically effective amount." Effective amounts for each purpose will depend on the severity of the disease or injury and the weight and general condition of the subject.

[0096] The subject for administration can be a human or a non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. Administration to humans is typical.

[0097] The pharmaceutical compositions of the present invention can be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by those skilled in the art, the route and / or mode of administration will vary depending on the desired results. Examples of routes of administration for the compounds or pharmaceutical compositions of the present invention include intravenous, intramuscular, intradermal, intraocular, intraperitoneal, subcutaneous, spinal, or other parenteral administration routes, such as by injection or infusion. As used herein, the term "parenteral administration" refers to modes of administration other than enteral and topical administration, usually by injection. Alternatively, the pharmaceutical compositions of the present invention can be administered via non-parenteral routes, such as topical, epidermal, or mucosal administration routes.

[0098] The appropriate dose of the pharmaceutical composition of the present invention may be determined by a skilled medical practitioner. The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be varied to obtain an amount of the active ingredient that is effective for a particular patient, composition, and mode of administration to achieve the desired therapeutic response without toxicity to the patient. The selected dosage level will depend on various pharmacokinetic factors, including the activity of the particular composition of the present invention employed, the route of administration, the time of administration, the excretion rate of the particular compound employed, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health, and medical history of the patient being treated, and similar factors well known in the medical field.

[0099] Dosage regimen can be adjusted to achieve optimal desired response, for example, therapeutic response.For example, if single dose can be administered, several divided doses can be administered over time, or dose can be proportionally reduced or increased as indicated by the exigencies of therapeutic situation.As used herein, dosage unit refers to a physically independent unit that is suitable as a single dosage for the subject to be treated; each unit contains a predetermined amount of active compound that is calculated to achieve desired therapeutic effect in association with required pharmaceutical carrier.

[0100] Administration may be in single or multiple doses. Multiple doses may be administered via the same or different routes and to the same or different locations. Alternatively, the dose may be in a sustained release formulation, in which case less frequent administration is required. Dosage and frequency may vary depending on the half-life of the pharmaceutical composition in the patient and the desired duration of treatment.

[0101] In certain preferred embodiments of the present invention, there is provided a pharmaceutical composition as described herein, comprising one or more (e.g., one) cell-penetrating amino acid sequences as described herein in combination with a pharmaceutically acceptable carrier.

[0102] In some embodiments, the cell is in vivo. In a further embodiment, the cell is a splenic cell. In yet a further embodiment, the cell is a dendritic cell (DC). In certain embodiments, the membrane-permeable construct for use described herein transports a cargo across a lipid membrane and then delivers the cargo to cells of the in vivo spleen, for example, to DCs in vivo. Thus, according to a further aspect of the present invention, there is provided a membrane-permeable construct described herein or a pharmaceutical composition described herein for use in transporting a cargo across a lipid membrane and then delivering the cargo to cells of the in vivo spleen, for example, to DCs in vivo. In particular, the use is provided for a membrane-permeable construct comprising a cell-permeable amino acid sequence comprising the sequence of SEQ ID NO: 2.

[0103] In another aspect of the present invention, there is provided a membrane permeable construct as described herein or a pharmaceutical composition as described herein for use in a method of eliciting an immune response in a subject. In a further aspect, there is provided a membrane permeable construct or a pharmaceutical composition for use in a method of vaccinating a subject, for example vaccinating the subject against an infectious disease.

[0104] As described hereinabove, the membrane-permeable constructs of the present invention have a particularly good ability to deliver mRNA cargo to the intracellular compartment of cells, specifically to cells in the spleen in vivo, such as DCs.Therefore, they are expected to be useful in eliciting an immune response in a subject, where the mRNA cargo encodes an antigenic protein or peptide.This is because such mRNA is efficiently delivered to the spleen in vivo, where the maximum number of antigen-presenting cells (APCs), such as DCs, can be found in mammals.Therefore, in some embodiments, the mRNA cargo encodes an antigenic protein or peptide for eliciting an immune response in a subject.

[0105] In a further aspect of the present invention, there is provided a method of eliciting an immune response in a subject, the method comprising administering to the subject a membrane permeable construct or pharmaceutical composition described herein. In another aspect, there is provided a method of vaccinating a subject, the method comprising administering to the subject a membrane permeable construct or pharmaceutical composition described herein. In one embodiment, the administration comprises a therapeutically effective amount as described herein above. In another embodiment, the administration comprises a prophylactically effective amount as described herein above.

[0106] In a further aspect of the present invention, there is provided a use of a membrane permeable construct or pharmaceutical composition described herein for the manufacture of a medicament. In certain embodiments, the medicament is a vaccine. Accordingly, in another aspect, there is provided a use of a membrane permeable construct or pharmaceutical composition described herein for the manufacture of a vaccine. In one embodiment, the vaccine is for eliciting an immune response in a subject. Accordingly, in certain embodiments, the vaccine comprises an mRNA cargo encoding an antigenic protein or peptide for eliciting an immune response in a subject. In yet a further embodiment, the vaccine comprises a therapeutically effective amount of the membrane permeable construct or pharmaceutical composition described herein. In another embodiment, the vaccine comprises a prophylactically effective amount of the membrane permeable construct or pharmaceutical composition described herein.

[0107] In some embodiments, the use or administration of the membrane permeable construct of the pharmaceutical composition may be therapeutic, as described herein above. Thus, in one embodiment, the subject may be suffering from a disease or disorder and may therefore be in need of the uses and / or methods for eliciting an immune response described herein. In a further embodiment, the subject is suffering from cancer. According to this embodiment, the elicited immune response is directed against a cancer antigen. In other embodiments, the use or administration may be prophylactic. Thus, in a further particular embodiment, the subject may be at risk of contracting a disease or disorder, for example, an infectious disease. According to this particular embodiment, the elicited immune response is directed against an antigen of a pathogen, for example, a virus (e.g., a viral surface protein). Thus, also according to this particular embodiment, the subject may be in need of vaccination against an infectious disease (e.g., a viral disease).

[0108] Thus, in a further aspect, there is provided a membrane permeable construct or pharmaceutical composition described herein for use in a method of eliciting an immune response against a viral surface protein in a subject. In a further aspect, there is provided a membrane permeable construct or pharmaceutical composition described herein for use in a method of vaccinating a subject against a viral surface protein. In another aspect, there is provided a method of eliciting an immune response against a viral surface protein in a subject, the method comprising administering to the subject a membrane permeable construct or pharmaceutical composition described herein. In a still further aspect, there is provided a method of vaccinating a subject against a viral surface protein, the method comprising administering to the subject a membrane permeable construct or pharmaceutical composition described herein. In certain embodiments of these aspects, the viral surface protein is a capsid and / or viral envelope protein, e.g., a viral glycoprotein.

[0109] (In vitro and ex vivo use) As described hereinabove, the membrane-permeable construct is particularly useful in vivo. However, in particular, the membrane-permeable construct of the second aspect of the present invention (i.e., comprising a cell-permeable amino acid sequence comprising SEQ ID NO: 2) can also be used to transfect cells in vitro or ex vivo, particularly for stable transfection. The membrane-permeable construct of the second aspect can be used to deliver siRNA, pDNA, mRNA, peptides and / or proteins, non-peptide pharmaceuticals, polysaccharides, lipids, small molecule drugs, and imaging agents to cells, including cells in vitro.

[0110] Such cells that can be transfected include, but are not limited to, cell lines and cell types: HeLa, NIH 3T3, HEK-293, CHO-K1, U2-OS, and COS-7. Some difficult-to-transfect cell lines, such as Jurkat, CaCo2, human adenocarcinoma cell lines, dendritic cells, and epidermal cells, can also be transfected using the membrane-permeable constructs described herein.

[0111] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by those skilled in the art to which this invention belongs. As used herein, the term "about" when used herein means up to 10% higher (inclusive) and up to 10% lower (inclusive) than the specified value, preferably up to 5% higher (inclusive) and up to 5% lower (inclusive) than the specified value, and particularly includes the specified value. As used herein, the term "between" includes the specified boundary value.

[0112] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise" and variations thereof, such as "comprises" and "comprising," will be understood to imply the inclusion of a stated integer, step, group of integers, or group of steps, but not the exclusion of any other integer, step, group of integers, or group of steps.

[0113] Furthermore, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a "cell-permeable amino acid sequence" includes two or more such sequences, or reference to "mRNA" includes two or more such mRNA molecules, etc.

[0114] It will be understood that all embodiments described herein may be applied to all aspects of the invention, and vice versa, and that such combinations will be readily apparent to those skilled in the art from the description provided herein.

[0115] Other features and advantages of the present invention will be apparent from the description provided herein. However, since various changes and modifications will become apparent to those skilled in the art, it should be understood that this description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only.

[0116] (Provisions) The set of clauses that define the present invention, its aspects and embodiments are as follows: 1. A membrane-permeable construct for use in transporting a cargo across a lipid membrane and subsequently delivering the cargo to a cell in vivo, the construct comprising a cell-permeable amino acid sequence and a fatty acid chain attached to the N-terminus of the amino acid sequence, wherein the cargo is mRNA, and wherein the cell-permeable amino acid sequence is [ka] (wherein X1 represents Lys(K), Orn(O), Dab, or Dap; where: * indicates that the peptide is optionally continued from a side chain amino group rather than from the α-amino group, wherein X2 represents KA or AK, and wherein the cell-penetrating amino acid sequence is optionally chemically modified at the C-terminus. The membrane-permeable construct comprises the sequence: 2. A membrane permeable construct for use according to clause 1, wherein said mRNA cargo encodes an antigenic protein or peptide for eliciting an immune response in a subject. 3. A membrane permeable construct for use according to clause 1 or clause 2, wherein said cell is a cell of the spleen, such as a dendritic cell (DC). 4. A membrane permeable construct for use according to any one of clauses 1 to 3, wherein said mRNA encodes an antigenic protein or peptide, such as a viral surface protein, in particular a capsid and / or viral envelope protein, such as a viral glycoprotein. 5. A membrane-permeable construct for transport of a cargo across a lipid membrane and subsequent delivery of the cargo to a cell, the construct comprising a cell-permeable amino acid sequence and a fatty acid chain attached to the N-terminus of the amino acid sequence, wherein the cell-permeable amino acid sequence is [ka] (wherein X1 represents Lys(K), Orn(O), Dab, or Dap; where: * indicates that the peptide continues from a side chain amino group rather than from the α-amino group, and where X2 represents KA or AK. The membrane-permeable construct comprises the sequence: 6. A membrane permeable construct or membrane permeable construct for use according to any one of clauses 1 to 5, wherein X1 is K, Orn(O) or Dab, in particular Dab. 7. The peptide is * 7. The membrane-permeable construct or membrane-permeable construct for use according to any one of clauses 1 to 6, wherein the amino acid sequence at position 1 continues from a side chain amino group rather than from the α-amino group. 8. The membrane permeable construct or membrane permeable construct for use according to any one of clauses 1 to 7, wherein X2 is KA. 9. The membrane permeable construct or membrane permeable construct for use according to any one of clauses 1 to 7, wherein X2 is AK. 10. A membrane-permeable construct or membrane-permeable construct for use according to any one of clauses 1 to 9, wherein said cell-permeable amino acid sequence comprises one or more further amino acid substitutions at positions 9 to 21 of SEQ ID NO: 1 or positions 9 to 19 of SEQ ID NO: 2. 11. A membrane-permeable construct for use in the membrane-permeable construct of any one of clauses 1 to 10, wherein the fatty acid chain has 16 to 22 carbon atoms, for example, 18 to 22 carbon atoms. 12. A membrane-permeable construct or membrane-permeable construct for use according to clause 11, wherein the fatty acid chain has 16, 18, 20 or 22 carbon atoms, for example 18, 20 or 22 carbon atoms, in particular 18 carbon atoms. 13. The membrane-permeable construct or membrane-permeable construct for use according to any one of clauses 1 to 12, wherein the cell-permeable amino acid sequence is chemically modified, for example amidated, at the C-terminus, for example the C-terminus is CONH2. 14. The cell-permeable amino acid sequence is [ka] especially: [ka] (where, *indicates that the peptide continues from the side chain amino group rather than from the α-amino group. Contains an array of : For example, wherein the membrane permeable construct is C16-(SEQ ID NO:3) (referred to herein as NF419); C18-(SEQ ID NO:3) (referred to herein as NF55); C20-(SEQ ID NO:3) (referred to herein as NF410); C22-(SEQ ID NO:3) (referred to herein as NF411); C16-(SEQ ID NO:4) (referred to herein as NF420); C18-(SEQ ID NO:4) (referred to herein as NF554); C20-(SEQ ID NO:4) (referred to herein as NF412); C22-(SEQ ID NO:4) (referred to herein as NF413); C16-SEQ ID NO:5) (referred to herein as NF422); C18-(SEQ ID NO:5) (referred to herein as NF553); C22-(SEQ ID NO:5) (referred to herein as NF423); C18-(SEQ ID NO:6) (referred to herein as NF559); C18-(SEQ ID NO:7) (referred to herein as NF550); C18-(SEQ ID NO:8) (referred to herein as NF54); C18-(SEQ ID NO:9) (referred to herein as NF430); C20-(SEQ ID NO:9) (referred to herein as NF437); C16-(SEQ ID NO: 10) (referred to herein as NF425); C18-(SEQ ID NO: 10) (referred to herein as NF424); C20-(SEQ ID NO: 10) (referred to herein as NF426); C18-(SEQ ID NO: 11) (referred to herein as NF436); or C20-(SEQ ID NO: 11) (referred to herein as NF438) especially: C22-(SEQ ID NO:3) (referred to herein as NF411); C18-(SEQ ID NO:4) (referred to herein as NF554); C18-(SEQ ID NO:5) (referred to herein as NF553); C18-(SEQ ID NO:8) (referred to herein as NF54); C18-(SEQ ID NO: 10) (referred to herein as NF424); C20-(SEQ ID NO: 10) (referred to herein as NF426); or C18-(SEQ ID NO: 11) (referred to herein as NF436) 14. The membrane permeable construct of any one of clauses 1 to 13, comprising: 15. The cell-permeable amino acid sequence is [ka] especially: [ka] (where, * indicates that the peptide continues from the side chain amino group rather than from the α-amino group. Contains an array of : For example, wherein the membrane permeable construct is C18-(SEQ ID NO:9) (referred to herein as NF430); C20-(SEQ ID NO:9) (referred to herein as NF437); C16-(SEQ ID NO: 10) (referred to herein as NF425); C18-(SEQ ID NO: 10) (referred to herein as NF424); C20-(SEQ ID NO: 10) (referred to herein as NF426); C18-(SEQ ID NO: 11) (referred to herein as NF436); or C20-(SEQ ID NO: 11) (referred to herein as NF438) especially: C18-(SEQ ID NO: 10) (referred to herein as NF424); C20 (SEQ ID NO: 10) (referred to herein as NF426); or C18-(SEQ ID NO: 11) (referred to herein as NF436) 14. The membrane permeable construct of any one of clauses 1 to 13, comprising: 16. A membrane-permeable construct according to any one of clauses 5 to 15 for use in transporting a cargo across a lipid membrane and subsequently delivering said cargo to a cell. 17. The membrane permeable construct or membrane permeable construct for use according to any one of clauses 1 to 16, wherein said construct further comprises a cargo covalently attached thereto. 18. A membrane permeable construct for use according to any one of clauses 1 to 16 or a complex comprising a membrane permeable construct and a cargo that interacts therewith non-covalently, for example via ionic interactions. 19. The membrane permeable construct, membrane permeable construct or complex for use according to any one of clauses 16 to 18, wherein said cargo is mRNA. 20. The membrane permeable construct, membrane permeable construct, or complex for use according to any one of clauses 16 to 19, wherein the cell is in vivo and / or the cell is a splenic cell, such as a dendritic cell (DC). 21. A membrane permeable construct, membrane permeable construct or complex for use according to clause 19 or clause 20, wherein the mRNA encodes an antigenic protein or peptide, such as a viral surface protein, in particular a capsid and / or viral envelope protein, such as a viral glycoprotein. 22. A pharmaceutical composition comprising the membrane-permeable construct or complex according to any one of clauses 5 to 21 and a pharmaceutically acceptable carrier, and optionally further comprising one or more adjuvants. 23. A membrane permeable construct, membrane permeable construct or complex for use according to any one of clauses 1 to 21, or a pharmaceutical composition according to clause 22, for use in a method of eliciting an immune response in a subject.

[0117] The invention will now be illustrated by the following non-limiting examples. [Example]

[0118] (Example) Example 1: Materials and Methods (Transfection reagent) Cell-penetrating peptides were synthesized on an automated peptide synthesizer (Biotage Initiator+ Alstra) using a fluorenylmethyloxycarbonyl (Fmoc) solid-phase peptide synthesis strategy with Rink-amide ChemMatrix resin (0.41 mmol / g loading) to yield C-terminally amidated peptides. Fatty acids were manually coupled to the N-terminus of the peptides using 5 equivalents of fatty acid at room temperature overnight. For the synthesis of "twisted" peptides, synthesis continued from the side-chain amino group using Boc-L-Orn(Fmoc)-OH, Boc-L-Lys(Fmoc)-OH, Boc-L-Dab(Fmoc)-OH, and Boc-L-Dap(Fmoc)-OH (Iris Biotech, Germany). Reactions were performed in DMF using HOBT / HBTU for manual synthesis or DIC / Oxima for machine synthesis as coupling reagents, with DIEA as the activator base. Cleavage was carried out at room temperature for 2 hours using trifluoroacetic acid, 2.5% triisopropylsilane, and 2.5% water. Peptides were purified by reversed-phase high-performance liquid chromatography on a C4 column (Phenomenex Jupiter C4, 5 μm, 300 Å, 250 × 10 mm) using a gradient of acetonitrile / water containing 0.1% TFA. Peptide molecular weights were analyzed by matrix-assisted laser desorption / ionization / time-of-flight mass spectrometry (Brucker Microflex LT / SH, USA). Where applicable, peptide concentrations were determined based on the dilution of accurately weighed material and the absorption of tyrosine.

[0119] (In vitro method:) (Maintenance of cell cultures) Adherent CHO-K1 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate, 100 U / ml penicillin, and 100 mg / ml streptomycin. Complete medium was supplemented with 10% (final) fetal bovine serum (FBS). Cells were maintained in a humidified incubator at 37°C and 5% CO2. Cell viability and confluence were assessed daily, and cell density was periodically reduced. For cell counting, we used a CytoSMART cell counter with 0.4% trypan blue staining before measurement. Multiwell plates containing cell cultures used in experiments were incubated in a humidified incubator at 37°C and 5% CO2.

[0120] (Preparation of transfection complexes) For in vitro experiments, 0.1 μg of nucleic acid (plasmid DNA (pDNA) or mRNA) was used per well of a 96-well plate. For CPP / NA complex formation, diluted NA was mixed with CPP in water. Complexes were formed with excess peptide at a charge ratio of 3:1, based on the theoretical charge ratio (CR) between the positive charge of the excess peptide and the negative charge of the nucleic acid backbone. The commercially available reagent Lipofectamine 3000 (LF3000) was used for transfection of adherent cells according to the manufacturer's recommendations.

[0121] Reporter quantification for in vitro experiments For evaluation of reporter luminescence (expressed from mRNA or plasmid pDNA encoding firefly luciferase) in adherent cell cultures, 10,000 cells were seeded per well of a 96-well plate in 100 μl of medium one day before transfection. Immediately before transfection, the cell medium was replaced with 100 μl of serum-containing (10% fetal bovine serum) DMEM medium. CPP and mRNA or pDNA were mixed in ultrapure water at a charge ratio of 3:1, incubated at room temperature, and then added to the cells in serum-containing medium. Cells were transfected with 0.1 μg mLuc per well.

[0122] Twenty-four hours after transfection, the medium was aspirated, the cells were washed with 1x PBS, and 30 μl of lysis buffer (0.1% Triton X100 in 1x PBS) was added. The cells were incubated with the lysis buffer for 20 minutes to lyse the cells. After adding the substrate luciferin (PerkinElmer) in the buffer, 20 μl of the lysate was transferred to a white-well 96-well plate with a black frame for luminescence measurement. Luminescence signals were detected using a GloMax® 96 microplate luminometer equipped with GloMax® 1.9.2 software (Promega). RLU values ​​were converted to RLU / mg by normalization to the total protein in the cell lysate, which reflects the total number of cells. The Pierce™ BCA Protein Assay Kit was used for protein detection.

[0123] (In vivo method:) (Preparation of transfection complexes) Transfection complexes (CPP / mRNA) were formed using 10 μg of mRNA mixed with each peptide at the indicated charge ratio (CR) in a final volume of 100 μl of MQ water. CR was calculated theoretically and took into account both the positive charge of the peptide and the negative charge of the nucleic acid. Transfection complexes (CPP / mRNA and CPP / pDNA) were formed using 50 μg of mRNA mixed with each peptide at the indicated charge ratio (CR) in a final volume of 168 μl of MQ water. The complexes were incubated at room temperature for 30 minutes and then mixed with an equal volume of 10% glucose solution (resulting in a final solution of 5% glucose with a total volume of 200 μl for the 10 μg dose group and 336 μl for the 50 μg dose group) and immediately injected intravenously via the tail vein.

[0124] (Reporter quantification from tissue homogenates) Reporter gene (luciferase) expression levels were assessed from postmortem tissue homogenates 16–24 h after a single injection.

[0125] (Reporter quantification using a whole-body imager) Mice were intraperitoneally injected with 100 μl of 15 mg / ml luciferin (PerkinElmer). After 10 minutes, the mice were imaged using an In Vivo Imaging System (IVIS Lumina II, PerkinElmer).

[0126] (Immunophenotyping of spleen cell subtypes) To analyze the CPP-transfected cell subtypes, 30 μg of mRNA encoding luciferase was mixed with CPP in CR2 in water and injected intravenously. 16 h after injection, the spleen was removed by centrifugation in ice-cold dissociation buffer (CaCl2 supplemented with 2 mM EDTA). 2+ and Mg 2+ After gentle mechanical dissociation by trituration, spleen cells were filtered through a 100 μm cell strainer and centrifuged at 300 × g for 5 min at +4°C. A 2-min incubation with 1 ml of RBC lysis buffer ACK (Thermo) was used for erythrocyte lysis. Flow cytometry buffer (dissociation buffer supplemented with 0.5% (w / v) BSA) was added, and the cells were centrifuged at 300 × g for 5 min at +4°C and counted. Twenty million cells were incubated with Truestain FcX anti-mouse CD16 / 32 (Biolegend) and stained with the antibodies and markers listed in Table 1. Cells were washed three times and sorted using a SONY MA900 sorter equipped with a 100 μm sorting tip. Cell subtypes were defined as follows: CD4+ / CD8+=T cells; Cd11c+=DC cells; Cd11b+ / F4+=macrophages. Table 1: Reagents and antibodies used for flow cytometry [Table 1]

[0127] Example 2: Delivery of reporter-expressing mRNA by cell-penetrating peptides (CPPs) in cell culture The present inventors have previously reported CPPs developed from the NickFect (NF) and PepFect (PF) families for plasmid (pDNA), siRNA, and miRNA delivery (see, e.g., WO 2020 / 144317, WO 2010 / 039088, and WO 2012 / 113846). The emergence of mRNA-based therapeutics led to the investigation of peptides for their ability to deliver mRNA. It was thought that CPPs that are efficient for siRNA and miRNA delivery, such as histidine-containing CPPs (Porosk et al., 2019), might be good candidates for mRNA delivery.

[0128] Therefore, CPPs from each major family (CPPs derived from NF55, PF14, and NF70) were tested for mRNA delivery / transfection. Naked mRNA-treated and untreated cells were used as controls. LF3000 was used to compare the transfection efficacy of liposome-based transfection reagents with CPPs (Figure 1).

[0129] As can be seen from the data in Figure 1 and Table 2, in contrast to the above-mentioned assumption that CPPs that are efficient for siRNA and miRNA delivery are also effective for mRNA, mRNA delivery follows a different logic than the delivery of other nucleic acids, such as pDNA, siRNA, and miRNA. Therefore, surprisingly, even if a CPP is an efficient transfection vector for siRNA, miRNA, or pDNA delivery, it cannot be assumed that it is also efficient for mRNA delivery. In particular, although PF14 and NF1 are known to be efficient delivery vectors for pDNA, the results herein show that they are not efficient for mRNA transfection. Furthermore, NF71 and NF70, which are highly efficient for siRNA and miRNA delivery, are not efficient for mRNA delivery.

[0130] In comparison with PF14, NF1, NF71, and NF70, we found that other CPPs were much more efficient at delivering mRNA. Of all the peptides tested, NF424, NF411, NF426, NF553, NF554, NF410, NF54, and NF55 were the most efficient (see Figure 1). Several other new peptides also showed very promising efficacy as mRNA delivery vectors.

[0131] These experiments revealed that the most efficient CPPs were not histidine-rich (e.g., NF70 and NF71) nor PF14-based, but rather NF55-based. Therefore, NF55-based peptides were investigated and further evaluated, particularly in vivo. Table 2: Delivery of reporter mRNA by CPPs in cell culture - assessment of reporter levels after transfection [Table 2]

[0132] Example 3: Comparison of reporter levels in cells after transfection with pDNA or mRNA delivered by CPPs CPPs from the PepFect, NickFect, and histidine-rich families were tested in parallel for pDNA and mRNA transfection (Fig. 2).

[0133] The results show that PF14 and NF71, highly efficient transfection reagents for pDNA delivery, were not effective tools for mRNA transfection / delivery. This is surprising because both mRNA and pDNA are nucleic acid polymers and encoded the same reporter (firefly luciferase) in these experiments. In contrast, NF55 was highly efficient for mRNA transfection but not for pDNA transfection (see Figure 2).

[0134] As previously highlighted, NF71 is highly efficient for siRNA and miRNA transfection. However, surprisingly, NF71 is not an efficient delivery vector for mRNA, even though siRNA, miRNA, and mRNA are all RNA-type cargoes and exert their activities in the same cellular compartment (the cytoplasm).

[0135] These results indicate that the ability of a CPP to deliver mRNA cannot be predicted by its corresponding efficacy to transfect pDNA, and that specific CPPs are required to deliver mRNA.

[0136] Example 4: Biodistribution of reporter levels after transfection of mRNA or pDNA with NF or PF in vivo To determine which organs in vivo mRNA could be delivered to using NickFect or PepFect CPP, mRNA complexes with NF55, NF71, and PF14 were formed and injected via the tail vein of Balb / c mice (Figure 3A). Organs were harvested 24 hours after the single injection, and reporter signals were measured from whole-tissue homogenates. Biodistribution of reporter levels was measured in the liver, spleen, lung, kidney, heart, and thymus.

[0137] Interestingly, for NF55, mRNA delivery to the spleen was 3.5-fold higher than delivery to the lung, far higher than any other organ (see Figure 3A and Table 3). At the same time, for PF14, no significant difference in measured reporter levels was observed in the spleen compared to lung tissue. This data demonstrates the selectivity of NF55 for transfecting mRNA into the spleen in vivo. NF71 also delivered mRNA primarily to the spleen, with very low transfection levels observed in other organs.

[0138] To compare the biodistribution profiles between harvested organs when either mRNA or pDNA was used as cargo, NF424 was used to transfect these two types of nucleic acids in vivo (Figure 3B and Table 4). These data show that when pDNA was transfected, primary expression was found in the lung and liver. However, in the case of mRNA delivery, the highest expression levels were found in the spleen (i.e., the organ of interest) of treated mice. Table 3: Biodistribution of reporter levels after transfection of mRNA or pDNA with NF or PF CPPs in vivo - mRNA delivery by NF55, PF14, and NF71. [Table 3] Table 4: Biodistribution of reporter levels after transfection of mRNA or pDNA with NF or PF CPPs in vivo - Delivery of mRNA and pDNA with NF424 [Table 4]

[0139] Example 5: Live animal imaging 1 to 48 hours after administration of NF424 / mRNA or NF424 / pDNA complexes Live animal imaging was used to track the time points of mRNA and pDNA transfection with NF (Figure 4). The CPP used was NF424, which was complexed with mRNA or pDNA in parallel and injected via the tail vein of BALB / c mice. Reporter gene expression levels were assessed using a whole-body imager at 1, 6, 12, 24, and 48 hours after injection.

[0140] It is noteworthy that the spleen contains the largest number of antigen-presenting cells (APCs) in the mammalian organism (see supra), which is why the spleen is an attractive target for immunization approaches.

[0141] As can be seen in Figure 4 (left panel), after 1 hour, it was already possible to detect a reporter signal in the spleen of mice injected with the NF424 / mRNA complex. For pDNA, the reporter signal was visible in the lungs just 6 hours after injection. Despite the injected dose of pDNA being 5-fold higher (50 μg) than the dose of mRNA (10 μg), the detected signal was much higher from the mRNA. This data indicates that NF424 delivers mRNA primarily to the spleen and pDNA to the lungs, but is much more effective at delivering mRNA than pDNA in vivo.

[0142] Example 6: In vivo delivery of mRNA by CPPs in the spleen Extending the in vitro results of Example 2, the most efficient CPP for mRNA delivery was tested in vivo. The CPP and mRNA complex was injected into the tail vein of BALB / c mice, the spleens were harvested 16 hours after the single injection, and reporter signals were measured from whole tissue homogenates (Figure 5 and Table 5).

[0143] The highest reporter levels after mRNA delivery to the spleen in vivo were achieved with NF436, NF424, and NF430. However, other vectors, such as NF426, NF54, NF425, NF419, NF411, and NF55, also delivered mRNA to the spleen in vivo with high efficiency. The best CPP, NF436, demonstrated 43-fold more efficient mRNA delivery to the spleen in vivo than PF1452 and 100-fold more efficient mRNA delivery to the spleen in vivo than NF70 (see Figure 5 and Table 5).

[0144] Therefore, while many CPPs can transfect nucleic acids, very few can efficiently deliver mRNA to the spleen in vivo. The results presented herein demonstrate several CPPs that are suitable for mRNA delivery, including delivery to the spleen in vivo, demonstrating the importance of selecting an appropriate CPP for in vivo delivery of mRNA. Table 5: Delivery of mRNA by CPPs in vivo - reporter levels in the spleen [Table 5]

[0145] Example 7: Immunophenotyping of splenocyte subtypes after NF55 mRNA transfection in vivo To determine which types of splenocytes were transfected with mRNA in vivo, cells collected from the spleens of transfected animals were sorted according to their subtype and reporter expression was measured. Luciferase-encoding mRNA (mRNA(luc)) was used as a reporter model. The majority of reporter signal was observed to arise from dendritic cells (DCs) sorted from the spleens of transfected animals (Figure 6). This data demonstrates the selectivity of NF55 for transfecting mRNA into splenic antigen-presenting cells in vivo.

[0146] (Sequence Listing) [Table 6] X1 represents K, Orn(O), Dab, or Dap; * indicates that the peptide is optionally followed (SEQ ID NOs: 1 and 2) or follows from the side chain amino group rather than from the α-amino group; X2 represents KA or AK; Dab refers to 2,4-diaminobutyric acid; and Dap refers to 2,3-diaminopropionic acid.

Claims

1. 1. A membrane-permeable construct for use in transporting a cargo across a lipid membrane and subsequently delivering the cargo to a cell in vivo, the construct comprising a cell-permeable amino acid sequence and a fatty acid chain attached to the N-terminus of the amino acid sequence, wherein the cargo is mRNA, and wherein the cell-permeable amino acid sequence is 【Chemical 1】 (where X １ represents Lys(K), Orn(O), Dab, or Dap; where: ＊ indicates that the peptide is optionally continued from a side chain amino group rather than from the α-amino group, Here, X ２ represents KA or AK, and wherein the cell-penetrating amino acid sequence is optionally chemically modified at the C-terminus. The membrane-permeable construct comprises the sequence:

2. the mRNA cargo encodes an antigenic protein or peptide for eliciting an immune response in a subject, and / or the cell is a splenic cell, e.g., a dendritic cell (DC); A membrane-permeable construct for use according to claim 1.

3. The membrane-permeable construct for use according to claim 1 or claim 2, wherein the mRNA encodes an antigenic protein or peptide, such as a viral surface protein, in particular a capsid and / or viral envelope protein, such as a viral glycoprotein.

4. 1. A membrane-permeable construct for transport of a cargo across a lipid membrane and subsequent delivery of the cargo to a cell, the construct comprising a cell-permeable amino acid sequence and a fatty acid chain attached to the N-terminus of the amino acid sequence, wherein the cell-permeable amino acid sequence is 【Chemistry 2】 (where X １ represents Lys(K), Orn(O), Dab, or Dap; where: ＊ indicates that the peptide continues from a side chain amino group rather than from the α-amino group, and Here, X ２ represents KA or AK) The membrane-permeable construct comprises the sequence:

5. X １ The membrane permeable construct or membrane permeable construct for use according to any one of claims 1 to 4, wherein is K, Orn(O) or Dab, in particular Dab.

6. The peptide is ＊ 6. The membrane-permeable construct for use according to any one of claims 1 to 5, wherein the amino acid sequence continues from a side chain amino group rather than from the α-amino group at the position β.

7. X ２ is KA or X ２ The membrane-permeable construct or membrane-permeable construct for use according to any one of claims 1 to 6, wherein is AK.

8. The membrane-permeable construct or membrane-permeable construct for use according to any one of claims 1 to 7, wherein the cell-permeable amino acid sequence comprises one or more additional amino acid substitutions at positions 9 to 21 of SEQ ID NO: 1 or positions 9 to 19 of SEQ ID NO:

2.

9. the fatty acid chain has 16 to 22 carbon atoms, for example, 18 to 22 carbon atoms, and Optionally, the fatty acid chain has 16, 18, 20, or 22 carbon atoms, for example, 18, 20, or 22 carbon atoms, particularly 18 carbon atoms. A membrane-permeable construct for use in a membrane-permeable construct according to any one of claims 1 to 9.

10. The cell-penetrating amino acid sequence is chemically modified at the C-terminus, for example, amidated, and the C-terminus is CONH ２ The membrane-permeable construct or membrane-permeable construct for use according to any one of claims 1 to 9, wherein

11. The cell-penetrating amino acid sequence is 【Chemistry 3】 especially: 【Chemistry 4】 (where, ＊ indicates that the peptide continues from the side chain amino group rather than from the α-amino group. Contains an array of: For example, wherein the membrane permeable construct is C16-(SEQ ID NO:3) (referred to herein as NF419); C18-(SEQ ID NO:3) (referred to herein as NF55); C20-(SEQ ID NO:3) (referred to herein as NF410); C22-(SEQ ID NO:3) (referred to herein as NF411); C16-(SEQ ID NO:4) (referred to herein as NF420); C18-(SEQ ID NO:4) (referred to herein as NF554); C20-(SEQ ID NO:4) (referred to herein as NF412); C22-(SEQ ID NO:4) (referred to herein as NF413); C16-SEQ ID NO:5) (referred to herein as NF422); C18-(SEQ ID NO:5) (referred to herein as NF553); C22-(SEQ ID NO:5) (referred to herein as NF423); C18-(SEQ ID NO:6) (referred to herein as NF559); C18-(SEQ ID NO:7) (referred to herein as NF550); C18-(SEQ ID NO:8) (referred to herein as NF54); C18-(SEQ ID NO:9) (referred to herein as NF430); C20-(SEQ ID NO:9) (referred to herein as NF437); C16-(SEQ ID NO: 10) (referred to herein as NF425); C18-(SEQ ID NO: 10) (referred to herein as NF424); C20-(SEQ ID NO: 10) (referred to herein as NF426); C18-(SEQ ID NO:11) (referred to herein as NF436); or C20—(SEQ ID NO: 11) (referred to herein as NF438) especially: C22-(SEQ ID NO:3) (referred to herein as NF411); C18-(SEQ ID NO:4) (referred to herein as NF554); C18-(SEQ ID NO:5) (referred to herein as NF553); C18-(SEQ ID NO:8) (referred to herein as NF54); C18-(SEQ ID NO: 10) (referred to herein as NF424); C20-(SEQ ID NO: 10) (referred to herein as NF426); or C18-(SEQ ID NO:11) (referred to herein as NF436) 11. The membrane-permeable construct for use according to any one of claims 1 to 10, comprising:

12. The cell-penetrating amino acid sequence is 【Chemistry 5】 especially: 【Chemistry 6】 (where, ＊ indicates that the peptide continues from the side chain amino group rather than from the α-amino group. Contains an array of: For example, wherein the membrane permeable construct is C18-(SEQ ID NO:9) (referred to herein as NF430); C20-(SEQ ID NO:9) (referred to herein as NF437); C16-(SEQ ID NO: 10) (referred to herein as NF425); C18-(SEQ ID NO: 10) (referred to herein as NF424); C20-(SEQ ID NO: 10) (referred to herein as NF426); C18-(SEQ ID NO:11) (referred to herein as NF436); or C20—(SEQ ID NO: 11) (referred to herein as NF438) especially: C18-(SEQ ID NO: 10) (referred to herein as NF424); C20 (SEQ ID NO: 10) (referred to herein as NF426); or C18-(SEQ ID NO:11) (referred to herein as NF436) 11. The membrane-permeable construct according to any one of claims 1 to 10, comprising:

13. A membrane-permeable construct according to any one of claims 4 to 12 for use in the transport of a cargo across a lipid membrane and subsequent delivery of said cargo to a cell.

14. A membrane-permeable construct or membrane-permeable construct for use according to any one of claims 1 to 13, wherein said construct further comprises a cargo covalently attached thereto.

15. A membrane-permeable construct for use according to any one of claims 1 to 13 or a complex comprising a membrane-permeable construct and a cargo that interacts with it non-covalently, for example via ionic interactions.

16. the cargo is mRNA, Optionally, wherein the mRNA encodes an antigenic protein or peptide, such as a viral surface protein, in particular a capsid and / or viral envelope protein, such as a viral glycoprotein. A membrane-permeable construct, membrane-permeable construct, or complex for use according to any one of claims 13 to 15.

17. The membrane permeable construct, membrane permeable construct, or complex for use according to any one of claims 13 to 16, wherein the cell is in vivo and / or the cell is a splenic cell, such as a dendritic cell (DC).

18. A pharmaceutical composition comprising the membrane-permeable construct or complex according to any one of claims 4 to 17 and a pharmaceutically acceptable carrier, and optionally further comprising one or more adjuvants.

19. A membrane permeable construct, membrane permeable construct, or complex for use according to any one of claims 1 to 17, or a pharmaceutical composition according to claim 18, for use in a method for eliciting an immune response in a subject.

Images