Peptide for the delivery of anionic materials

Abstract

The invention provides a peptide or a salt or amide thereof, for use as a cell delivery agent, comprising, or consisting of: (Xaa1)a-(Xaa2)b-LYRLFRKS-(Xaa3)c-(Xaa4)d-(Xaa4)e-NLKPFERHARAC, wherein: a,

Description

FIELD OF THE INVENTION The present invention is directed to an improved peptide for the delivery of anionic materials, such as therapeutic agents, across a biological barrier and methods of use thereof. BACKGROUND The complex design and difficulty in predicting functionality of alpha helical, amphipathic, cellpenetrating peptides (CPP) for the delivery of anionic cargo can be challenging. Artificial intelligence (Al) has emerged as a promising alternative which can augment the design process, for example by determining physiochemical characteristics, secondary structure, stability, disorder and flexibility, as well as predicting in vivo behaviour such as toxicity and peptidase degradation. Other more recent tools utilise supervised machine learning (ML) to predict the penetrative ability of an amino acid (AA) sequence, but this can also be difficult as the cell membrane is constantly in a dynamic state of flux. For example, Tran et al. trained a ML model with peptide sequences from 4 databases up to 52AA. With removal of non-natural amino acids, 16,648 peptides were examined using a recurrent neural network (RNN) with two connected gated recurrent units (GRUs) that included positive data and a permeability tracker with 256 hidden variables. The upper limit was set to 20 AA, and the top 10 were Identified from their mechanistic score. The 10 peptides were then prioritised by molecular dynamics with Pep-MD (YRGWHCRGITKNGIIFDIKW) resulting in successful membrane penetration.1 Specific amino acids (AA) have a role to play in CPPs. For the delivery of nucleic acids, cationicity is required at a physiological pH; therefore lysine or arginine are key components. Of the two, Wender et al. showed that the guanidium group found in arginine formed bidentate bonds with negatively charged groups such as phosphates or carboxylates, which resulted in cellular entry as a result of the membrane potential.2 3 However, hydrophobic AAs such as Tryptophan (W) have been shown to play a key role in membrane interactions aiding cellular entry. With the largest aromatic side-chain compared to phenylalanine (F) and Tyrosine (Y), W locates in the water-bilayer of membranes and can accept or donate hydrogen bonds.4 Of interest is the research by Kiessling and Diehl (2021) who showed that aromatic AA can form hydrophobic C-H-tt interactions which are critical in glycan / protein interactions.5 6 Given that proteoglycans are found in the extracellular matrix and the outer regions of cells, and that W is typically present at a higher frequency than other amino acids, within 0.4 nm between proteins and non-covalent glycans, it can be concluded that it is key in membrane dynamics.7 Chakravarty also demonstrated anion-n interactions between W residues and phospholipid head groups in SERCA1, a membrane protein SERCA1.8 Furthermore, when Wis included within an oligoarginine CPP, increased cellular uptake was demonstrated in the present of glycosaminoglycans (GAG).9 Condensation of nucleic acids via arginine, and improved cellular entry via size (<200 nm), charge (cationic) and the presence of an aromatic AA, are only part of the journey. If the cellular entry is energy-dependent, then escaping the endosome becomes a key consideration. Histidine-rich peptides are usually endosomolytic in nature and can facilitate endosomal escape through either the proton sponge or ‘flip-flop’ effects, according to the number of histidine residues or the arrangement of the residues in a peptide.10 With a neutral charge at a pH of 8, histidine then becomes protonated at a pH of 6 to give a +1 charge. Therefore, having histidine within a CPP can confer a conditional charge within the endosome that facilitates the escape of the nucleic acid cargo.11 In addition, however, it is also critical that the electrostatic forces that bind the nucleic acid cargo to the CPP are broken intracellularly to ensure efficient release. Many studies have shown that Cysteine (C) has a role to play in this. The intracellular concentration of glutathione (GSH) reduces the disulphide bonds formed by cysteine residues.12 Within the CPP, the presence of C can facilitate the presence of multimers and significantly improves the translational efficiency of the nucleic acid. For example, Lo et al. showed that when histidine was added to the TAT peptide (TAT-H10), gene expression increased 7000-fold. Then when C was added to give CH5TATH5C, there was a further 1000-fold upregulation in expression overTAT-Hio.13 In this invention, peptides have been rationally designed to create a novel cell-penetrating peptide which can bind and package nucleic acids into nanoparticles (NPs) for delivery into cells, resulting in a homogenous population of NPs with impressive transfection, while limiting the cost of production. The NPs are extremely safe and cost-effective. STATEMENTS OF THE INVENTION 1. According to a first general aspect of the invention, there is provided a peptide, or a salt or amide thereof, comprising, or consisting of: (Xaa1)a-(Xaa2)b-LYRLFRKS-(Xaa3)c-(Xaa4)d-(Xaa4)e-NLKPFERHARAC, in which a is 0 or 1 and, if a is 1, Xaa1 is selected from an Ala residue, a Vai residue or a Gly residue; in which b is independently 0 or 1 and, if b is 1, Xaa2 is selected from an Ala residue, a Vai residue or a Gly residue; in which c is independently 0 or 1 and, if c is 1, then Xaa3 is an aromatic residue selected from a His or a Trp residue; in which d is 1 and Xaa4 is an aromatic residue selected from a Trp ora His residue; in which e is 1 and Xaa5 is a His or a Trp residue. The peptide comprises a) A consensus sequence of two parts ‘LYRLFRKS’ and ‘NLKPFER’ that must be split by other amino acids for functionality as a cell-penetrating peptide. b) Inclusion of a minimum of two Lysine residues; c) Inclusion of a minimum of four Arginine residues; d) Inclusion of a minimum of four Alanines to increase the hydrophobicity and to readily pass across the cell membrane; e) Introduction of a minimum of three aromatic residues from tryptophan or phenylalanine to improve hydrophobic interaction with cell membranes; f) Introduction of a minimum of two Histidines to enhance conditional endosomal escape; g) One Cysteine residue positioned at the C-terminus to enhance stability and cargo release once inside cell; and h) One proline to enhance cellular entry and safety. The peptide comprises, or consists of, no more than 30 amino acids, such as 25 amino acids. Ideally, the peptide comprises b) 5%-25%, c) 5%-25%, d)15%-30% e)10%-30%, f) 5%-20%. Optionally, Xaa3 is a Trp residue; and Xaa4 is a His residue. Alternatively or additionally, c is 0 and Xaa3 is absent. Optionally, the peptide comprises AALYRLFRKSWHNLKPFERHARAC, or a salt or amide thereof. According to a second aspect of the invention, there is provided a cell delivery agent comprising the peptide of the first aspect of the invention in association with an anionic cargo. Optionally, the anionic cargo is an mRNA and the cell delivery agent has a polydispersity index of 0.025 to 0.35; and / or a mean hydrodynamic size (nm) of 50 to 450 nm; and / or a charge of 17.5 to 20 mV. According to a third aspect of the invention, there is provided a peptide of the first aspect of the invention for use as a cell delivery agent. According to a fourth aspect of the invention, there is provided use of a peptide of the first aspect of the invention for the delivery of an anionic cargo to a cell or subject in need thereof. According to a further aspect of the invention, there is provided a peptide of the first aspect of the invention for use in inducing an immune response in a subject in need thereof, or a method for inducing an immune response in a subject comprising the administration of the claimed formulation to a subject in need thereof. According to a further aspect of the invention, there is provided a peptide of the first aspect of the invention for use in gene therapy in a subject in need thereof. According to a further aspect of the invention, there is provided a peptide of the first aspect of the invention for use in the treatment and / or prophylaxis of an infection, cancer, wounds in a subject in need thereof, or a method for the treatment and / or prophylaxis of an infection, cancer, wounds comprising the administration of the claimed formulation to a subject in need thereof. DETAILED DESCRIPTION Rational peptide design was carried out by determining consensus sequences (C1 &C2) in proteins that readily entered cells. These peptides C1 and C2 were then screened against other sequences, and C3 was found as a hit. However, C3 is not a Cell-Penetrating Peptide, so a series of modifications were added to improve functionality. Modifications were based on each terminus and in the middle of C3. The sequences of the novel amphipathic peptides (Table 1), characteristics (Table 2) and non-covalent bonds (Table 3) are shown. Table 1: Sequences of novel amphipathic peptides and rationale Peptide ID Sequence Rationale Peptide C1 LYKLFK (SEQ ID NO: 1) A 60% partial hit with human plexin B2 isoform (Figure 1). Peptide C2 NLKPFER (SEQ ID NO: 2) A 71 %-100% hit with a range of dynamin binding proteins (Figure 2). Peptide C3 LYRLFRKSNLKPFER (SEQ ID NO: 3) Residues 452-466 in Chain A of the Spik Glycoprotein (Figure 3). Peptide C4 AALYRLFRKSNLKPFER (SEQ ID NO: 4) Two Alanines were added to the N-Terminus to assist with membrane bindin and transport (Figure 4 &5). Peptide C5 LYRLFRKSWHNLKPFER (SEQ ID NO: 5) A tryptophan was added to improve membrane binding and histidine for endosomal escape (Figure 4 &5). Peptide C6 AALYRLFRKSWHNLKPFER (SEQ ID NO: 6) Modifications of C4 and C5 were combine into C6 (Figure 4 &5). Peptide C7 LYRLFRKSNLKPFERHARAC (SEQ ID NO: 7) The HARAC sequence was added to give an extra R for nucleic acid binding, H for endosome escape, interspaced A for membrane transport and a C for dimer formation (Figure 4 &5). Peptide C8 LYRLFRKSWHNLKPFERHARAC (SEQ ID NO: 8) Modifications of C7 and C5 were added t( C8 (Figure 4 &5). Peptide C9 (HAWC) AALYRLFRKSWHNLKPFERHARAC (SEQ ID NO: 9) Modifications of C4, C5 and C7 were combined to give C9, the optimal HAWC peptide (Figure 4 &5). Peptide C9.1 AALYRLFRKSHWHNLKPFERHARAC (SEQ ID NO: 10) An additional H to enhance endosomal escape (Figure 4 &5). Peptide C9.2 AALYRLFRKSWHNLKPFERHARAS (SEQ ID NO: 11) The Cysteine replaced with Serine to produce monomers (Figure 4 &5). Peptide C9.3 AALYRLFRKSWHNLRPFERHARAC (SEQ ID NO: 12) The K in position 15 is replaced with R (Figure 4 &5). Peptide C9.4 AALYRLFRRSWHNLRPFERHARAC (SEQ ID NO: 13) In addition to 9.3, the K in position 9 is replaced with R (Figure 4 &5). CPP design can involve systematic alteration of the physiochemical properties of the sequence.12 Note that the PDI of HAWC is very homogenous for non-covalent nanoparticles. Instability index is a measure of how stable the peptide is in vitro in relation to its primary structure and is calculated by assigning a weight value of instability to all 400 dipeptides (DIWV) conformations.49 50 Instability index is calculated using the formula: Instability Index = 10 v z . 1=1 Where L is the length of the peptide, DIWV is the dipeptide instability weight value in position i Ideally, an instability index of <40 ensures the peptides will be stable at ambient conditions.2 However, as can be found in Table 2, only those peptides with an instability index of >45 and classified as unstable were functional (bolded). This was unexpected. Table 2: The physiochemical characteristics with transfection efficiency of the 13 peptides. Peptide Peptide sequence Instability Stability % Transfection Polydispersity Mean Charge (mV) ID Index NCTC-929 Index hydrodynamic size (nm) C1 LYRLFRKS -12.39 stable 0.6±0.21 0.16 ±0.10 164.33 ± 15.33 -17.87 ±4.84 C2 NLKPFER 13.19 stable 0.5+0.03 0.57 + 0.05 611.10 ±175.31 0.57 ±0.05 C3 LYRLFRKSNLKPFER 0.21 stable 0.7±0.32 0.31 ±0.14 1128.83 ±371.85 3.59 ±1.29 C4 AALYRLFRKSNLKPFER 1.36 stable 0.6±0.21 0.20 ±0.12 1809.33 ±438.50 0.78± 0.67 C5 LYRLFRKSWHNLKPFER 29.22 stable 0.5±0.03 0.13 ±0.01 770.33 ±140.47 6.73 ±1.09 C6 AALYRLFRKSWHNLKPFER 27.20 stable 0.7±0.32 0.31 ±0.10 2405.33 ±405.45 6.73 ±1.09 C7 LYRLFRKSNLKPFERHARAC 34.26 stable 53.5±3.86 0.20 ±0.02 57.88 ± 12.81 29.22 ±2.05 C8 lyrlfrkswhnlkpferharac 53.58 unstable 11.3±4.23 0.30 ±0.13 506.67± 151.17 17.47 ±6.68 C9 AALYRLFRKSWHNLKPFERHARAC 49.95 unstable 74.9±7.99 0.06 ± 0.001 103.17±11.13 18.93 ±1.51 HAWC C9.1 AALYRLFRKSHWHNLKPFERHARAC 47.20 unstable 40.2±0.02 0.20 ±0.02 91.2 ±0.58 19.12 ±1.52 C9.2 AALYRLFRKSWHNLKPFERHARAS 31.64 stable 0.6±0.34 0.07 ±0.02 183.50 ±41.87 16.39 ±1.10 C9.3 AALYRLFRKSWHNLRPFERHARAC 72.68 unstable 62.8±26.09 0.24 ±0.08 276.80±43.46 19.55 ±0.33 C9.4 AALYRLFRRSWHNLRPFERHARAC 114.85 unstable 67.5±15.21 0.24 ± 0.02 458.06 ±48.13 17.47 ±1.34 Table 3: The theoretical non-covalent bonds between Monomers and Dimers (Intra and Inter) of the 13 peptides. Peptide ID Peptide sequence Monomer Intrachain Dimer Intrachain Dimer Interchain H Bonds Van der Waals n-n H Bonds Van der Waals rr-TT H Bonds Van der Waals TT—IT Disulphide tt-H Ionic C1 LYRLFRKS 6 10 0 10 4 0 0 4 2 0 0 0 C2 NLKPFER 1 1 0 0 0 0 3 8 1 0 0 0 C3 LYRLFRKSNLKPFER 6 10 0 14 16 2 0 3 1 0 0 0 C4 AALYRLFRKSNLKPFER 11 8 0 14 14 2 2 1 0 0 0 2 C5 LYRLFRKSWHNLKPFER 8 9 1 24 18 4 0 3 1 0 0 1 C6 AALYRLFRKSWHNLKPFER 13 11 0 24 23 0 0 1 4 0 0 0 C7 LYRLFRKSNLKPFERHARAC 16 16 0 36 31 2 0 8 2 1 0 0 C8 LYRLFRKSWHNLKPFERHARAC 20 12 0 48 29 3 0 6 0 1 0 0 C9 HAWC AALYRLFRKSWHNLKPFERHARAC 25 13 0 46 33 2 0 13 5 1 0 0 C9.1 AALYRLFRKSHWHNLKPFERHARAC 24 12 1 46 30 0 1 7 7 1 0 0 C9.2 AALYRLFRKSWHNLKPFERHARAS 20 13 1 47 32 4 0 9 3 0 0 0 C9.3 AALYRLFRKSWHNLRPFERHARAC 21 21 0 43 44 4 0 6 1 1 1 0 C9.4 AALYRLFRRSWHNLRPFERHARAC 22 21 0 43 34 4 0 9 1 1 1 0 ANIONIC CARGO In a general context, the anionic cargo may be selected from a nucleic acid or small molecule agent such as a phosphonate drug or phosphate drug, preferably a bisphosphonate, a diphosphate, triphosphate drug. For example, the anionic cargo may be an organophosphate with a molecular weight between 0.15 and 3 kDa. Optionally, the anionic cargo may be an alkali metal bisphosphate, diphosphate, or triphosphate, preferably an alkaline earth metal bisphosphate, diphosphate, or triphosphate. In one aspect, the bisphosphonate may be etidronate, clodronate, tiludronate, pamidronate, neridronate, olpadronate, alendronate, ibandronate, risedronate, zoledronate, or any derivative of these compounds. In another aspect, the diphosphate may be based on a nucleotide ora nucleoside such as didanosine, vidarabine, emtricitabine, lamivudine, zalcitabine, abacavir, acyclovir, entecavir, stavudine, telbivudine, zidovudine, idoxuridine, trifluridine, ganciclovir, valganciclovir, ciclovir, aciclovir, azacitidine, decitabine, zebularine, cytarabine, gemcitabine, troxacitibine, CNDAC (2-C-cyano-2'-deoxy-1-p-D-arabino-pentofuranosylcytosine), fludarabine, cladribine, clofarabine, pentostatin, forodesine, azidothymidine, edoxudine, and / or any derivative of these compounds. For example, the diphosphate may be selected from one or more of the following isopentenylpyrophosphate (IPP), (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), (E)-5-hydroxy-4-methylpent-3-enyl pyrophosphate (HDMAPP), Dimethylallyl pyrophosphate (DMAPP), ethyl pyrophosphate (EPP), and associated metabolites such as IPP’s triphosphoric acid 1-adenosin-5'-yl ester 3-(3-methylbut-3-enyl) ester (Apppl). In another aspect, the triphosphate may be based on a nucleotide or nucleoside such as didanosine, vidarabine, emtricitabine, lamivudine, zalcitabine, abacavir, acyclovir, entecavir, stavudine, telbivudine, zidovudine, idoxuridine, trifluridine, ganciclovir, valganciclovir, ciclovir, aciclovir, azacitidine, decitabine, zebularine, cytarabine, gemcitabine, troxacitibine, CNDAC (2'-C- cyano-2'-deoxy-1-p-D-arabino-pentofuranosylcytosine), fludarabine, cladribine, clofarabine, pentostatin, forodesine, azidothymidine, edoxudine, or any derivative of these compounds. For example, the triphosphate may be based on a diphosphate or pyrophosphate such as isopentenyl pyrophosphate (IPP), (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), (E)- 5-hydroxy-4-methylpent-3-enyl pyrophosphate (HDMAPP), Dimethylallyl pyrophosphate (DMAPP), ethyl pyrophosphate (EPP), and associated metabolites such as IPP’s triphosphoric acid 1-adenosin- 5'-yl ester 3-(3-methylbut-3-enyl) ester (Apppl). In another aspect, the anionic cargo is a nucleic acid selected from DNA, RNA, mRNA, siRNA, shRNA, saRNA, miRNA, ncRNA, tRNA, rRNA, snRNA, snoRNA, piRNA, IncRNA, a plasmid vector, or a nucleic acid aptamer. USES It will be understood that the claimed formulation may be used as a cell delivery agent. Additionally, the claimed formulation may be for use in inducing an immune response in a subject in need thereof. Still additionally, the claimed formulation may be for use in gene therapy in a subject in need thereof. Still further additionally, the claimed formulation may be for use in the treatment and / or prophylaxis of an infection or cancer in a subject in need thereof. Finally, the claimed formulation could be used to deliver therapies from polymeric medical devices. FIGURE LEGENDS Figure 1: Characterisation of consensus sequence C1. A) NCBI blasts of the sequence showing presence of the sequence in PSD4 protein. The sequence was searched on https: / / blast.ncbi.nlm.nih.qov / Blast.cqi?PAGE=Proteins under the blastp setting; B) Structure of the PSDM4 protein using AlphaFold server 3 and PyMOL; C) DLS spectra of complexes formulated with peptide C1 and GFP mRNA cargo (SEQ ID NO:14). Complexes were formulated at room temperature at a N:P ratio of 10 using 1 pg of mRNA in a final volume of 50 pL. Particle size was measured using a Malvern Zetasizer Nano ZS. Results show N=3; D) Transfection images of C1 complexes in NCTC-929 cells. Transfections were carried out in 96-well plates using a seeding density of 20k cells / well for 5 h in serum-free media. Images were taken 24 h after transfection. Figure 2: Characterisation of consensus sequence C2. A) NCBI blasts of the sequence showing presence of the sequence in DNMB protein. The sequence was searched on https: / / blast.ncbi. nlm.nih.gov / Blast.cqi?PAGE=Proteins under the blastp setting; B) Structure of the DNMBP protein using AlphaFold server 3 and PyMOL; C) DLS spectra of complexes formulated with peptide C2 and GFP mRNA cargo. Complexes were formulated at room temperature at a N:P ratio of 10 using 1 pg of mRNA in a final volume of 50 pL. Particle size was measured using a Malvern Zetasizer Nano ZS. Results show N=3; D) Transfection images of C2 complexes in NCTC-929 cells. Transfections were carried out in 96-well plates using a seeding density of 20k cells / well for 5 h in serum-free media. Images were taken 24 h after transfection. Figure 3: Characterisation of consensus sequence C3, present within Chain A of the Spike Glycoprotein of SARS-CoV-2. A) Details of the accession and PubMed details obtained from https: / / www.ncbi.nlm.nih.gov / protein; B) Structure of the spike glycoprotein using AlphaFold servers and PyMOL; C) DLS spectra of complexes formulated with peptide C3 and GFP mRNA cargo. Complexes were formulated at room temperature at an N:P ratio of 10 using 1 pg of mRNA in a final volume of 50 pL. Particle size was measured using a Malvern Zetasizer Nano ZS. Results show N=3; D) Transfection images of C3 complexes in NCTC-929 cells. Transfections were carried out in 96-well plates using a seeding density of 20k cells / well for 5 h in serum-free media. Images were taken 24 h after transfection. Figure 4: Peptide nanoparticle formulations with mRNA. A) Structure of the mRNA GFP molecule used to formulate NPs; DLS spectra of nanoparticles formulated with peptides B) C4; C) C5; D) C6; E) C7; F) C8; G) C9; H) C9.1; I) C9.2; J) C9.3 and K) C9.4. Nanoparticles were formulated at a N:P ratio of 10 using 1 pg of mRNA in a final volume of 50 pL. Mean hydrodynamic size, PDI and zeta potential were measured on the Malvern Zetasizer Nano ZS. Results show N=3. Figure 5: Peptide nanoparticle transfections in NCTC-929 mouse fibroblast cells. A) Representative fluorescence microscopy images of cells 24 h after transfection using peptide NPs as indicated; B) Quantitative analysis of transfection efficiency using flow cytometry. Cells were trypsinised and resuspended in FACS buffer, followed by analysis on an Accuri C6 plus flow cytometer; C) Cell viability 24 h following transfections determined by Alamar Blue assay. Results show N=3 + / - SEM. Figure 6: Characterisation of HAWC / mRNA nanoparticles. A) DLS spectra of HAWC / mRNA nanoparticles formulated at a range of N:P ratios. NPs were formulated at room temperature using 1 pg of mRNA in a final volume of 50 pL. Particle size was measured using a Malvern Zetasizer Nano ZS. Results show N=3; B) Mean hydrodynamic size and zeta potential of HAWC / mRNA nanoparticle obtained using a Malvern Zetasizer Nano ZS. Results show N=3 + / - SEM; C) Polydispersity index of the HAWC / mRNA nanoparticles from B; D) Encapsulation efficiency of HAWC / mRNA nanoparticles. Following formulation, free mRNA was quantified using the RiboGreen assay to calculate encapsulation; E) Ion exchange chromatography of HAWC / mRNA nanoparticles. NPs were passed through an anionic Sephadex column to elute free mRNA which was quantified using a Nanodrop spectrophotometer; F) TEM images of HAWC / mRNA nanoparticles formulated at N:P 10 and loaded onto a carbon-coated copper 400 mesh grid. Images were captured on a JEM-1400Plus transmission electron microscope; G) Representative fluorescence microscopy images of cells 24 h post transfection with HAWC / mRNA NPs at a range of N:P ratios; Analysis of H) NCTC-929 and I) DC2.4 cells 24 h following transfection with HAWC / mRNA NPs showing (i) Transfection efficiency using Flow cytometry and (ii) Cell viability through Alamar blue assay 24 h post transfection; All results show N=3 + / - SEM. Figure 7: Characterisation of HAWC / miRNA nanoparticles. A) Structure of the microRNA molecules used to formulate nanoparticle; B) DLS spectra of HAWC / miRNA nanoparticles formulated at a range of N:P ratios using a blend of miR-31 (SEQ ID NO: 15) and miR-132 (SEQ ID NO: 16). NPs were formulated at room temperature using 0.5 pg of miRNA in a final volume of 50 pL. Particle size was measured using a Malvern Zetasizer Nano ZS. Results show N=3; C) Encapsulation efficiency of HAWC / miRNA nanoparticles. Following formulation, free miRNA was quantified using the RiboGreen assay to calculate encapsulation; D) Ion exchange Chromatography of HAWC / miRNA nanoparticles. NPs were passed through an anionic Sephadex column to elute free miRNA which was quantified using a Nanodrop spectrophotometer; E) TEM images of HAWC / miRNA nanoparticles were formulated at N:P 12 and loaded onto a carbon-coated copper 400 mesh grid. Images were captured on a JEM-1400Plus transmission electron microscope; F) Cell viability of NCTC-929 cells measured using Alamar blue assay 24 h post transfection with HAWC / miRNA NPs at a range of N:P ratios; RT-PCR results showing fold increase in the expression of G) miR-31 and H) miR-132 in NCTC-929 cells 24 h post transfection; All results show N=3 + / - SEM. Figure 8: Characterisation of HAWC / pDNA nanoparticles. A) Plasmid map of the pDNA used to formulate nanoparticles; B) DLS spectra of HAWC / pDNA nanoparticles formulated at N:P 10. NPs were formulated at room temperature using 1 pg of pDNA in a final volume of 50 pL. Particle size was measured using a Malvern Zetasizer Nano ZS. Results show N=3; C) Encapsulation efficiency of HAWC / pDNA nanoparticles. Following formulation, free pDNA was quantified using the PicoGreen assay to calculate encapsulation; D) Ion exchange Chromatography of HAWC / pDNA nanoparticles. NPs were passed through an anionic Sephadex column to elute free pDNA which was quantified using a Nanodrop spectrophotometer; E) TEM images of HAWC / pDNA nanoparticles formulated at N:P 10 and loaded onto a carbon-coated copper 400 mesh grid. Images were captured on a JEM-1400Plus transmission electron microscope. Figure 9: Mechanism of cellular entry for HAWC NPs. A) Schematic diagram of the HAWC peptide mechanism of cell entry; B) Representative fluorescence microscopy images of NCTC-929 cells 24 h following transfection with HAWC / mRNA NPs in the presence of different pathway blockers or vehicle control as indicated; C) Quantitative analysis of transfection efficiency in the presence of pathway blockers using flow cytometry. Cells were trypsinised and resuspended in FACS buffer, followed by analysis on an Accuri C6 plus flow cytometer; D) Cell viability of NCTC-929 cells 24 h following transfection with HAWC / mRNA NPs using propidium iodide staining and analysis using flow cytometry. MATERIALS AND METHODS GENERATION OF PEPTIDES All peptides were produced commercially by Shanghai RoyoBiotech Co. Ltd (China) and supplied as a lyophilised powder which required reconstitution before use. Peptides were supplied in the acetate salt form and were of >95% purity. Lyophilised peptides were reconstituted in Ultrapure DNAse / RNAse free water (Invitrogen, UK) and stored in aliquots at -80°C. GENERATION OF NUCLEIC ACID CARGO Messenger RNA (mRNA) An in vitro transcription (IVT) plasmid encoding green fluorescent protein (GFP) was procured from Vector builder Inc. (USA). mRNA was synthesized using an HiScribe® T7 High Yield RNA Synthesis Kit (New England Biolabs, UK) following the manufacturer’s protocol. The synthesized RNA was purified using a Monarch® RNA Cleanup Kit (New England Biolabs, UK) and quantified using a Nanodrop spectrophotometer (Thermo Scientific, MA, USA) at 260 nm. The IVT mRNA was stored at -80°C until further use. Plasmid DNA (pDNA) pEF-GFP was purchased from Addgene (USA). Plasmids were propagated in MAX Efficiency® DH5a™ Competent Cells (Life Technologies, UK) and purified using PureLink®HiPure Plasmid Filter Maxiprep Kit (Life Technologies, UK). The purified plasmid was then quantified using the Nanodrop spectrophotometer at 260 nm and stored in -20°C until further use. Micro RNA (miRNA) miR 132 and miR 31 mimics were procured commercially from Horizon Discovery Ltd (UK). The miRs were designed to mimic the endogenous miRNA duplex resulting from Dicer processing and are delivered as lyophilised powder. Lyophilised miRNAs were reconstituted with Ultrapure DNAse / RNAse free water to a concentration of 1 mg / mL and stored at -20°C until further use. Cell Lines NCTC-929 murine fibroblasts cells (Merck, Germany) were maintained as monolayers in Dulbecco's Modified Eagle's Medium (DMEM) (Invitrogen, UK), supplemented with 10% foetal calf serum (FCS). Cells were passaged after reaching 80% confluency and were not used for experimental protocols beyond a passage number of 10. Cells were maintained in an incubator at 37°C with 5% CO2 atmosphere and subjected to mycoplasma testing routinely. All cell lines were authenticated by short tandem repeat (STR) profiling carried out by the suppliers. N:P Ratio Calculation for Peptide / Nucleic Acid Nanoparticles N:P ratio is widely used to describe the contents of peptide- / protein-based nucleic acid nanoparticles. It is defined as the molar ratio of positively-charged nitrogen atoms in the amino acids of the peptide / protein to the negatively-charged phosphates within the nucleic acid backbone, or more simply the mass of peptide required to neutralise 1 pg of Nucleic Acid (NA). N:P ratio can be calculated as follows: N:P ratio= Mpeptide MNucleic acid Cn:P where Mpeptide is the mass of the peptide in the nanoparticle, MNucieicacid is the mass of the NA cargo in the nanoparticle and Cn p is the N:P constant. The N:P constant is the ratio of the positive charge density of the amino acid chain to the negative charge density of the NA, where charge density can be calculated as the net charge divided by the molecular mass. Based on the presence of the positively-charged amino acids and the knowledge that the mass and charge of the bases in the NA backbone are constant, the N:P constant can be calculated with only the mass and the charge of the peptide / protein as variables. Cnp is calculated as follows: QNucleic acid MWpeptide Cn :P = —------rrcr;-------- QPeptide MVVNucleic acid Where QNucieicacid is the charge of the nucleic acid molecule, Qpeptideis the net charge of the peptide molecule, MWNucieicacid is the average molecular weight of the nucleic acid molecule, and MWpeptide is the molecular mass of peptide. This can be simplified as following for mRNA cargo and HAWC peptide as: No. of strands in mRNA x MW hawc 1 x 2971.45 HAWC Cnp—-—,.——-------= —z—-r—-— = 1.7 Av MW 1 pg mRNA x Charge hawc 340x5 Formulation of Peptide / mRNA GFP, pGFP or miR Nanoparticles Nucleic acid cargo was complexed by peptide using a range of N:P ratios via electrostatic interactions. Depending upon N:P ratio, an appropriate quantity of peptide was added to 1 pg of nucleic acid cargo in Ultrapure water to a final volume of 50 pL. Peptide nanoparticle formulations are presented in Table 4. Table 4 Nanoparticles formed at N:P ratio 10 for each peptide with mRNA. Peptide Volume of mRNA (pL) [1mg / mL] Mass of peptide for N:P 1 (pg) Volume of peptide forN:P 10 (pL) [1mg / mL] Volume H2O (pl) to give 50 pl Total C1 1 1.06 10.6 38.4 C2 1 2.66 26.6 22.4 C3 1 1.45 14.5 34.5 C4 1 1.55 15.5 33.5 C5 1 1.64 16.4 32.6 C6 1 1.75 17.5 31.5 C7 1 1.47 14.7 34.3 C8 1 1.63 16.3 32.7 C9 (HAWC) 1 1.71 17.1 31.9 C9.1 1 1.76 17.6 31.4 C9.2 1 1.67 16.7 32.3 C9.3 1 1.73 17.3 31.7 C9.4 1 1.75 17.5 31.5 Nanoparticle Size Measurement using Malvern Zetasizer Peptide complexes were prepared at a range of N:P ratios. A Malvern Zetasizer Nano ZS instrument with DLS software (Malvern Instruments, UK) was used to measure the mean hydrodynamic particle size of nanoparticles. A volume of 50 pL was used in a disposable microcuvette to measure the mean size by intensity of nanoparticles formed by Dynamic Light Scattering (DLS) at 25°C. Results were reported as mean ± SEM, and all measurements were performed in triplicate. Determination of Nanoparticle Zeta Potential using Malvern Zetasizer Following size measurement, 50 pL of the nanoparticles sample was subsequently made up to 1000 pL with Ultrapure water and added to a folded capillary zeta cell (Malvern Instruments, UK). Zeta potential was measured by Laser Doppler Velocimetry using a Malvern Zetasizer Nano ZS instrument at 25°C. Results were reported as mean ± SEM and measured in triplicate. Transmission Electron Microscopy (TEM) Peptide-nucleic acid complexes were formulated at a concentration of 0.1 mg / mL. Nanoparticles were loaded onto a carbon-coated copper 400 mesh grid (TAAB Laboratories, UK) and allowed to dry overnight. Following drying, the samples were stained with 5% uranyl acetate in methanol at room temperature for 5 min and allowed to dry again overnight. Nanoparticles were imaged using a JEM-1400Plus Transmission Electron Microscope (Joel, USA) at voltage of 80 kV. Ion Exchange Chromatography 10 mL of 1M NaCI was added to 0.5 g of SP-Sephadex (Sigma-Aldrich, SPC25120, GER), which was incubated overnight at room temperature (1g requires >7 mL NaCI for swelling volume). To remove residual ionic solvent, the supernatant was discarded, and the resin was rinsed three times in 10 mL ultrapure (DNase / RNase free) water. A frit column was filled under pressure with 2 mL of washed resin. 20 pL of free cargo solution or Peptide: cargo complex at >20 mg / mL was loaded onto the column and eluted with 3 mL of ultrapure (DNase / RNase free) H2O. The eluted fractions were collected in 0.5 mL centrifuge tubes and analysed using a Nanodrop spectrophotometer (Thermo Scientific, MA, USA) at 260 nm. Complexation Efficiency Peptide complexes were prepared at a range of N:P ratios. Quant-iT™RiboGreen® Reagent or PicoGreen® Reagent (Life Technologies, UK) was diluted 1:200 in TAE buffer, and 50 pL was added to each sample. Sample fluorescence was analysed by excitation at 480 nm, and the fluorescence emission intensity measured at 520 nm using a FLUOstar Omega Multimode Plate Reader (BMG Labtech, UK). Fluorescence intensity of a naked cargo control was taken as 100% fluorescence and 0% complexed, and any fluorescence detected from samples was taken to be un-complexed. The percentage of un-complexed nucleic acid in each sample was then used to calculate the percentage of complexed mRNA. Transfection with Peptide Delivering Nucleic Acid Cargo Transfections with HAWC / NA were carried out in 96-well tissue culture plates. NCTC-929s were seeded at a density of 2.0x104 cells per well and allowed to adhere overnight. Medium was replaced with OptiMEM (Invitrogen, UK) 2 h prior to transfection, and the cells returned to the incubator. Nanoparticles were prepared at a range of N:P ratios before being added to the appropriate wells (1 pg / 25pL per well). Cells were incubated with the nanoparticles for 5 h, before being replaced with complete media. Untreated cells, and cells treated with mRNA, only served as negative controls. Endosomal Escape and Cellular Uptake Pathway Studies Cells were treated with cellular uptake pathway inhibitors including Dynasore (an inhibitor of clathrln-mediated endocytosis), genistein (an inhibitor of caveolae mediated inhibition) and Ethylisopropyl amiloride (EIPA) (an inhibitor of micropinocytosis). For cellular uptake studies, cells were pre-treated with 100 pM of Dynasore, Genistein or EIPA in OptiMEM for 2 h before transfection. Fluorescence Microscopy To facilitate qualitative analysis of GFP expression correlating to transfection efficiency, cells were visualised and imaged 24 h following transfection under fluorescent light using an EVOS FL Cell Imaging System (Life Technologies). Flow Cytometry Analysis of Transfection Efficiency Transfected cells were washed with phosphate buffered saline (PBS) and 2x trypsin used to detach cells at 37°C in 5% CO2 atmosphere. Complete media was then added, and the cells were centrifuged at 2300 g for 10 min. An Accuri C6 Plus (BD Bioscience, UK) was used for the detection of green fluorescent protein (GFP) expressing cells using Flow cytometry buffer (eBioscience™, thermofisher, UK). 10 pL of Propidium iodide at a concentration of 0.01 mg / mL was added to the cells to assess cell viability. Results are reported as mean ± SEM, n=3. Cell Viability The in vitro cytotoxicity of peptide nucleic acid NPs cells was analysed using the alamarBlue™ Cell Viability Reagent, 24 h following transfection, Alamar Blue was added to the cells to a final concentration of 10% and incubated for 2 h. The absorbance of the samples was then measured at 570 nm using a FLUOstar Omega microplate reader (BMG Labtech, Germany). Cell viability was calculated as a relative percentage to the untreated control. Real-Time PCR of for miRNA Quantification RNA was extracted by phase separation using a miRNeasy kit (Qiagen, UK) according to the manufacturer's protocols. Reverse transcription reactions were conducted in polypropylene tubes according to manufacturer’s protocol (TaqMan Small RNA assay, Invitrogen, UK). Reverse transcription master mix was prepared using 0.15 pL 100mM dNTPs, 1 pL Multiscribe Reverse Transcriptase, 1.5 pL 10X Reverse transcription buffer, 0.19 pL RNase Inhibitor, 4.16 pL Nuclease-free H2O to give a final total volume of 7 pL / reaction. 5 pL of RNA sample (containing 10 ng of RNA) and 3 pL of 5XRT primer (miR-31, miR-132 or U6) were then added to the relevant tube to give a final reaction volume of 15 pL. Reverse transcription was conducted in a thermal cycler. The following cycling parameters were used: 16°C for 30 min, 42°C for 30 min, 85°C for 5 min, hold at 4°C. qRT-PCR was conducted in transparent 96-well plates (Roche, UK). Reactions were prepared for each set of probes (miR-31, miR-132 and U6) according to manufacturer’s protocols (TaqMan Small RNA assay, Invitrogen, UK). Briefly, 0.5 pL of TaqMan Small RNA assay (20X); 5 pL of TaqMan Universal PCR Mastermix Mix II (2X), no UNG; 3.84 pL Nuclease-free H2O; 0.67 pL cDNA (from RT reaction) were prepared to give a total reaction volume of 10 pL / well. qRT-PCR was conducted using a Lightcycler 480 II (Roche, UK) using the following cycling parameters: 95°C for 10 min, then 40 X cycles of 95°C for 15 s, and 60°C for 60 s. The Ct values generated were used to quantify miRNA expression relative to U6 using the AACt method. Results are reported as fold change relative to control. Modelling of peptides: The peptides were modelled to check the monomeric and dimeric structures using AlphaFold 3 model which is a Google DeepMind and Isomorphic Labs collaboration (https: / / alphafoldserver.com / ). The results were downloaded and visualised using visualisation softwares such as PyMOL and RING ( https: / / ring.biocomputinqup.it / ) to check the intrachain and interchain bonds including hydrogen bonds, tt-tt stacking, disulphide linkages and Van der Waals. CONCLUSION Thirteen peptide sequences were designed from initially identifying two consensus sequences in the PSD4 protein and in the dynamin-binding protein isoform A (Figure 1 &2). The EG within the LYRLEGFRKS was not included in the first part of the sequence, as glutamic acid can reduce the charge of the peptide, and a net positive charge of 5 is required. Glycine can also increase hydrophilic spacing and flexibility, and given there was a serine in the second consensus sequence C2, one flexible residue was modelled to be sufficient. It was clear from Figure 1 that, although LYRLFRKS formed nanoparticles with mRNA to give a PDI <0.16, there was no transfection. This could be attributed to the net negative charge of-17.87 mV, and clearly more amino acids were required for functionality. The C2 consensus sequence NLKP and KPFER were identified in two locations within the dynamin-binding protein (DNMBP), and both C1 and C2 play a significant role in clathrin-independent and clathrin-dependent endocytosis respectively. Yet Figure 2 indicates that NLKPKPFER neither forms nanoparticles with a size of 611 nm nor transfects cells. Given that it would be advantageous to enter cells by as many pathways as possible, coupled with the fact that the C3 sequence LYRLFRKSNLKPFER was found within the SARS-CoV-2 Chain A Spike Glycoprotein, this indicates that it is important for cellular entry cross species (human and viral). Nevertheless, as clearly indicated in Figure 3, the C3 sequence cannot form nanoparticles (1128 nm), has a large PDI >0.3 and cannot transfect cells. It is therefore concluded that, while C3 may have an important role in cellular entry, it is not a cell-penetrating peptide. Therefore, multiple modifications are required. Figure 4 reveals the variations that were added to the C3 sequence to produce a CPP. In summary, neither the addition of two alanines on the N-terminus (C4), a WH in the middle of C1 and C2 (C5), nor the combination of AA and WH produced nanoparticles or transfection (Figure 5). However, when HARAC was added to the C-terminus, nanoparticles formed at 57.88 nm with a charge of 29 mV and transfection at 54%. Although this was positive, the DLS spectra was not as uniform, as indicated by a PDI of 0.2 with variations between measurements. When WH was added to 07 to give 08, the size increased to 506 nm with a reduction in transfection to 11 % (Figure 5). However, when the AA was added to the N-terminus of the C3 peptide along with the WH to split the sequence and the HARAC on the C-Terminus, the results were entirely unexpected. According to bio-informatic analysis, peptide C9 should be unstable; yet nanoparticles were formed with a size of 103 nm and charge of 19 mV. Apart from an exceptional level of transfection in NCTC-929s of 75%, the polydispersity index was incredibly uniform at 0.06, and these nanoparticles were made manually and not via microfluidics. C9 was therefore termed the HAWC peptide that, through the addition of those amino acids, is a new CPP. The addition of another H in the middle of HAWC to give 9.1 did not improve the transfection and, indeed, when a serine was added to the end of HAWC instead of C 9.2, no transfection occurred even though nanoparticles were made with a PDI of 0.07. Finally, the addition of one R at position 15 and then another at position 9 only served to increase the size to 277 nm and 458 nm for C9.3 and C9.4 with a reduction in transfection efficiency to 63% and 67% respectively. The spectra of C9 HAWC peptide was far superior to all others (Figures 4 &5) but was also deemed unstable (Table 3) along with all variants of C9 which was unexpected. In addition, it is of note that the HAWC peptide also has the highest number of hydrogen bonds in the monomeric state (25) which have been shown to increase the stability of the structure (Table 3).14 The HAWC peptide also has the highest number of Van der Waals bonds between the dimeric interchains with 13 (Table 3). Van der Waals are indicative of molecular interactions and could account for the compactness of the structure measured when complexed with mRNA.15 There is also tt-tt stacking in both the intra- and inter-dimer HAWC structures which is facilitated via the presence of the three aromatic amino acids present. The tt-tt stacking will also enable nano-assembled structures.16 Taken together, the combination of all of these non-covalent bonds as a consequence of amino acid position could account for the superior spectra, PDI, functionality and safety of the HAWC peptide (Figures 4 &5). The HAWC peptide was then analysed to determine the optimal N:P ratio in Figure 6 along with PDI, encapsulation efficiency and transfection efficiency in two cell lines, NCTC-929 and the dendritic cell line DC2.4. Figure 6A indicated that the size of the HAWC / mRNA nanoparticle decreased with increasing Peptide concentration but that the Charge and PDI were inversely correlated to N:P ratio (Figure 6 B&C). Encapsulation was uniform from N:P 8-20 which averaged out at 95% following ionic exchange chromatography showing minimal difference to water through the column (Figure 6E). In Figure 6 G&H, the goal was to find the optimal N:P ratio for each cell line and go to high enough N:P ratios to observe a reduction in efficiency. For NCTC-929s, the optimal transfection of 81 % occurrent at N:P10 reducing to 43% by N:P20. For DC2.4s, the optimal transfection was found at a higher N:P ratio of 14 of 63% reducing to 38% by N:P20. The amount of peptide required for transfection can vary between cell lines, which is linked to the amount of glycoproteins on the cell surface, so studies to determine the optimal N:P ratio are required. Figure 7 details that HAWC can also complex miRs into extremely compact uniform nanoparticles with similar DLS spectra to mRNA, with >90% transfection efficiency and a minimal reduction in cell viability >90% across all N:P ratios. Two miRs were used in the nanoparticle in a blended formation to illustrate that HAWC can deliver more than one nucleic acid cargo in a nanoparticle. As can be found in Figure 7 G &H, following transfection, miR31 and miR132 were upregulated in NCTC-929s. The basal levels of miR-31 are much higher in NCTC-929S than miR-132 which accounts for the fold difference between the two miRs.17 Figure 8 shows similar results with a larger 5kb double stranded plasmid DNA that is readily condensed into NPs by the HAWC peptide with a size of 112 nm, PDI <0.2 and charge of+17 mV. Encapsulation via pico-green is >80% and via IEC >95% with spherical nanoparticles formed. Finally, as the original C1 is involved in clathrin-independent entry such as micropinocytosis and C2 in clathrin-mediated cell entry, we wanted to demonstrate that the HAWC peptide could enter cells using both pathways. Figure 9 A) illustrates the findings of 9 B) &C) where the use of dynasore to block clathrin-mediated cell entry and EIPA to block micropinocytosis significantly reduced HAWC / mRNA transfection efficiency to 13% and 25% respectively. Of note, the addition of Genistein which blocks caveolin-dependent endocytosis had no effect on transfection efficiency with the HAWC / mRNA NPs, indicating that this is not the mechanism of cell entry. The HAWC peptide can complex nucleic acids with a superior compactness as evidenced via the spectra which results in a uniform population of nanoparticles. Although evaluated as unstable, the position of the amino acids confers a number of non-covalent bonds that indicate otherwise. In addition, the levels of transfection are very high with minimal cytotoxicity. The two mechanisms of cellular entry from the consensus sequence have been retained, but the cell-penetrating capability is a result of the three modifications added at the N, C and middle of the consensus peptide. This peptide will be further developed as a platform technology for the delivery of nucleic acids across a range of biomedical applications. REFERENCES 1. Tran DP, Tada S, Yumoto A, Kitao A, Ito Y, Uzawa T, Tsuda K. Using molecular dynamics simulations to prioritize and understand Al-generated cell penetrating peptides. Sci Rep. 2021 May 20;11(1):10630. doi: 10.1038 / s41598-021-90245-z. PMID: 34017051; PMCID: PMC8137933. 2. Wender PA, Galliher WC, Goun EA, Jones LR, Pillow TH. The design of guanidinium-rich transporters and their internalization mechanisms. Adv Drug Deliv Rev. 2008 Mar 1 ;60(4-5):452-72. doi: 10.1016 / j.addr.2007.10.016. Epub 2007 Nov 9. PMID: 18164781; PMCID: PMC2533582. 3. Stanzl EG, Trantow BM, Vargas JR, Wender PA. Fifteen years of cell-penetrating, guanidinium-rich molecular transporters: basic science, research tools, and clinical applications. Acc Chern Res. 2013 Dec 17;46(12):2944-54. doi: 10.1021 / ar4000554. Epub 2013 May 22. PMID: 23697862; PMCID: PMC3796152. 4. Khemaissa S, Walrant A, Sagan S. Tryptophan, more than just an interfacial amino acid in the membrane activity of cationic cell-penetrating and antimicrobial peptides. Q Rev Biophys. 2022 Aug 18;55:e10. doi: 10.1017 / S0033583522000105. PMID: 35979810. 5. Kiessling LL, Diehl RC. CH-tt Interactions in Glycan Recognition. ACS Chern Biol. 2021 Oct 15;16(10):1884-1893. doi: 10.1021 / acschembio.1c00413. Epub 2021 Oct 6. PMID: 34615357; PMCID: PMC9004545. 6. Gabius HJ, Andre S, Jimenez-Barbero J, Romero A, Solis D. From lectin structure to functional glycomics: principles of the sugar code. Trends Biochem Sci. 2011 Jun;36(6):298-313. doi: 10.1016 / j.tibs.2011.01.005. Epub 2011 Apr1. PMID: 21458998. 7. Hudson KL, Bartlett GJ, Diehl RC, Agirre J, Gallagher T, Kiessling LL, Woolfson DN. Carbohydrate-Aromatic Interactions in Proteins. J Am Chern Soc. 2015 Dec 9;137(48):15152-60. doi: 10.1021 / jacs.5b08424. Epub 2015 Nov 30. PMID: 26561965; PMCID: PMC4676033. 8. Chakravarty S, Ung AR, Moore B, Shore J, Alshamrani M. A Comprehensive Analysis of Anion-Quadrupole Interactions in Protein Structures, Biochemistry. 2018 Mar 27;57(12):1852-1867. doi: 10.1021 / acs.biochem.7b01006. Epub 2018 Mar 9. PMID: 29482321; PMCID: PMC6051350. 9. Walrant A, Bauza A, Girardet C, Alves ID, Lecomte S, lllien F, Cardon S, Chaianantakul N, Pallerla M, Burlina F, Frontera A, Sagan S. Ionpair-K interactions favor cell penetration of arginine / tryptophan-rich cell-penetrating peptides. Biochim Biophys Acta Biomembr. 2020 Feb 1 ;1862(2):183098. doi: 10.1016 / j.bbamem.2019.183098. Epub 2019 Oct 30. PMID: 31676372. 10. Mason AJ, Leborgne C, Moulay G, Martinez A, Danos O, Bechinger B, Kichler A. Optimising histidine rich peptides for efficient DNA delivery in the presence of serum. J Control Release. 2007 Mar 12:118(1):95-104. doi: 10.1016 / j.jconrel.2006.12.004. Epub 2006 Dec 8. PMID: 17254661. 11. He J, Xu S, Mixson AJ. The Multifaceted Histidine-Based Carriers for Nucleic Acid Delivery: Advances and Challenges. Pharmaceutics. 2020 Aug 14;12(8):774. doi: 10.3390 / pharmaceuticsl 2080774. PMID: 32823960; PMCID: PMC7465012. 12. Lushchak VI. Glutathione homeostasis and functions: potential targets for medical interventions. J Amino Acids. 2012:2012:736837. doi: 10.1155 / 2012 / 736837. Epub 2012 Feb 28. PMID: 22500213; PMCID: PMC3303626. 13. Lo SL, Wang S. An endosomolytic Tat peptide produced by incorporation of histidine and cysteine residues as a nonviral vector for DNA transfection. Biomaterials. 2008 May;29(15):2408-14. doi: 10.1016 / j.biomaterials.2008.01.031. Epub 2008 Mar 4. PMID: 18295328. 14. Tan KP, Singh K, Hazra A, Madhusudhan MS. Peptide bond planarity constrains hydrogen bond geometry and influences secondary structure conformations. Curr Res Struct Biol. 2020 Dec 8;3:1-8. doi: 10.1016 / j.crstbi.2020.11.002. PMID: 34382009; PMCID: PMC8261469. 15. Sung SS. Peptide folding driven by Van der Waals interactions. Protein Sci. 2015 Sep;24(9):1383-8. doi: 10.1002 / pro.2710. Epub 2015 Jun 11. PMID: 26013298; PMCID: PMC4570533. 16. Sasidharan S, Ramakrishnan V. Chapter Five - Aromatic interactions directing peptide nanoassembly, Editor(s): Rossen Donev. Advances in Protein Chemistry and Structural Biology. Academic Press. 2022 Volume 130: 119-160. ISSN 1876-1623; https: / / doi.Org / 10.1016 / bs.apcsb.2022.01.001; ISBN 9780323992299. 17. Bombin ADJ, Dunne N, McCarthy HO. Delivery of a peptide / microRNA blend via electrospun antimicrobial nanofibres for wound repair. Acta Biomater. 2023 Jan 1;155:304-322. doi: 10.1016 / j.actbio.2022.10.059. Epub 2022 Nov 3. PMID: 36334906.

Claims

2. A peptide, or a salt or amide thereof, comprising, or consisting of:(Xaa1)a-(Xaa2)b-LYRLFRKS-(Xaa3)c-(Xaa4)d-(Xaa4)e-NLKPFERHARAC,in which a is 0 or 1 and, if a is 1, Xaa1 is selected from an Ala residue, a Vai residue or a Gly residue; in which b is independently 0 or 1 and, if b is 1, Xaa2 is selected from an Ala residue, a Vai residue or a Gly residue;in which c is independently 0 or 1 and, if c is 1, then Xaa3 is an aromatic residue selected from a His or a Trp residue;in which d is 1 and Xaa4 is an aromatic residue selected from a Trp ora His residue;in which e is 1 and Xaa5 is a His or a Trp residue.

3. The peptide of Claim 1,wherein the peptide includes a minimum of two Lys residues;wherein the peptide includes a minimum of four Arg residues;wherein the peptide includes a minimum of four Ala residues to increase the hydrophobicity and to readily pass across the cell membrane;wherein the peptide includes a minimum of three aromatic residues selected from Tryptophan or phenylalanine to improve hydrophobic interaction with cell membranes;wherein the peptide includes a minimum of two His residues to enhance conditional endosomal escape;wherein the peptide includes a Cys residue at, or adjacent, the C-terminus to enhance stability and cargo release once inside cell;and wherein the peptide includes a Pro residue to enhance cellular entry and safety.

4. A peptide according to Claim 1 or 2, in which the peptide comprises, or consists of no more than 30 amino acids.

5. A peptide according to Claim 3, comprising 5%-25% Lysine, 5%-25% Arginine, 15%-30% Alanine, 10%-30% aromatic residues selected from tryptophan or phenylalanine, and I or 5%-20% Histidines.

6. The peptide of any one of Claims 1 to 4, in which Xaa3 is a Trp residue; and Xaa4 is a His residue.

7. The peptide of any one of Claims 1 to 5, in which c is 0 and Xaa3 is absent.

8. A peptide according to any one of Claims 1 to 6, comprising:AALYRLFRKSWHNLKPFERHARAC, or a salt or amide thereof.

9. A cell delivery agent comprising the peptide of any one of Claims 1 to 7 in association with an anionic cargo.

10. The cell delivery agent of Claim 8, in which the anionic cargo is an mRNA and the cell delivery agent has a polydispersity index of 0.025 to 0.35.

11. The cell delivery agent of Claim 8, in which the anionic cargo is an mRNA and the cell delivery agent has a mean hydrodynamic size (nm) of 50 to 450 nm.

12. The cell delivery agent of Claim 8, in which the anionic cargo is an mRNA and the cell delivery agent has a charge of 17.5 to 20 mV.

12. A peptide according to any one of Claims 1 to 7 for use as a cell delivery agent.

13. Use of a peptide according to any one of Claims 1 to 7 for the delivery of an anionic cargo to a cell or subject in need thereof.

14. A peptide according to any one of Claims 1 to 7 for use in inducing an immune response in a subject in need thereof or a method for inducing an immune response in a subject comprising the administration of the peptide to a subject in need thereof.

15. A peptide according to any one of Claims 1 to 7 for use in gene therapy in a subject in need thereof.

16. A peptide according to any one of Claims 1 to 7 for use in the treatment and / or prophylaxis of an infection, cancer, wounds in a subject in need thereof or a method forthe treatment and / or prophylaxis of an infection, cancer, wounds comprising the administration of the peptide to a subject in need thereof.Application No: GB2413378.7Examiner:Dr Daniel WatkinsClaims searched: 1-16Date of search: 19 February 2025Patents Act 1977: Search Report under Section 17Documents considered to be relevant:Category Relevant to claims Identity of document and passage or figure of particular relevance A A - Infection, Genetics and Evolution, vol. 85, 2020, Hemmati Shiva et al., Decoding the proteome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) for cell-penetrating peptides involved in pathogenesis or applicable as drug delivery vectors, article no. 104474. See esp. abstract and sequences 'SCV2-CPP199' and 'SCV2-CPP202' on page 5. US 2023 / 0143215 Al (MELIEF et al.) See esp. SEQ ID NO: 29 on page 23.Categories:X Document indicating lack of novelty or inventive step A Document indicating technological background and / or state of the art. Y Document indicating lack of inventive step if P Document published on or after the declared priority date but combined with one or more other documents of same category. before the filing date of this invention. & Member of the same patent family E Patent document published on or after, but with priority date earlier than, the filing date of this application.Field of Search:International Classification:Subclass Subgroup Valid From C07K 0019 / 00 01 / 01 / 2006 A61K 0038 / 16 01 / 01 / 2006 A61K 0047 / 64 01 / 01 / 2017 A61K 0047 / 69 01 / 01 / 2017 A61K 0048 / 00 01 / 01 / 2006 C07K 0014 / 00 01 / 01 / 2006 C07K 0014 / 165 01 / 01 / 2006 C12N 0015 / 85 01 / 01 / 2006

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