Formulation method

Abstract

The present invention is directed to a method for preparing a cell penetrating peptide-cargo formulation, said method comprising providing the cell penetrating peptide in ethanol without an aprotic solvent, associating the cell penetrating peptide with a nucleic acid cargo in an association buffer comprising 30-90% sucrose (in particular 50-90% sucrose) at a ratio of 3:1 to the cell penetrating peptide in ethanol, and storing the cell penetrating peptide-cargo formulation at -70°C or below, wherein associating is performed in a volume between 0.5x and 0.1x the final volume at which the formulation is prepared for administration. The formulation may also be lyophilised. Also provided are cell penetrating peptide-cargo formulations obtainable and / or obtained by the method herein, pharmaceutical compositions comprising the formulations, and said formulations and pharmaceutical compositions for use in the transport of the cargo across a lipid membrane and delivery of the cargo into a cell or for use in raising an immune response in a subject.

Description

[0001] FORMULATION METHOD

[0002] FIELD OF THE INVENTION

[0003] The present invention is directed to a method for preparing a cell penetrating peptide-cargo formulation, said method comprising providing the cell penetrating peptide in ethanol without an aprotic solvent, associating the cell penetrating peptide with a nucleic acid cargo in an association buffer comprising 30-90% sucrose (in particular 50-90% sucrose) at a ratio of 3:1 to the cell penetrating peptide in ethanol, and storing the cell penetrating peptide-cargo formulation at -70°C or below, wherein associating is performed in a volume between 0.5x and O.lx the final volume at which the formulation is prepared for administration. The formulation may also be lyophilised. Also provided are cell penetrating peptide-cargo formulations obtainable and / or obtained by the method herein, pharmaceutical compositions comprising the formulations, and said formulations and pharmaceutical compositions for use in the transport of the cargo across a lipid membrane and delivery of the cargo into a cell or for use in raising an immune response in a subject.

[0004] BACKGROUND TO THE INVENTION

[0005] Cell penetrating amino acid sequences (also referred to as cell penetrating peptides or “CPPs”) are relatively short peptides that have the ability to gain access into the cell and mediate the delivery of cargo covalently or non-covalently attached to them.

[0006] Cell penetrating peptides have been used to deliver a multitude of different molecules including, but not limited to, nucleic acids. The cell penetrating amino acids sequences typically contain several positively charged amino acids (arginine, lysine, histidine and non- proteogenic amino acid ornithine) and are thus able to associate with the negatively charged backbone of nucleic acid. They form a non-covalent complex mainly via electrostatic interactions, although hydrophobic interactions may play a role in the stability of the formed complexes. The non-covalent cell penetrating amino acid sequence / cargo complexation strategy allows for a simple complex formation with a high mixing versatility, but the limitations of using this approach may include heterogeneity and low stability of the complexes, and the premature or limited dissociation of the cargo from the complexes.

[0007] When associated with a cargo, the cell penetrating amino acid sequence / cargo complexes mainly enter via endosomal pathways and end up in the endosomal compartments or in the lysosomes. In order to take its intended effect, the internalisation has to be promptly followed by the release of complexes from these organelles. The cell membrane has an amphiphilic nature. To enhance cell penetrating amino acid membrane interactions, several cell penetrating amino acid sequences have been designed to have both hydrophilic and hydrophobic regions and / or moieties in their sequence. NickFect55 (NF55) is a cell penetrating peptide, useful as a plasmid DNA (pDNA) delivery vector for nucleic acid delivery applications in vitro and in vivo. It has an N-terminal fatty acid and a non-proteinogenic amino acid, ornithine in the aa? position. It was initially designed for the delivery of pDNA with an optimal CPP / pDNA complex stability and positive charges distributed along theoretical alpha-helix. Several amphipathic CPPs have previously been used to efficiently deliver siRNAs into the mammalian cells. mRNA therapeutics are a class of drugs that allow two types of therapeutic approaches: mRNA-based vaccination; and mRNA as protein replacement therapy. The COVID-19 pandemic (caused by the SARS-CoV2 virus) has granted mRNA vaccines unprecedented attention (Dolgin E. (2021) Nature, 589(7841): 189-191 , doi: https: / / doi.Org / 10.1038 / d41586- 021 -00019-w) and although frequently presented as a “new technology” they are actually the result and combination of past technological breakthroughs and discoveries from various disciplines Pardi et al. (2018) Nat Rev Drug Discov, 17(4):261-279, doi: https: / / doi.org / 10.1038 / nrd.2017.243). The most critical of those technological components is 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) and it is currently the bottleneck (Sahin et al. (2014) Nat Rev Drug Discov, 13(10):759-80, doi: https: / / d0i.0rg / l 0.1038 / nrd4278) that defines the potential of the whole platform. Current mRNA vaccine technology is based on liposomes (LNPs; Corbett et al. (2020) Nature, 586(7830):567-571 , doi: htps: / / doi . orq / 10.1038 / s41586-020-2622-0) and impressive development in the past 10 years has advanced these to in vivo systemic use (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: htps: / / doi.org / 10.1073 / pnas.1322937111). Nevertheless, the greatest controversy and challenge for the LNPs have been: (i) their side effects; and (ii) the fact that nucleic acid delivery - albeit effective - is restricted to the liver tissue. In order to fully tackle the possibilities of the mRNA therapeutics, the delivery technology has to be improved. For example, the current SARS-CoV2 vaccination regime requires local administration of the LNP and a low dose of mRNA, which is 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-druqs-and-vaccines). However, in order to treat chronic lifethreatening diseases, the delivery technology has to be: (i) significantly more efficient; and (ii) less toxic. CPPs have previously been developed from the NickFect (NF) and PepFect (PF) families for plasmid (pDNA), siRNA and miRNA delivery. In the PepFect family, PF14 has been used successfully for pDNA (Veimann et al. (2013) Mol. Pharmaceutics, 10(1): 199-210, doi: htps: / / doi.org / 10.1021 / mp3003557) and siRNA delivery (Sirmane et al. (2018) Peptides, 104(6):62-69, doi: htps: / / doi.Org / 10.1016 / j.peptides.2018.04.015), both in vitro and in vivo. The present inventors have also previously shown that PF1452 (Kurrikoff et al. (2017) Sci. Rep., 7(1):17056, doi: htps: / / doi.org / 10.1038 / s41598-Q17-17316-y) is even more efficient than PF14 for the delivery of pDNA in vivo. NF55 (Freiman et al. (2016) J of Control. Release, 241 (10): 135-143, doi: htps: / / doi.org / 10.1016 / j.jconrel.2016.09.022) is an excellent vector for the delivery of DNA in vivo. In Porosk et al. (2019, Biomater. Sci., 7:4363-4374, doi: htps: / / doi.org / 10.1039 / c9bm00688e), the present 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 efficient than NF55. In Carreras-Badosa et al. (2020, Biomaterials 262:120316, doi: htps: / / doi.org / 10.1016 / j. biomaterials.2020.120316), different NF and PF family CPPs (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: htps: / / doi.Org / 10.3390 / pharmaceutics13101618). NF411 , NF554, NF553, NF54, NF424, NF426 and NF436 have all been shown to be effective in the delivery of mRNA, in particular in vivo (Porosk et al. (2023) Pharmaceutics., 15(3): 952, doi: https: / / doi.org / 10.3390 / pharmaceutics 15030952; and WO 2024 / 074553, the results of which for these peptides are specifically incorporated by reference), as has PF14 (Periyasamy et al. (2023) Front. Pharmacol., 14:1219761 , doi:

[0008] The conditions of formulation for and solvent used to dissolve PF14 for example have been shown to be crucial for complexation with nucleic acid cargo (Biswas et al. (2023) Pharmaceutics, 15(2): 396 (doi: https: / / doi.org / 10.3390 / pharmaceutics15020396). Therefore, while good CPPs for numerous cargoes (in particular nucleic acid cargoes) have been developed to date, methods for preparing formulations in which they are complexed / associated with the cargo are needed. To date, such methods have not produced formulations that are satisfactorily suitable for freezing and / or lyophilisation, as required for storage prior to their use, such as by administration in vivo to a subject.

[0009] SUMMARY OF THE INVENTION

[0010] According to a first aspect of the invention, there is provided a method of preparing a formulation comprising a cell penetrating peptide and a nucleic acid cargo, said method comprising the steps of: (i) providing a cell penetrating peptide in ethanol without an aprotic solvent;

[0011] (ii) associating the cell penetrating peptide with a nucleic acid cargo in an association buffer, thereby forming a cell penetrating peptide-cargo formulation, wherein the nucleic acid cargo and association buffer are at a ratio amount of 3:1 to the cell penetrating peptide in ethanol and wherein the association buffer comprises between 30% and 90% sucrose; and

[0012] (iii) storing the cell penetrating peptide-cargo formulation at -70°C or below, and optionally lyophilising the cell penetrating peptide-cargo formulation, wherein associating step (ii) is performed in a volume between 0.5x and 0.1x the final volume at which the formulation is prepared for administration.

[0013] In some embodiments, the cell penetrating peptide may comprise the sequence of: AGYLLGXINLKALAALAKAIL (SEQ ID NO: 1), or the sequence of SEQ ID NO: 1 having one or more modifications selected from:

[0014] (i) an N9L amino acid substitution;

[0015] (ii) an A14K amino acid substitution;

[0016] (iii) a deletion of the alanine residue at position 1 , or a deletion of the alanine 20 residue at position 1 and a deletion of the glycine residue at position 2;

[0017] (iv) a substitution of the ornithine residue at position 7 with Dab or Dap; and

[0018] (v) one or two amino acid substitutions at positions 9 to 21 of SEQ ID NO: 1 in addition to or instead of N9L and / or A14K, wherein the cell penetrating peptide comprises two or more histidine residues by substitution to the N-terminal part and / or addition to the N-terminus, optionally wherein the cell penetrating peptide is chemically modified at the C- terminus, such as by amidation and / or wherein the C-terminus is CONH2.

[0019] In other embodiments, the cell penetrating peptide may comprise the sequence of:

[0020] AGYLLG Xi* INLKALAALA X2IL (SEQ ID NO: 2); or AGYLLG Xi* LKALAALA X2IL (SEQ ID NO: 3), wherein Xi represents Lys (K), Orn (O), Dab or Dap, wherein * indicates that the peptide optionally continues from the side chain amino group and not from the a-amino group, wherein X2represents KA or AK, and wherein the cell penetrating amino acid sequence is optionally chemically modified at the C-terminus.

[0021] In further embodiments, the nucleic acid cargo is mRNA, plasmid DNA (pDNA), smallinterfering RNA (siRNA) or micro RNA (miRNA). In yet further embodiments, the association buffer comprises between 50% and 90% sucrose and 10mM HEPES, in particular about 60% sucrose and 10mM HEPES. In still further embodiments, associating step (ii) is performed in a volume 0.1x the final volume at which the formulation is prepared for administration.

[0022] According to a further aspect of the invention, there is provided a cell penetrating peptide- cargo formulation obtainable and / or obtained by the method described herein.

[0023] In a yet further aspect, there is provided the cell penetrating peptide-cargo formulation or pharmaceutical composition described herein for use in the transport of the nucleic acid cargo across a lipid membrane and subsequent delivery of said cargo into a cell, optionally wherein the cell is in vivo, and / or wherein the cell is a cell of the spleen and / or lymph node, such as a dendritic cell (DC).

[0024] In a still further aspect, there is provided the cell penetrating peptide-cargo formulation or pharmaceutical composition described herein for use in a method of raising an immune response in a subject.

[0025] BRIEF DESCRIPTION OF THE FIGURES

[0026] Figure 1 : Transfection efficiency of cell penetrating peptide NF424 complexed with fLuc-encoding mRNA cargo according to various formulation methods as described in Example 2. NF424-cargo formulations were injected into the tail vein of BALB / c mice. The reporter gene expression levels were evaluated after a single injection from the whole tissue homogenate of spleen (A) and lymph nodes (B) 16 hours postmortem. FMQ: Formulation in water, peptide (NF424) in water, total volume of the formulation is 100pl. For in vivo application sugar solution in water was added prior injection. FSOL: Formulation similar to a method in WO 2023 / 277779. Formulation in HEPES, peptide (NF424) in ethanol, total volume of the formulation is 10Opl. For in vivo application sugar solution in water was added prior injection. CF: Concentrated formulation. Formulation in HEPES containing 60% sucrose, peptide (NF424) in ethanol, buffer: peptide ratio = 3:1 , total volume of the formulation is 10pl. For in vivo applications 90pl HEPES was added prior injection.

[0027] Figure 2: Comparison of immediately prepared formulations by the present method (“fresh”) and following freezing, reconstituted after 24h, 1-4 weeks and 2-3 months storage at -80°C as described in Example 3. A) Traces showing 24h-3 weeks time periods. B) Traces showing 2 weeks-3 months time periods. Dynamic light scattering, measured on Zetasizer Nano ZS, Malvern Instruments.

[0028] Figure 3: Transfection efficiency of cell penetrating peptide NF424 complexed with fLuc-encoding mRNA cargo according to the present formulation method (CF) following reconstitution at 24h, 1-4 weeks and 2-3 months storage at -80°C as described in Example 4. NF424-cargo concentrated formulations were stored at -80°C and injected after thawing at the indicated timepoints into the tail vein of the BALB / c mice. The reporter gene expression levels were evaluated after a single injection from the whole tissue homogenate of spleen (A) and lymph nodes (B) 6 hours postmortem. Immediately prepared formulation (“fresh”) was used as a positive control.

[0029] Figure 4: Transfection efficiency of cell penetrating peptide NF424 complexed with fLuc-encoding mRNA cargo according to the present formulation method (CF) following 4 freeze / thaw cycles as described in Example 4. NF424-cargo concentrated formulations were frozen at -80°C and thawed 4 times. Thereafter injected into the tail vein of the BALB / c mice. The reporter gene expression levels were evaluated after a single injection from the whole tissue homogenate of spleen (A) and lymph nodes (B) 6 hours postmortem. Immediately prepared formulation (“fresh”) was used as a positive control.

[0030] Figure 5: Comparison of immediately prepared formulations by the present method (“fresh”) and following 4 freezing at -80C° and thawing cycles as described in Example 3. Dynamic light scattering, measured on Zetasizer Nano ZS, Malvern Instruments.

[0031] Figure 6: Comparison of in vitro transfection efficiencies for formulations prepared in HEPES associated buffers comprising various sucrose concentrations as described in Example 5. Formulations were prepared with varying concentrations of sucrose in HEPES buffer and applied to HEK (left panel) and CHO cells (right panel) in vitro for 24 hours, luciferase expression was measured the following day.

[0032] Figure 7: Comparison of in vivo transfection efficiencies for formulations prepared in HEPES associated buffers comprising various sucrose concentrations as described in Example 5. Luciferase expression in spleen depending on administered formulations with varying concentration of sucrose in HEPES buffer. The reporter gene expression levels were evaluated after a single injection from the whole tissue homogenate of spleen 6 hours postmortem.

[0033] Figure 8: In vivo transfection efficiency in spleen and lymph nodes of cell penetrating peptide NF424 complexed with fLuc-encoding mRNA cargo according to the present formulation method (CF) following lyophilisation and reconstitution for injection as described in Example 4. Freshly prepared complexes were used as a control. The reporter gene expression levels were evaluated after a single injection from the whole tissue homogenate 6 hours postmortem.

[0034] Figure 9: Comparison of lyophilised and immediately prepared formulations by the present method (“fresh”) as described in Example 3. Dynamic light scattering, measured on Zetasizer Nano ZS, Malvern Instruments. DETAILED DESCRIPTION OF THE INVENTION

[0035] According to a first aspect of the invention, there is provided a method of preparing a formulation comprising a cell penetrating peptide and a nucleic acid cargo, said method comprising the steps of:

[0036] (i) providing a cell penetrating peptide in ethanol without an aprotic solvent;

[0037] (ii) associating the cell penetrating peptide with a nucleic acid cargo in an association buffer, thereby forming a cell penetrating peptide-cargo formulation, wherein the nucleic acid cargo and association buffer are at a ratio amount of 3:1 to the cell penetrating peptide in ethanol and wherein the association buffer comprises between 30% and 90% sucrose; and

[0038] (iii) storing the cell penetrating peptide-cargo formulation at -70°C or below, and optionally lyophilising the cell penetrating peptide-cargo formulation, wherein associating step (ii) is performed in a volume between 0.5x and 0.1x the final volume at which the formulation is prepared for administration.

[0039] The inventors have found that cell penetrating peptide-cargo formulations prepared according to this method are stable and retain a similar nanoparticle size compared to freshly prepared formulations and an acceptable polydispersity index following freezing at -70°C or below or following lyophilisation, as required for storage. This is in contrast to formulations prepared in water, or formulations where the cell penetrating peptide is provided in ethanol with an aprotic solvent but the association buffer does not comprise 30-90% or 50-90% sucrose and / or association is not performed in a volume 0.5-0.1x the final volume at which the formulation is to be prepared for administration (e.g. as described in WO 2023 / 277779 and Biswas et al. (2023)). Such previously described preparation methods do not yield formulations with maintained consistent nanoparticle sizes or polydispersity indexes following freezing compared to freshly prepared formulations, nor are the resulting formulations suitable for lyophilisation (see Table 1 herein). As such, the present invention provides an improved method for preparing cell penetrating peptide-cargo formulations. The resulting formulations can be frozen at -70°C or below and / or lyophilised for storage, and retain a consistent nanoparticle size and polydispersity index following said freezing or lyophilisation, as compared to freshly prepared formulation.

[0040] Thus, the cell penetrating peptide-cargo formulations prepared according to the present method may be frozen at -70°C or below, such as at -80°C. Freezing the formulation may be for storage or as a prerequisite for lyophilisation (also known as “freeze drying” and “cryodesiccation”). As demonstrated herein, formulations prepared according to the present method can be frozen and retain a consistent nanoparticle size and polydispersity index, and are thus suitable for possible subsequent lyophilisation. Thus, in embodiments the method herein comprises step (iii) of storing the cell penetrating peptide-cargo formulation at -70°C or below. In a further embodiment, freezing is at -80°C. The frozen formulation may be stored and subsequently thawed and diluted as described herein for administration, or diluted without thawing such that addition of a volume of diluent / buffer as described herein thaws the frozen formulation. Thus, in a yet further embodiment step (iii) herein may comprise storing the cell penetrating peptide-cargo formulation at -70°C or below (e.g. at -80°C) without lyophilisation. In another embodiment, step (iii) comprises lyophilising the cell penetrating peptide-cargo formulation. Said lyophilisation is performed after freezing at -70°C or below (e.g. at -80°C). Lyophilisation is preferred for long term storage and transport as lyophilised formulation may be stored more easily and at higher / easier to maintain temperatures than non-lyophilised formulation, such as at -20°C. Methods of lyophilisation are well known in the art. Lyophilised formulation may be resuspended / reconstituted in a buffer as described herein, such as in a buffer to a total volume of between 2x and 10x the volume at which step (ii) is performed, in particular to a total volume of 10x that at which step (ii) is performed. Suitable buffers for resuspension / reconstitution are described herein, such as a saline buffer and / or a buffer comprising HEPES, in particular a buffer comprising 10mM HEPES without sucrose.

[0041] Alternatively, the cell penetrating peptide-cargo formulations prepared according to the present method may be used after preparation without being frozen for storage, i.e. they are used ‘fresh’ and wherein step (iii) of the method herein is optional. Thus, in some embodiments step (iii) of the method herein is omitted. According to these embodiments, it will be appreciated that step (ii) is still performed in a volume between 0.5x and 0.1x the final volume at which the formulation is prepared for administration, such that the associated formulation is diluted as described herein following step (ii) in a total volume between 2x and 10x the volume at which step (ii) is performed, in particular 10x.

[0042] Thus, in a further aspect of the invention there is provided a method of preparing a formulation comprising a cell penetrating peptide and a nucleic acid cargo, said method comprising the steps of:

[0043] (i) providing a cell penetrating peptide in ethanol without an aprotic solvent; and

[0044] (ii) associating the cell penetrating peptide with a nucleic acid cargo in an association buffer, thereby forming a cell penetrating peptide-cargo formulation, wherein the nucleic acid cargo and association buffer are at a ratio amount of 3:1 to the cell penetrating peptide in ethanol and wherein the association buffer comprises between 30% and 90% sucrose, wherein associating step (ii) is performed in a volume between 0.5x and 0.1x the final volume at which the formulation is prepared for administration. The method described herein prepares cell penetrating peptide-cargo formulations in the form of stable nanoparticles. Thus, in one embodiment the associated cell penetrating peptide- cargo formulation is in the form of a nanoparticle. As will be readily understood, said nanoparticles comprise the cell-penetrating peptide and the cargo complexed / associated together. Without being bound by theory, it is believed that the cell penetrating peptides form a nanoparticle surrounding the cargo (i.e. a nucleic acid) in the lumen, thus facilitating passage of the cargo across a cell membrane. It is thus required that methods of preparing such formulations correctly complex the cell penetrating peptides with the cargo such that nanoparticles are formed, such as described herein. Cell penetrating peptide-cargo formulations may therefore also be referred to as membrane permeable constructs.

[0045] The cell penetrating peptides described herein comprise an amino acid peptide sequence with a fatty acid chain attached to the N-terminus of said amino acid sequence. The formulated constructs described herein may also further comprise a cargo, in particular a nucleic acid cargo, and thus the formulations prepared according to the present method comprise a cargo, in particular a nucleic acid cargo.

[0046] A property of the formulations described herein is their ability to translocate the lipid membrane and facilitate the delivery of various molecular cargoes to the cytoplasm or to an organelle of a cell or to an intracellular cell surface. Thus, cell penetrating peptide-cargo formulations described herein deliver a cargo to the cytoplasm of a cell, such that wherein the cargo is for example mRNA it is available to the translation machinery (e.g. ribosomes etc.) to generate the protein or peptide encoded by said mRNA, such as an antigenic protein or peptide.

[0047] Cell Penetrating Peptide Sequences

[0048] Cell penetrating peptides, also referred to as cell penetrating amino acid sequences or “CPPs”, are short amino acid sequences that transport different types of cargo molecules across a lipid membrane and facilitate cellular uptake of the cargo molecules. Cell penetrating peptide sequences may comprise between 19 to 25 or 25 to 30 amino acids. Cell penetrating peptide sequences may comprise 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 peptide sequences may comprise 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids. Thus, in some embodiments the cell penetrating peptide sequence comprises 19 or more, 20 or more or 21 or more amino acids. In further embodiments, the cell penetrating peptide sequence comprises 19, 20 or 21 amino acids. A property of cell penetrating peptides described herein is their ability to translocate the lipid membrane and facilitate the delivery of various molecular cargoes to the cytoplasm or to an organelle of a cell or to an intracellular cell surface.

[0049] In one embodiment, the cell penetrating peptide comprises the sequence of: AGYLLGXINLKALAALAKAIL (SEQ ID NO: 1), or the sequence of SEQ ID NO: 1 having one or more modifications selected from:

[0050] (i) an N9L amino acid substitution;

[0051] (ii) an A14K amino acid substitution;

[0052] (iii) a deletion of the alanine residue at position 1 , or a deletion of the alanine 20 residue at position 1 and a deletion of the glycine residue at position 2;

[0053] (iv) a substitution of the ornithine residue at position 7 with Dab or Dap; and

[0054] (v) one or two amino acid substitutions at positions 9 to 21 of SEQ ID NO: 1 in addition to or instead of N9L and / or A14K, wherein the cell penetrating peptide comprises two or more histidine residues by substitution to the N-terminal part and / or addition to the N-terminus, optionally wherein the cell penetrating peptide is chemically modified at the C- terminus, such as by amidation and / or wherein the C-terminus is CONH2.

[0055] Dab refers to 2,4-diaminobutanoic acid. Dap refers to 2,3-diaminopropionic acid.

[0056] In a further embodiment, the cell penetrating amino acid sequence comprises SEQ ID NO: 1 , or comprises the sequence of SEQ ID NO: 1 having one or more modifications selected from: i) an N9L amino acid substitution;

[0057] (ii) an A14K amino acid substitution;

[0058] (iii) a deletion of the alanine residue at position 1 or a deletion of the alanine residue at position 1 and a deletion of the glycine residue at position 2; and

[0059] (iv) a substitution of the ornithine residue at position 7 with Dab or Dap, wherein the cell penetrating amino acid sequence comprises two or more histidine residues by substitution to its N-terminal part and / or addition to its N-terminus, and wherein the cell penetrating amino acid sequence is optionally chemically modified (e.g. amidated) at the C terminus (e.g. the C terminus is CONH2).

[0060] The natural proteogenic amino acids and the non-proteogenic (also known as non-natural) amino acids of the cell penetrating peptide may be in the naturally occurring L-enantiomer configuration (which may be represented by upper case letters). A general formula representing the sequence of certain cell penetrating peptides described herein is as follows:

[0061] HnYHHGO*ILLKALKALAKAIL (SEQ ID NO: 17), wherein * indicates that the peptide continues from the side chain amino group and not from the a-amino group.

[0062] The variable n refers to the number of histidine residues and n may equal one, two, three, four, five, six, seven, eight, nine or ten, preferably one, two, three, four, six, eight or ten, more preferably two, three, four, six, eight or ten, especially two, four, six, eight or ten.

[0063] Throughout the present disclosure, a residue and / or position number in the cell penetrating amino acid sequence refers to a position in the sequence by reference to the numbering of the relevant sequence as appropriate, it being understood that the said sequences may have any or none of the modifications described herein. Therefore, by way of example only, the numbering of residues / positions within the cell penetrating peptide sequences described herein can be represented as follows:

[0064] Ai G2 Y3 L4 L5 Ge O7* Is N9 L10 Ki 1 A12 L13 A14 A15 Lie A17 Kis A19 I20 L21 (SEQ ID NO: 4); or

[0065] A1 G2 Y3 L4 L5 Ge O7* Ls K9 A10 Ln A12 A13 L14 A15 Kw A17 I is L19 (SEQ ID NO: 10).

[0066] The cell penetrating peptide may have a N9L amino acid substitution and / or an A14K amino acid substitution and / or a deletion of the alanine residue at position 1 or a deletion of the alanine residue at position 1 and a deletion of the glycine residue at position 2 and / or a substitution of the ornithine residue at position 7 with Dab or Dap and / or one or two amino acid substitutions at positions 9 to 21 of SEQ ID NO: 1 in addition to or instead of N9L and / or A14K. In an embodiment, the cell penetrating peptide sequence does not comprise one or two amino acid substitutions at positions 9 to 21 of SEQ ID NO: 1 in addition to or instead of N9L and / or A14K.

[0067] The “N-terminal part” referred to herein comprises the amino acid residues at the ‘end’ of the cell penetrating peptide which is the N-terminus. Thus, in one embodiment the N-terminal part comprises positions 1 to 6 of e.g. SEQ ID NO: 1 , SEQ ID NO: 2 or SEQ ID NO: 3. In another embodiment, the N-terminal part comprises positions 1 to 5 of any of SEQ ID NO: 1 , SEQ ID NO: 2 or SEQ ID NO: 3. The N-terminal part 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 peptide sequence. Substitution refers to the replacement of an amino acid with another different amino acid.

[0068] The cell penetrating peptide may comprise two or more histidine residues by substitution to its N-terminal part. By way of particular example, two or more of the wild type amino acids in the N-terminal part of the cell penetrating peptide sequence may be substituted or replaced with two or more histidine residues. Two or more, three or more, four or more, five or more, or six of the wild type amino acids in the N-terminal part of the cell penetrating peptide sequence may be substituted or replaced with two or more, three or more, four or more, five or more, six histidine residues. Thus, the cell penetrating peptide may comprise two, three, four, five or six histidine residues in the N-terminal part.

[0069] The N-terminus of the cell penetrating amino acid sequence normally refers to the amino acid at position 1 of the amino acid sequence. For example, the N-terminus of SEQ ID NO: 1 refers to the amino acid at position 1 of SEQ ID NO: 1 . When the amino acid at position 1 of SEQ ID NO: 1 is deleted, however, the N-terminus refers to the amino acid at position 2 of the amino acid sequence. When the amino acids at positions 1 and 2 of SEQ ID NO: 1 are deleted, the N-terminus refers to the amino acid at position 3 of the amino acid sequence. The N-terminus refers to the first amino acid present in the amino acid sequence of the cell penetrating peptide.

[0070] The cell penetrating peptide may comprise one or more histidine residues added to the N-terminus of the cell penetrating peptide sequence. In one embodiment, two or more histidine residues are added to the N-terminus of the cell penetrating peptide sequence. One or more, two or more, three or more, four or more, five or more, six or more, seven or more, or eight or more histidine residues may be added to the N-terminus of the cell penetrating peptide sequence. Histidine residues referred to herein as “N-terminus histidine residues” are those added to the N-terminus of the cell penetrating peptide.

[0071] In some embodiments, the cell penetrating peptide comprises between three and eight histidine residues substituted in its N-terminal part and / or added at its N-terminus, preferably three, four, five, six or eight histidine residues. The cell penetrating peptide sequence may comprise one, two, three, four, five, six, seven, eight, nine or ten histidine residues substituted in its N-terminal part and / or added at its N-terminus.

[0072] The cell penetrating peptide of SEQ ID NO: 1 may comprise histidine amino acid substitutions at positions four and / or five, preferably at positions four and five of the cell penetrating amino acid sequence. SEQ ID NO: 1 may comprise histidine amino acid substitutions at positions four and / or five, preferably at positions four and five, when the amino acids at positions one and / or two are deleted.

[0073] In a further embodiment, the cell penetrating peptide of SEQ ID NO: 1 comprises at least three histidine residues in its N-terminal part and / or added to its N terminus, arranged such that one histidine residue is substituted at position two of SEQ ID NO: 1 , two histidine residues are substituted at positions four and five of SEQ ID NO: 1 and optionally one or more histidine residues are added to its N-terminus.

[0074] In another embodiment, the cell penetrating peptide comprises three histidine residues in its N-terminal part, arranged such that one histidine residue is substituted at position two of SEQ ID NO: 1 and two histidine residues are substituted at positions four and five of SEQ ID NO: 1.

[0075] In a further embodiment, the cell penetrating peptide comprises at least four histidine residues in its N-terminal part and / or added to its N terminus, arranged such that two histidine residues are substituted at positions one and two of SEQ ID NO: 1 , two histidine residues are substituted at positions four and five of SEQ ID NO: 1 and optionally one or more histidine residues are added to its N-terminus.

[0076] In yet a further embodiment, the cell penetrating peptide comprises four histidine residues in its N-terminal part, arranged such that two histidine residues are substituted at positions one and two of SEQ ID NO: 1 and two histidine residues are substituted at positions four and five of SEQ ID NO: 1.

[0077] In another embodiment, the cell penetrating peptide comprises four histidine residues in its N- terminal part, arranged such that two histidine residues are substituted at positions one and two of SEQ ID NO: 1 , two histidine residues are substituted at positions four and five of SEQ ID NO: 1 and also one histidine residue is added to its N-terminus.

[0078] In yet another embodiment, the cell penetrating peptide comprises four histidine residues in its N-terminal part, arranged such that two histidine residues are substituted at positions one and two of SEQ ID NO: 1 , two histidine residues are substituted at positions four and five of SEQ ID NO: 1 and also two histidine residues are added to its N-terminus.

[0079] In a further embodiment, the cell penetrating peptide comprises four histidine residues in its N-terminal part, arranged such that two histidine residues are substituted at positions one and two of SEQ ID NO: 1 , two histidine residues are substituted at positions four and five of SEQ ID NO: 1 and also four histidine residues are added to its N-terminus.

[0080] In yet a further embodiment, the cell penetrating peptide comprises at least five histidine residues in its N-terminal part and / or added to its N terminus, arranged such that five histidine residues are substituted at positions one to five of SEQ ID NO: 1 , and optionally one or more histidine residues are added to its N-terminus.

[0081] In another embodiment, the cell penetrating peptide comprises five histidine residues in its N- terminal part, arranged such that five histidine residues are substituted at positions one to five of SEQ ID NO: 1 and also one histidine residue is added to its N-terminus.

[0082] In a further embodiment, the cell penetrating peptide comprises two or more histidine residues substituting two or more amino acids at any of positions 1 to 5 of SEQ ID NO: 1 , SEQ ID NO: 2 or SEQ ID NO: 3. In a yet further embodiment, said substitution is at any of positions 2 to 5 of SEQ ID NO: 1 , SEQ ID NO: 2 or SEQ ID NO: 3, in particular at any of positions 3 to 5.

[0083] In some embodiments, the cell penetrating peptide may have the sequence of any one of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16. Cell penetrating peptides of SEQ ID NOs: 1 and 13-17 herein are described in WO 2020 / 144317 (as SEQ ID NOs: 1-6 therein), the sequences and results of which are hereby specifically incorporated by reference.

[0084] In particular alternative embodiments of the present invention, the cell penetrating peptide comprises or consists of:

[0085] AGYLLG Xi* INLKALAALA X2IL (SEQ ID NO: 2).

[0086] According to these embodiments:

[0087] Xi represents Lys (K), Orn (O), Dab or Dap,

[0088] * indicates that the peptide optionally continues from the side chain amino group and not from the a-amino group, and

[0089] X2represents KA or AK.

[0090] In a further embodiment, Xi is K, Orn (O) or Dab. In a particular embodiment Xi is Dab. In a yet further embodiment, the peptide continues from the side chain amino group and not from the a-amino group at the * position. In a still further embodiment, X2is KA.

[0091] In another embodiment, the cell penetrating peptide sequence comprises or consists of: AGYLLG Xi* LKALAALA X2IL (SEQ ID NO: 3).

[0092] According to this embodiment:

[0093] Xi represents K, Orn (O), Dab or Dap,

[0094] * indicates that the peptide continues from the side chain amino group and not from the a-amino group, and

[0095] X2represents KA or AK.

[0096] In a further embodiment, Xi is Orn (O) or Dab. In a particular embodiment Xi is Dab. In a particular embodiment, X2is KA. In another particular embodiment, X2is AK.

[0097] As previously described in WO 2024 / 074553, the inventors have previously found that cell penetrating peptides according to SEQ ID NOs: 2 and 3 have surprisingly good ability to deliver mRNA cargo into the intracellular compartment, in particular into cells of the spleen in vivo. Such delivery was predicted to be efficient for raising an immune response in a subject, such as a priming or vaccination response, since professional antigen presenting cells (APCs), such as dendritic cells (DCs), relocate to secondary lymphoid organs like the spleen and lymph nodes following exposure to antigen and the spleen and lymph nodes contain the largest number of APCs (including DCs) in mammals.

[0098] Furthermore and also as described in WO 2024 / 074553, the inventors have previously found that cell penetrating peptides according to SEQ ID NO: 3 have good ability to deliver cargo into an intracellular compartment, such as cargoes selected from: mRNA, plasmid DNA (pDNA), small-interfering RNA (siRNA), micro RNA (miRNA), or any other coding or noncoding nucleic acid, as well as peptides, proteins, non-peptidic pharmaceutical agents, polysaccharides, lipids (including lipoproteins and glycolipids), small molecule drugs and imaging agents. Such delivery is predicted to be efficient for raising an immune response in a subject, such as a priming or vaccination response, since professional antigen presenting cells (APCs), such as dendritic cells (DCs), relocate to secondary lymphoid organs like the spleen and lymph nodes following exposure to antigen and the spleen an lymph nodes contain the largest number of APCs (including DCs) in mammals, in particular when the cargo is mRNA encoding an antigenic protein or peptide.

[0099] Therefore, the formulations and their uses prepared according to the method described herein are likely to be greatly improved compared to commonly used liposome (LNP) delivery systems which target cargo to liver tissue and comprise polyethylene glycol (PEG), and thus have side effects and toxicity after systemic administration (Semple et al. (2010); and Dong et al. (2014)). By effectively delivering nucleic acid cargo (e.g. mRNA) to the spleen and lymph nodes, the presently prepared formulations yield higher efficiency and lower toxicity than liposomes / LNPs. Furthermore, because of their reduced toxicity, the formulations may be used to deliver higher doses of cargo, in particular mRNA, than liposomes / LNPs which to date have used a low dose due to their toxicity, side effects and liver-restricted delivery. As such, formulations prepared according to the method described herein may also find utility in methods of treating chronic diseases.

[0100] Thus, all formulations prepared according to the method described herein find particular utility in delivering nucleic acid constructs which are large in size and linear, such as messenger RNA (mRNA), and which encode an antigenic protein or peptide. It is therefore expected that the formulations prepared according to the method described herein may be effectively used for delivering mRNA encoding antigenic proteins or peptides in methods of raising an immune response to the antigenic proteins / peptides in a subject, or may be administered to a subject as part of a method for raising such an immune response in said subject.

[0101] In some embodiments, the cell penetrating peptide sequence comprises one or more further amino acid substitution at positions 9 to 21 of SEQ ID NO: 2 or at positions 9 to 19 of SEQ ID NO: 3.

[0102] Thus, in some embodiments the cell penetrating peptide comprises or consists of the sequence of:

[0103] AGYLLGO*INLKALAALAKAIL (SEQ ID NO: 4); AGYLLGK*INLKALAALAKAIL (SEQ ID NO: 5);

[0104] AGYLLGDab*INLKALAALAKAIL (SEQ ID NO: 6); AGYLLGDabINLKALAALAKAIL (SEQ ID NO: 7);

[0105] AGYLLGDap*INLKALAALAKAIL (SEQ ID NO: 8); or AGYLLGO*INLKALAALAAKIL (SEQ ID NO: 9), wherein * indicates that the peptide continues from the side chain amino group and not from the a-amino group.

[0106] In further embodiments, the cell penetrating peptide comprises the sequence of: AGYLLGO*LKALAALAKAIL (SEQ ID NO: 10);

[0107] AGYLLGDab*LKALAALAKAIL (SEQ ID NO: 11); or AGYLLGDab*LKALAALAAKIL (SEQ ID NO: 12), wherein * indicates that the peptide continues from the side chain amino group and not from the a-amino group, In particular embodiments, the cell penetrating peptide comprises or consists of the sequence of:

[0108] AGYLLGO*INLKALAALAKAIL (SEQ ID NO: 4);

[0109] AGYLLGK*INLKALAALAKAIL (SEQ ID NO: 5);

[0110] AGYLLGDab*INLKALAALAKAIL (SEQ ID NO: 6); or AGYLLGO*INLKALAALAAKIL (SEQ ID NO: 9).

[0111] In a further particular embodiment, the cell penetrating peptide comprises or consists of the sequence of:

[0112] AGYLLGDab*LKALAALAKAIL (SEQ ID NO: 11).

[0113] As demonstrated in WO 2024 / 074553, penetrating peptide sequences according to these embodiments provide better or equivalent delivery of an mRNA cargo than the existing cell penetrating peptides, NF55 and NF54. In particular, certain cell penetrating peptides comprising sequences of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9 or SEQ ID NO: 11 provided significantly better delivery of an mRNA cargo, leading to significantly increased activity of the expressed protein (i.e. increased expression of said protein), compared to NF55.

[0114] In a further embodiment, the cell penetrating peptide is optionally chemically modified at the C-terminus. Such C-terminal modifications include amidation, such as wherein the C-terminus is CONH2. Thus, in a particular embodiment the C-terminus of the cell penetrating peptide is CONH2.

[0115] The cell penetrating peptide may be further modified to add specific targeting elements, such as for targeting the membrane-permeable constructs described herein to specific cells or organ(s). In certain embodiments, the targeting element may be for a surface receptor or ligand present on cells of the spleen. In further certain embodiments, the targeting element may be for a surface receptor or ligand present on cells of the lymph nodes. Such targeting elements may be for a surface receptor found on cells of both the spleen and lymph nodes. In a particular embodiment, the targeting element is for a surface receptor or ligand on dendritic cells. Targeting elements include and may therefore be selected from any one or more of the following: peptides and / or proteins (including antibodies and fragments thereof, such as antigen binding fragments (Fabs) and heavy chain only / VHH fragments) and aptamers. According to the present invention, the cell penetrating peptide is provided in ethanol (EtOH). As has been previously described, formulations prepared in water form large elongated micelles and a substantial number of peptide aggregates which do not form nanoparticles. By contrast, such aggregates are avoided if the peptide is provided in an ethanol solution prior to association / complexation. The avoidance of large aggregates is desired as these are unable to form nanoparticle complexes / formulations with the cargo and may lead to adverse effects in vivo upon administration. Avoiding the formation of aggregates also prevents the need to optionally remove them by filtration, which adds time and cost to a preparation method and can lead to a loss of material. Other suitable dry alcohols may be used as the solvent in place of ethanol, such as any branched chain alcohol, including methanol, ethanol, 1- propanol, 1 -butanol, 1 -pentanol, 1 -hexanol, 2-propanol, 2-butanol, 2-pentanol, 2-hexanol, tertbutanol, and 2-ethyl 1 -hexanol. The identity of the alcohol may also depend on the fatty acid present at the N-terminus of the cell penetrating peptide, for example wherein the longer the fatty acid, the longer the alcohol chain.

[0116] The cell penetrating peptides herein are also provided free from / without an aprotic solvent. Whilst the use of aprotic solvents has been previously described (WO 2023 / 277779), they carry risk of adverse reaction upon administration. The terms “free from” or “without” herein refer to wherein the cell penetrating peptide in ethanol is essentially or substantially free from aprotic solvent. That is that no aprotic solvent is added to the peptide in ethanol or during the dissolving / preparation of the peptide in ethanol. The cell penetrating peptide in ethanol may therefore comprise less than 10% aprotic solvent, less than 5%, or up to or less than 1%, such as 0% or no aprotic solvent.

[0117] Fatty Acids

[0118] A fatty acid is a carboxylic acid with a long aliphatic chain which is either saturated or unsaturated. Short chain fatty acids are fatty acids with aliphatic tails of five or fewer carbon atoms. Medium chain fatty acids are fatty acids with aliphatic tails of six to twelve carbon atoms. Long chain fatty acids are fatty acids with aliphatic tails of 13 to 21 carbon atoms. Very long chain fatty acids are fatty acids with aliphatic tails of 22 carbon atoms or more.

[0119] The cell penetrating peptides comprised in the formulations described herein may, in particular embodiments comprise a fatty acid chain attached to the N-terminus. The fatty acid chain may be a saturated carbon chain or an unsaturated carbon chain, but preferably is a saturated carbon chain. Saturated fatty acids have no carbon to carbon double bonds. Unsaturated fatty acids have one or more (e.g. one to four such as one or two) carbon to carbon double bonds. The carbon to carbon double bonds can give either cis or trans isomers. A c / s configuration means that two hydrogen atoms adjacent to the double bond protrude out on the same side of the chain. The rigidity of the double bond freezes its conformation and in the case of the c / s isomer, causes the chain to bend and restricts the conformational freedom of the fatty acid. The more double bonds the chain has in the c / s configuration, the less flexibility it has.

[0120] A trans configuration means that the adjacent two hydrogen atoms lie on opposite sides of the chain. Consequently, they do not cause the chain to bend much, and their shape is similar to straight saturated fatty acids.

[0121] A saturated fatty acid chain having 16 carbon atoms is also referred to as palmitic acid. A saturated fatty acid chain having 18 carbon atoms is also referred to as stearic acid. A saturated fatty acid chain having 20 carbon atoms is also referred to as arachidic acid. A saturated fatty acid chain having 22 carbon atoms is also referred to as behenic acid.

[0122] An example of an unsaturated fatty acid having 16 carbon atoms is palmitoleic acid or sapienic acid. An example of an unsaturated fatty acid having 18 carbons atoms is oleic acid or elaidic acid. An example of an unsaturated fatty acid having 20 carbon atoms is arachidonic acid or eicosapentaenoic acid. An example of an unsaturated fatty acid having 22 carbon atoms is erucic acid or docosahexaenoic acid.

[0123] The cell penetrating peptides herein have fatty acid chains attached thereto. The fatty acid chain may also be 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, the fatty acid is attached to the N-terminus of the cell penetrating peptide. In a still preferred embodiment, the fatty acid is attached to the N-terminal amine of the amino acid at position 1 , such as position 1 of SEQ ID NO: 1 , SEQ ID NO: 2 or SEQ ID NO: 3.

[0124] A linker may separate the peptide and the fatty acid chain. The linker may be a chemical moiety that contains two reactive groups / functional groups, one of which can react with the peptide and the other with the fatty acid chain. The two reactive / functional groups of the linker are linked via a linking moiety or spacer wherein 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 made up of amino acids linked together by peptide bonds. 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 a certain embodiment, the fatty acid chain has 16, 18 or 20 carbon atoms. In a particularly preferred embodiment, the fatty acid chain has 18 carbon atoms.

[0125] Thus, in some embodiments the cell penetrating peptide comprises or consists of:

[0126] C16-(SEQ ID NO: 4) (also referred to herein as NF419);

[0127] C18-(SEQ ID NO: 4) (also referred to herein as NF55);

[0128] C20-(SEQ ID NO: 4) (also referred to herein as NF410);

[0129] C22-(SEQ ID NO: 4) (also referred to herein as NF411);

[0130] C16-(SEQ ID NO: 5) (also referred to herein as NF420);

[0131] C18-(SEQ ID NO: 5) (also referred to herein as NF554);

[0132] C20-(SEQ ID NO: 5) (also referred to herein as NF412);

[0133] C22-(SEQ ID NO: 5) (also referred to herein as NF413);

[0134] C16-SEQ ID NO: 6) (also referred to herein as NF422);

[0135] C18-(SEQ ID NO: 6) (also referred to herein as NF553);

[0136] C22-(SEQ ID NO: 6) (also referred to herein as NF423);

[0137] C18-(SEQ ID NO: 7) (also referred to herein as NF559);

[0138] C18-(SEQ ID NO: 8) (also referred to herein as NF550);

[0139] C18-(SEQ ID NO: 9) (also referred to herein as NF54);

[0140] C18-(SEQ ID NO: 10) (also referred to herein as NF430);

[0141] C20-(SEQ ID NO: 10) (also referred to herein as NF437);

[0142] C16-(SEQ ID NO: 11) (also referred to herein as NF425);

[0143] C18-(SEQ ID NO: 11) (also referred to herein as NF424);

[0144] C20-(SEQ ID NO: 11) (also referred to herein as NF426);

[0145] C18-(SEQ ID NO: 12) (also referred to herein as NF436); or C20-(SEQ ID NO: 12) (also referred to herein as NF438).

[0146] In particular, the cell penetrating peptide may comprise or consist of:

[0147] C22-(SEQ ID NO: 4) (also referred to herein as NF411);

[0148] C18-(SEQ ID NO: 5) (also referred to herein as NF554);

[0149] C18-(SEQ ID NO: 6) (also referred to herein as NF553);

[0150] C18-(SEQ ID NO: 9) (also referred to herein as NF54);

[0151] C18-(SEQ ID NO: 11) (also referred to herein as NF424);

[0152] C20-(SEQ ID NO: 11) (also referred to herein as NF426); or C18-(SEQ ID NO: 12) (also referred to herein as NF436).

[0153] In one embodiment, the cell penetrating peptide comprises or consists of the sequence of SEQ ID NO: 4 and a fatty acid chain having 22 carbon atoms, e.g. wherein the fatty acid is behenic acid. This cell penetrating peptide is also referred to herein as NF411.

[0154] In another embodiment, the cell penetrating peptide comprises or consists of the sequence of SEQ ID NO: 5 and a fatty acid chain having 18 carbon atoms, e.g. wherein the fatty acid is stearic acid. This cell penetrating peptide is also referred to herein as NF554.

[0155] In another embodiment, the cell penetrating peptide comprises or consists of the sequence of SEQ ID NO: 6 and a fatty acid chain having 18 carbon atoms, e.g. wherein the fatty acid is stearic acid. This cell penetrating peptide is also referred to herein as NF553.

[0156] In another embodiment, the cell penetrating peptide comprises or consists of the sequence of SEQ ID NO: 9 and a fatty acid chain having 18 carbon atoms, e.g. wherein the fatty acid is stearic acid. This cell penetrating peptide is also referred to herein as NF54.

[0157] In another embodiment, the cell penetrating peptide comprises or consists of the sequence of SEQ ID NO: 11 and a fatty acid chain having 18 carbon atoms, e.g. wherein the fatty acid is stearic acid. This cell penetrating peptide is also referred to herein as NF424.

[0158] In another embodiment, the cell penetrating peptide comprises or consists of the sequence of SEQ ID NO: 11 and a fatty acid chain having 20 carbon atoms, e.g. wherein the fatty acid is arachidic acid. This cell penetrating peptide is also referred to herein as NF426.

[0159] In another embodiment, the cell penetrating peptide comprises or consists of the sequence of SEQ ID NO: 12 and a fatty acid chain having 18 carbon atoms, e.g. wherein the fatty acid is stearic acid. This cell penetrating peptide is also referred to herein as NF436.

[0160] In other embodiments, the cell penetrating peptide comprises or consists of:

[0161] C18-(SEQ ID NO: 10) (also referred to herein as NF430);

[0162] C20-(SEQ ID NO: 10) (also referred to herein as NF437);

[0163] C16-(SEQ ID NO: 11) (also referred to herein as NF425);

[0164] C18-(SEQ ID NO: 11) (also referred to herein as NF424);

[0165] C20-(SEQ ID NO: 11) (also referred to herein as NF426);

[0166] C18-(SEQ ID NO: 12) (also referred to herein as NF436); or C20-(SEQ ID NO: 12) (also referred to herein as NF438).

[0167] In particular, the cell penetrating peptide may comprise or consist of:

[0168] C18-(SEQ ID NO: 11) (referred to herein as NF424); C20 (SEQ ID NO: 11) (referred to herein as NF426); or C18-(SEQ ID NO: 12) (referred to herein as NF436).

[0169] In one embodiment, the cell penetrating peptide comprises or consists of the sequence of SEQ ID NO: 11 and a fatty acid chain having 18 carbon atoms, e.g. wherein the fatty acid is stearyl. This cell penetrating peptide is also referred to herein as NF424.

[0170] In another embodiment, the cell penetrating peptide comprises or consists of the sequence of SEQ ID NO: 11 and a fatty acid chain having 20 carbon atoms, e.g. wherein the fatty acid is arachidic acid. This cell penetrating peptide is also referred to herein as NF426.

[0171] In another embodiment, the cell penetrating peptide comprises or consists of the sequence of SEQ ID NO: 12 and a fatty acid chain having 18 carbon atoms, e.g. wherein the fatty acid is stearic acid. This cell penetrating peptide is also referred to herein as NF436.

[0172] Cell penetrating peptides of SEQ ID NOs: 2-12 herein are described in WO 2024 / 074553 (as SEQ ID NOs: 1-11 therein), the sequences and results of which are hereby specifically incorporate by reference.

[0173] Cargo

[0174] A cargo molecule may be a substance associated with a cell penetrating peptide intended to be transported into a cell. The cargo molecule may be associated with the cell penetrating peptide either through chemical linkage via covalent bonds, or through non-covalent bond or ionic bonds or non-covalent interactions or ionic interactions. The cargo associated with the cell penetrating peptide may be transported from outside of the cell, across the membrane of the cell and enter into the cell. The cargo may then be released into the cytoplasm of the cell, directed to an intracellular organelle or presented at the intracellular or extracellular cell surface. The cargo associated with the cell penetrating peptide (forming a construct or complex or nanoparticle) may enter the cell via the endosomal pathway. The construct, complex or nanoparticle may then be encapsulated in an intra-organelle, preferably in an endosomal compartment or in a lysosome, and subsequently released into the intracellular compartment or environment. Cargoes include peptides, proteins, non-peptidic pharmaceuticals, polysaccharides, lipids (including combinations thereof including lipoproteins and glycolipids), nucleic acids (e.g. DNA, messenger RNA (mRNA), 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 may comprise one or more peptides, polypeptides or proteins linked together. The peptide may be selected from a group consisting of, but not limited to, a cell or tumour targeting peptide, an aptamer, a receptor ligand, a peptide ligand, a cytotoxic peptide, a bioactive peptide, an antibody, and a diagnostic agent. When the cargo molecule is a nucleic acid, the nucleic acid may comprise one or more nucleic acids where each one encodes one peptide or polypeptide. The cargo molecule may be a combination of a protein, a lipid and / or a polysaccharide including lipoproteins and glycolipids. The cargo may be selected from a group consisting of, but not limited to, oligonucleotides including single-stranded oligonucleotides (e.g. DNA, RNA, PNA, LNA and their analogues), double-stranded oligonucleotides (e.g. siRNA, shRNA, microRNA and decoyDNA) and cyclic DNA (e.g. plasmids, such as pDNA). Thus, in certain embodiments of the present invention the cargo is a nucleic acid. In further certain embodiments, the cargo is mRNA.

[0175] In one embodiment, the cell penetrating peptide described herein comprises a cargo covalently attached thereto. In the case of covalent attachment, a linker moiety may optionally be present between the cell penetrating peptide and the cargo molecule, i.e. such a linker may link or connect the cell penetrating peptide and the cargo molecule together. The covalent attachment e.g. the linker moiety may be biodegradable to facilitate release of the cargo into the intracellular compartment or environment.

[0176] In a further embodiment, the formulations / complexes herein comprise the cell penetrating peptide and a cargo non-covalently interacting therewith, for example via ionic interactions. Such cargo suitably is ionic and carries negative charges. In a certain embodiment, the formulation / complex forms a nanoparticle. The complex self-assembles into a nanoparticle. The nanoparticle of the present invention comprises the amphiphilic cell penetrating peptide sequence and associated cargo, the nanoparticle being intact upon entry into the cell. The nanoparticle of the present invention may for example be 20 to 400nm in diameter, preferably 80 to 300nm in diameter, more preferably 90 to 200nm in diameter. In particular as shown in Table 1 herein, the formulation in the form of a nanoparticle may be around 100 to 200nm in diameter, such as around 150nm in diameter, both when freshly prepared and following freezing according to the method described herein. However, as will be readily appreciated the final exact size of the nanoparticle will depend on the nucleic acid (e.g. mRNA; in particular its size / length) associated / complexed with the cell penetrating peptide. Upon entry into the cell, the nanoparticle may undergo phase transition, for example inside endosomes / lysosomes, which provide a sufficiently low pH environment, thereby releasing the associated cargo into the cell.

[0177] In a preferred embodiment, the cargo non-covalently interacts (e.g. through ionic interactions) with the peptide and forms a complex. In one embodiment, an siRNA cargo non-covalently interacts (e.g. via ionic interactions) with the peptide and forms a complex. In a further embodiment, the complex formed of siRNA non-covalently interacting (e.g. ionically interacting) with the peptide, forms a nanoparticle. In a particular embodiment, an mRNA cargo non-covalently interacts (e.g. via ionic interactions) with the peptide and forms a complex. In a further embodiment, the complex formed of mRNA non-covalently interacting (e.g. ionically interacting) with the peptide, forms a nanoparticle.

[0178] In certain embodiments, an mRNA cargo may encode an antigenic protein or peptide. mRNA encoding an antigenic protein or peptide delivered as described herein is predicted to be particularly useful for raising an immune response in a subject, such as a mammalian subject (e.g. a human). Thus, the cargo may form part of an mRNA vaccine, such that the formulation described herein with an mRNA cargo is an mRNA vaccine. In a further embodiment, the mRNA cargo encodes an antigenic protein or peptide for raising an immune response in a subject. In certain embodiments, the antigenic protein or peptide is a surface protein of a pathogen, such as a virus or bacteria surface protein. Thus, in a particular embodiment the antigenic protein or peptide is a virus or bacteria surface protein. Virus surface proteins include those which make up the capsid or virus envelope, e.g. a capsid protein and / or a virus envelope protein. In a particular embodiment, the antigenic protein or peptide is a vial glycoprotein. Such glycoproteins include the spike and haemagglutinin proteins, e.g. the spike protein of a coronavirus, in particular CARS-CoV2. Thus, in one embodiment the antigenic protein or peptide is the spike protein of a coronavirus, in particular CARS-CoV2. In other embodiments, the antigenic protein or peptide is a surface protein, such as the spike protein, of influenza, respiratory syncytial virus (RSV) or tick-borne encephalitis (TBE). In further other embodiments, the antigenic protein or peptide is a bacterial surface protein. In still other embodiments, the antigenic protein or peptide is a surface protein of a parasite.

[0179] In an alternative embodiment, the antigenic protein or peptide may be an antigen associated with cancer, e.g. a cancer marker protein. Such cancer-associated antigens may be suitably identified and selected depending on the particular cancer to be targeted. Thus, in further embodiments formulations prepared according to the method of the present invention may be for use in a method of raising an anti-cancer immune response in a subject. Thus, the subject may be suffering from cancer.

[0180] According to the present method, associating the cell penetrating peptide with a cargo is performed in an association buffer, thereby forming the cell penetrating peptide-cargo formulation. The association buffer will therefore be appreciated to be a suitable solution for providing and optionally storing the cargo prior to association / complexation or into which the cargo is transferred from storage for association / complexation. A particular example of an association buffer for use according to the present method is a buffer containing HEPES, optionally a saline buffer comprising HEPES. In one embodiment, the association buffer comprises around 10mM HEPES, such as 10mM HEPES. HEPES (4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid) is a zwitterionic sulfonic acid buffering agent and maintains physiological pH. In further embodiments, the association buffer comprises and / or additionally comprises salts, such as wherein the association buffer is a HEPES buffered saline (HBS) or in the form of phosphate buffered saline (PBS).

[0181] The association buffer herein comprises a sugar at a concentration between 30% and 90%, in particular between 50% and 90% (weight / weight (w / w), weight / volume (w / v) or volume / volume (v / v)). As demonstrated herein, an association buffer comprising sugar provides good association / complexation of the cell penetrating peptide with cargo in the present method, yielding small and uniform formulation nanoparticles which are suitable for freezing and optionally lyophilisation, as required for storage. The 30-90%, in particular 50-90% sugar (e.g. sucrose) may act as a crowding agent to increase the association / complexation efficiency of the cargo with cell penetrating peptide and the forming of nanoparticles. Any suitable crowding agent and / or sugar may be used, such as and in particular sucrose. Furthermore, the sugar present in the association buffer may act as a cryoprotective agent when the formulation is frozen and optionally lyophilised. Any suitable cryoprotective agent and / or sugar may be used, such as and in particular sucrose. Thus, in certain embodiments the association buffer comprises between 30% and 90% sucrose, in particular between 50% and 90% sucrose. For example, the association buffer may comprise sucrose at a concentration of 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, between 30% and 90%, between 30% and 80%, between 30% and 75%, between 30% and 70%, between 30% and 65%, between 30% and 60%, between 40% and 90%, between 40% and 80%, between 40% and 75%, between 40% and 70%, between 40% and 65%, between 40% and 60%, between 45% and 90%, between 45% and 80%, between 45% and 75%, between 45% and 70%, between 45% and 65%, between 45% and 60%, between 50% and 80%, between 50% and 75%, between 50% and 70%, between 50% and 65%, between 50% and 60%, between 55% and 90%, between 55% and 80%, between 55% and 75%, between 55% and 70%, between 55% and 65%, between 55% and 60%, between 60% and 90%, between 60% and 80%, between 60% and 75%, between 60% and 70%, between 60% and 65%, around 55%, around 60%, around 65%, around 70% or around 75%. In particular, the association buffer may comprise around 60% sucrose, such as 60% sucrose. Thus, in a certain embodiment the association buffer comprises about 60% sucrose and 10mM HEPES. As demonstrated in herein, formulations prepared using an association buffer comprising 60% sucrose provided improved transfection efficiency compared to formulations prepared in association buffers with no or lower concentrations of sucrose. Alternatively in particular, the association buffer may comprise around 65% sucrose, such as 65% sucrose. Alternatively in particular, the association buffer may comprise around 70% sucrose, such as 70% sucrose. Alternatively in particular, the association buffer may comprise around 75% sucrose, such as 75% sucrose. Thus, in further certain embodiments the association buffer comprises about 65%, about 70% or about 75% sucrose in 10mM HEPES. Association buffers comprising such concentrations of sucrose are demonstrated herein to provide formulations with good transfection efficiency compared to formulations prepared in association buffers with no or alternative sucrose concentrations.

[0182] Also according to the present method, the cargo and association buffer (i.e. cargo in association buffer) are used at a volume between 2x and 4x that of the cell penetrating peptide in ethanol, in particular at a volume 3x that of the cell penetrating peptide in ethanol. In other words, the cargo and association buffer are used at a ratio amount of between 2 and 4 parts to 1 part cell penetrating peptide in ethanol, in particular 3 parts cargo and association buffer to 1 part cell penetrating peptide in ethanol. Alternatively, the volume of cell penetrating peptide in ethanol is between 0.5x and 0.25x that of the cargo in association buffer, in particular 0.33x that of the cargo in association buffer. In other words, the cargo and association buffer are at a ratio amount of 2-4:1 , in particular 3:1 , to the cell penetrating peptide in ethanol (cargo in association bufferCPP in ethanol), or the cell penetrating peptide in ethanol is at a ratio amount of 1 :2-4, in particular 1 :3, to the cargo in association buffer (CPP in ethanokcargo in association buffer). Said ratio amount is preferably v / v, such that the cargo and association buffer are preferably at a volume ratio of 2-4:1 , in particular 3:1 , to the cell penetrating peptide in ethanol, or such that the cell penetrating peptide in ethanol is at a volume ratio of 1 :2-4, in particular 1 :3, to the cargo in association buffer. By way of non-liming example, described herein is the association / complexation of 2.5pl cell penetrating peptide in ethanol with 7.5pl nucleic acid cargo in association buffer (10mM HEPES and 60% sucrose). The volume of associating step (ii) herein is between 0.5x and 0.05x the final volume at which the formulation is expected to be prepared and / or resuspended for subsequent administration, in particular between 0.5x and 0.1x the final volume. In other words, associating step (ii) is performed at between half and 1 / 20thof the final volume, in particular between half and 1 / 10thof the final volume, more particularly 1 / 10thof the final volume of administration. The small volume in which associating step (ii) is performed results in a very low concentration of or no ethanol in the frozen or lyophilised formulation, as well as in the final formulation following dilution or resuspension of the formulation as described herein. Due to the known toxicity of ethanol in formulations for administration in vivo, as low concentration in the final diluted or resuspended formulation for administration as possible is desired. The final concentration of sugar (e.g. sucrose) in the frozen, lyophilised, diluted or resuspended formulation is also low. By way of non-limiting example, described herein are ‘fresh’ and frozen nanoparticle formulations comprising final ethanol concentrations of 2.5% and lyophilised formulations comprising no or 0% ethanol. Also described herein are said formulations comprising a final sucrose concentration of 3-4%. The low final ethanol concentration achieved in frozen and lyophilised formulations by the present method is in contrast to ‘fresh’ formulations prepared according to the method described in WO 2023 / 277779 which contain around 1 % final concentration ethanol and cannot be frozen lyophilised as described hereinbefore. The use of a small volume is also hypothesised to increase the crowding effect of the cell penetrating peptide with the cargo and thus increase the efficiency of association / complexation and the forming of nanoparticles. Thus, in embodiments associating step (ii) is performed in a volume between 0.5x and 0.05x the final volume at which the formulation is prepared for administration. In certain embodiments, associating step (ii) is performed in a volume between 0.5x and 0.1x the final volume at which the formulation is prepared for administration. In a particular embodiment, associating step (ii) is performed in a volume 0.1x the final volume at which the formulation is prepared for administration. “Final volume for administration” as used herein refers to the volume in which the formulation is diluted (e.g. following association step (ii) without freezing or following freezing without lyophilisation) or resuspended (e.g. following lyophilisation) after storage, such as in preparation for administering to a subject according to the methods of and uses in raising an immune response or vaccination described herein. Thus, in further embodiments the method may additionally comprise step (iv) of diluting the cell penetrating peptide-cargo formulation or resuspending / reconstituting the lyophilised formulation in a total volume between 2x and 20x the volume at which step (ii) is performed. In certain embodiments, the formulation is diluted or resuspended / reconstituted in a total volume between 2x and 10x the volume at which step (ii) is performed. In particular, the formulation is diluted or resuspended / reconstituted in a total volume 10x that at which step (ii) is performed. Said dilution or resuspension / reconstitution may be in any suitable buffer as will be recognised by the skilled person, such as a saline buffer and in particular a buffer comprising HEPES, for example 10mM HEPES. Dilution or resuspension / reconstitution may further comprise any pharmaceutically acceptable carriers, diluents and / or excipients as described herein. In one embodiment, the formulation is diluted or resuspended / reconstituted in a buffer comprising 10mM HEPES without sucrose.

[0183] Cell Penetrating Peptide-Cargo Formulations, Pharmaceutical Compositions and Therapeutic Uses / Methods Thereof

[0184] According to one aspect of the present invention, there is provided a cell penetrating peptide- cargo formulation obtainable and / or obtained by the preparation method described herein.

[0185] The formulations of the invention may be formulated for delivery in pharmaceutical compositions. Thus, in another aspect there is provided a pharmaceutical composition comprising the cell penetrating peptide-cargo formulation as described herein. In certain embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents and / or excipients. In further embodiments, the pharmaceutical composition optionally further comprises one or more adjuvants. Suitable adjuvants, excipients and / or diluents will be readily recognised and selected by the skilled person based on their common general knowledge and the planned route of administration for the formulation and / or pharmaceutical composition.

[0186] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The carrier may be suitable for parenteral (e.g. topical), oral, nasal, intravenous, intramuscular, intradermal, intracranial, intraocular, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. For parenteral administration, the carrier preferably comprises water and may contain buffers for pH control, stabilising agents (e.g. surfactants and amino acids) and tonicity modifying agents (e.g. salts and sugars). If the formulation / composition is intended to be provided in lyophilised form for dilution at the point of use, it may contain a lyoprotectant, e.g. sugars such as trehalose. For oral administration, any of the above carriers or a solid carrier, such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose and magnesium carbonate, may be employed. Alternatively, the carrier may be suitable for non-parenteral administration, such as a topical, epidermal or mucosal route 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.

[0187] Thus, formulations and compositions of the invention may comprise buffers (e.g. neutral buffered saline, phosphate buffered saline or HEPES), carbohydrates (e.g. glucose, mannose, sucrose or dextrans), 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 a recipient, suspending agents, thickening agents and / or preservatives. Alternatively and in particular, formulations and compositions of the invention may be formulated as a lyophilizate.

[0188] The pharmaceutical compositions of the invention may include 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 any undesired toxicological effects. Examples of such salts include acid addition salts and base addition salts. Such salts may 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).

[0189] Pharmaceutically acceptable carriers comprise aqueous carriers or diluents. Examples of suitable aqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, buffered water and saline. Examples of other carriers include ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. In many cases, it will be desirable to include isotonic agents, for example sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.

[0190] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome or other ordered structure suitable to high drug concentration. Pharmaceutical compositions of the invention may comprise additional active ingredients. In therapeutic applications, compounds are administered to a subject already suffering from a disorder or condition as described above, in an amount sufficient to cure, alleviate or partially arrest the condition or one or more of its symptoms. Such therapeutic treatment may result in a decrease in severity of disease symptoms, or an increase in frequency or duration of symptom-free periods. An amount adequate to accomplish this is defined as a "therapeutically effective amount". In prophylactic applications, formulations are administered to a subject at risk of a disorder or condition as described herein, in an amount sufficient to prevent or reduce the subsequent effects of the condition 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 as well as the weight and general state of the subject.

[0191] A subject for administration may be a human or 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.

[0192] A pharmaceutical composition of the present invention may 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 the skilled person, the route and / or mode of administration will vary depending upon the desired results. Examples of routes of administration for compounds or pharmaceutical compositions of the invention include intravenous, intramuscular, intradermal, intraocular, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase “parenteral administration” as used herein mean modes of administration other than enteral and topical administration, usually by injection. Alternatively, the pharmaceutical composition of the invention can be administered via a non-parenteral route, such as topical, epidermal or mucosal route of administration.

[0193] A suitable dose of the pharmaceutical composition of the invention may be determined by a skilled medical practitioner. Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desire therapeutic response for a particular patient, composition and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts. Dosage regimens may be adjusted to provide the optimum desired response, for example a therapeutic response. For example, as single dose may be administered, several divided doses maybe administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0194] 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 location. Alternatively, doses can be via 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 duration of treatment desired.

[0195] In certain preferred embodiments of the present invention, pharmaceutical compositions as described herein are provided which comprise one or more (e.g. one) cell penetrating peptide- cargo formulations prepared according to the method described herein in combination with a pharmaceutically acceptable carrier.

[0196] The cell penetrating peptide-cargo formulation is membrane-permeable. Thus, it may pass from the extracellular environment across or through a membrane (e.g. a lipid membrane) into a cell or intracellular environment. Membranes may be single or multiple layer structures (e.g. a lipid bilayer). The CPP may transport the cargo across a membrane and deliver the cargo into the cytoplasm of the cell. The CPP may also transport the cargo across a membrane and deliver the cargo to an organelle in the cell. Alternatively or additionally, the CPP may transport cargo across a membrane and deliver the cargo to an intracellular surface. The CPP may enter the cell via an endosomal pathway. The CPP may be encapsulated in an intraorganelle, preferably in an endosomal compartment or in a lysosome, and subsequently be released into the intracellular compartment or environment.

[0197] A membrane may be an artificial membrane such as an artificially constructed complex membrane formed of, for example, lipids, phospholipids or molecules having both hydrophilic and hydrophobic compounds or structures. Alternatively, a membrane may be a biological membrane, including but not limited to eukaryotic cell membranes and prokaryotic cell membranes. A membrane may be a lipid bilayer or phospholipid bilayer. A membrane may be a lipid membrane of a phospholipid membrane. A membrane may be a plasma membrane, such as the plasma membrane of a cell. Thus, in one aspect of the invention the formulations and pharmaceutical compositions disclosed herein are for transport of cargo across a lipid membrane and subsequent delivery of said cargo to a cell. In another aspect, the formulations and pharmaceutical compositions are for use in such transport. Eukaryotic cell membranes include, but are not limited to, membranes of: immune cells, such as white blood cells, red blood cells, monocytes, macrophages, neutrophils, T cells, B cells or dendritic cells; epithelial cells; endothelial cells; keratinocytes; muscle cells; skin cells; nerve cells and fat cells.

[0198] In certain embodiments, the cell is in vivo. The cell may be a cell of the spleen and / or lymph nodes, such as a dendritic cell (DC). As such, the cell penetrating peptide-cargo formulations for use as described herein may transport cargo across a lipid membrane and subsequently deliver said cargo into a cell of the spleen and / or lymph nodes in vivo, such as an in vivo DC. Thus, according to a further aspect of the invention there is provided the formulation or pharmaceutical composition as described herein for use in the transport of cargo across a lipid membrane and subsequent delivery of said cargo into a cell of the spleen and / or lymph nodes in vivo, such as an in vivo DC. The ability of cell penetrating peptides comprised in the formulations of the present invention to effectively deliver cargo in vivo is demonstrated in WO 2024 / 074553, from which the sequences and their uses are specifically incorporated herein by reference.

[0199] In another aspect of the invention, there is provided the formulation or pharmaceutical composition as described herein for use in a method of raising an immune response in a subject. In a further aspect, there is provided the membrane-permeable construct or the pharmaceutical composition for use in a method of vaccinating a subject, such as vaccinating said subject against an infectious disease.

[0200] As demonstrated in WO 2024 / 074553, the cell penetrating peptides comprised in the formulations of the present invention have particularly good ability to deliver mRNA cargo in the intracellular compartment of cells, specifically into cells of the spleen and / or lymph nodes in vivo, such as DCs. Thus, it is predicted that the present formulations comprising said cell penetrating peptides will find utility in the raising of an immune response in a subject, wherein the mRNA cargo encodes for an antigenic protein or peptide. This is due to the efficient delivery of such mRNA to the spleen and lymph nodes in vivo as demonstrated herein where the largest number of antigen presenting cells (APCs), such as DCs, may be found in mammals. Thus, in certain embodiments an mRNA cargo encodes an antigenic protein or peptide for raising an immune response in a subject. In a further aspect, there is provided a method of raising an immune response in a subject, said method comprising administering the formulation or pharmaceutical composition as described herein to said subject. In another aspect, there is provided a method of vaccinating a subject, said method comprising administering the formulation or pharmaceutical composition as described herein to said subject. In one embodiment, administering comprises a therapeutically effective amount as described hereinbefore. In another embodiment, administering comprises a prophylactically effective amount as described hereinbefore.

[0201] In a further aspect, there is provided a use of the formulation or pharmaceutical composition as described herein for the manufacture of a medicament. Thus, in a still further aspect there is provided use of the method of preparing a formulation comprising a cell penetrating peptide and a cargo in the manufacture of a medicament. In a particular embodiment, the medicament is a vaccine. Thus, in another aspect there is provided a use of the formulation, pharmaceutical composition or method of preparation as described herein for the manufacture of a vaccine. In one embodiment, the vaccine is for raising an immune response is a subject. Thus, in certain embodiments the vaccine comprises an mRNA cargo encoding an antigenic protein or peptide for raising an immune response in a subject. In a yet further embodiment, the vaccine comprises a therapeutically effective amount of the membrane-permeable construct or pharmaceutical composition as described herein. In another embodiment, the vaccine comprises a prophylactically effective amount of the membrane-permeable construct or pharmaceutical composition as described herein.

[0202] In some embodiments, the use or administration of the formulations or pharmaceutical compositions may be therapeutic as described hereinbefore. Thus, in one embodiment the subject may be suffering from a disease or disorder and thus in need thereof of the uses in and / or methods of raising an immune response described herein. In a further embodiment, the subject is suffering from cancer. According to this embodiment the raised immune response is 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 suffering from a disease or disorder, such as an infectious disease. According to this particular embodiment, the raised immune response is against an antigen of the pathogen, such as a virus (e.g. a virus surface protein). Also according to this particular embodiment, the subject may thus require vaccination against the infectious disease (e.g. a viral disease).

[0203] Thus, in a further aspect there is provided the formulation or pharmaceutical composition as described herein for use in a method of raising an immune response against an infectious agent surface protein in a subject, such as against a virus or bacterial surface protein. In a further aspect, there is provided the formulation or pharmaceutical composition as described herein for use in a method of vaccinating a subject against a virus or bacteria surface protein. In another aspect, there is provided a method of raising an immune response against a virus or bacteria surface protein in a subject, said method comprising administering the formulation or pharmaceutical composition as described herein to said subject. In a yet further aspect, there is provided a method of vaccinating a subject against a virus or bacteria surface protein, said method comprising administering the formulation or pharmaceutical composition as described herein to said subject. In a particular embodiment of these aspects, the virus surface protein is a capsid and / or viral envelope protein, such as a viral glycoprotein.

[0204] In Vitro and Ex Vivo Uses

[0205] As described, the present formulations find particular utility in vivo. However, they may also be used to transfect cells in vitro or ex vivo, in particular for stable transfection. The formulations may be used to deliver siRNA, pDNA, mRNA, peptides and / or proteins, non- peptidic pharmaceuticals, polysaccharides, lipids, small molecule drugs and imaging agents to cells, including cells in vitro, such as in culture.

[0206] Such cells that may be transfected include, but are not limited to, the cell lines and types: HeLa, NIH 3T3, HEK-293, CHO-K1 , U2-OS and COS-7. Several hard-to-transfect cell lines, such as Jurkat, CaCo2, a human adenocarcinoma cell lines, dendritic cells and epidermis cells, may also be transfected using the membrane-permeable constructs described herein.

[0207] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. As used herein, the term “about” when used herein includes up to and including 10% greater and up to and including 10% lower than the value specified, suitably up to and including 5% greater and up to and including 5% lower than the value specified, especially the value specified. The term “between” as used herein includes the values of the specified boundaries.

[0208] Throughout the specification and the claims which 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 to the exclusion of any other integer, step, group of integers or group of steps.

[0209] In addition, as used herein and 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 penetrating peptide” includes two or more such peptides, or reference to “a cargo” or “an mRNA” include two or more such cargoes or mRNA molecules and the like.

[0210] It will be understood that all embodiments described herein may be applied to all aspects of the invention and vice versa, and such combinations would be readily apparent from the description provided herein and to those skilled in the art.

[0211] Other features and advantages of the present invention will be apparent from the description provided herein. It should be understood, however, that the description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications will become apparent to those skilled in the art. The invention will now be described using the following, non-limiting examples:

[0212] EXAMPLES

[0213] Example 1: Analysis of Nanoparticle Size and Polydispersity Index of Formulations

[0214] To compare the cell-penetrating peptide-cargo formulations resulting from the preparation method described herein with those from methods known in the art and previously described, nanoparticle sizes and polydispersity indexes were measured using Dynamic Light Scattering (DLS) immediately after preparation (“fresh”) and after storage at -80°C overnight (Frozen). The results are shown in Table 1.

[0215] Table 1 : Analysis of Formulations Prepared According to the Present Method, in Water, or

[0216] According to a Method Similar to in WO 2023 / 277779

[0217] “NF424” = cell penetrating peptide NickFect424 as described herein and in WO 2024 / 074553, the sequence of which is specifically incorporated by reference.

[0218] FMQ: Formulation in water. Peptide (NF424) in water. Total volume of the formulation is 100pl.

[0219] FSOL: Formulation similar to a method in WO 2023 / 277779. Formulation in HEPES. Peptide (NF424) in ethanol. Total volume of the formulation is 10Opl.

[0220] CF: Concentrated formulation. Formulation in HEPES containing 60% sucrose. Peptide (NF424) in ethanol. Buffer: peptide ratio 3:1 . Total volume of the formulation is 10pl. After nanoparticle formation 90pl HEPES is added.

[0221] As can be seen in the results of Table 1 , while formulations prepared in water (“FMQ - formulation in water”) have good nanoparticle size and an acceptable polydispersity index immediately after preparation, following freezing they aggregate (represented by increased size and polydispersity index). The same is seen for formulations prepared according to a method similar to that described in WO 2023 / 277779 (“FSOL” formulation in buffer, peptide in EtOH without aprotic solvent), wherein the cell penetrating peptide is provided in ethanol without an aprotic solvent (c.f. the method of WO 2023 / 277779 in which aprotic solvent is used) and a buffer containing only HEPES is used in a larger relative volume than described herein. Freezing is a prerequisite for lyophilisation and storage.

[0222] By contrast, formulations prepared according to the present method (“CF” formulation in 0.1x volume, buffer with 60% sucrose, peptide in EtOH, 3:1 buffer: peptide ratio) show little / no aggregation following freezing, with consistent size and polydispersity index compared to immediately after preparation. Furthermore, by being suitable for freezing for storage, the present formulations can be subsequently lyophilised and again have consistent size and polydispersity index after lyophilisation compared to freshly prepared formulations or after freezing. Thus, unlike formulations prepared in water or according to a method similar to that of WO 2023 / 277779, the present method allows the preparation of cell-penetrating peptide- cargo formulations which can be frozen and optionally further lyophilised for storage.

[0223] Example 2: Analysis of Transfection Activity of Formulations

[0224] To compare the cell-penetrating peptide-cargo formulations resulting from the preparation method described herein (CF) with those from methods known in the art and previously described, formulations transfection activity was measured. Results are shown in Figure 1. Figure 1 demonstrates that transfection of mRNA to the spleen and lymph nodes with formulations prepared in water (FMQ) is relatively low. The data also confirms that the transfection efficacy of formulations prepared according to a method similar to that described in WO 2023 / 277779 “FSOL” is 10 times lower compared to the herein described method without freezing (CF). Furthermore, the high transfection level of mRNA to the spleen and lymph nodes was achieved with nanoparticles formulated by the described method and frozen overnight at -80°C. Thus, this data demonstrates the ability of the formulations prepared according to the present method to efficiently transfect and deliver an mRNA cargo in vivo, including after freezing as required for storage and possible lyophilisation.

[0225] Example 3: Storage, Freeze-Thaw and Lyophilisation Stability of Nanoparticles by Size and Polydispersity Index

[0226] To evaluate the stability of nanoparticles formulated by the current method (CF) to freezing and storage at -80°C, nanoparticle sizes and polydispersity indexes were measured. The results are shown in Figure 2 and Table 2. The stability and homogeneity of formulations subjected to multiple freeze-thaw cycles and following lyophilisation and reconstitution were also tested by DLS (Figures 5 and 9 and Tables 3 and 4).

[0227] Table 2: Analysis of Formulations after Freezing at -80°C

[0228] Table 3: Analysis of Formulations after Freezing at -80°C and Freeze-Thaw Cycles

[0229] Table 4: Analysis of Formulations after Lyophilisation

[0230] As can be seen from the data in Figure 2 and Table 2, nanoparticles formulated according to the present method (CF) can be frozen and stored for at least 3 months at -80°C without aggregation or dissociation. Furthermore, nanoparticles formulated according to the present method (CF) remain stable following multiple freeze-thaw cycles (Figure 5 and Table 3) and following lyophilisation and reconstitution (Figure 9 and Table 4), with comparable DLS results to “fresh” formulations not subjected to freeze-thawing or lyophilisation. Storage, freeze-thaw and lyophilisation stability are prerequisites for transport and application by the pharma industry.

[0231] Example 4: Storage, Freeze-Thaw and Lyophilisation Stability of Nanoparticles by Biological Activity

[0232] To evaluate the stability of formulations prepared according to the present method (CF) to freezing and storage at -80°C as well as multiple freeze-thaw cycles and lyophilisation, the nanoparticle biological activity by in vivo transfection was measured. The results are shown in Figures 3, 4 and 8.

[0233] As can be seen in Figure 3, nanoparticles formulated by the present method can be stored frozen for at least 3 months without loss of bioactivity in vivo. As is shown in Figures 4 and 8, nanoparticles formulated by the present method which are subjected to multiple freeze-thaw cycles and / or lyophilisation also retain bioactivity with in vivo transfection efficiencies comparable to freshly prepared nanoparticles. In combination with the results of Example 3 (see Table 2 and Figures 2, 5 and 9), these data demonstrate the ability to store nanoparticles formulated by the present method at -80°C for at least 3 months without aggregation or dissociation and with an expected retention of bioactivity over this period, even following multiple freeze-thaw cycles or lyophilisation and reconstitution as would be required. Example 5: Comparison of Formulations Prepared with Varying Concentrations of Sucrose in HEPES Association Buffer

[0234] To determine the optimal sucrose concentration of association buffer for the preparation of formulations as described herein, the transfection efficiency both in vitro and in vivo was tested for formulations prepared in HEPES with 0%-90% sucrose. The results are shown in Figures 6 and 7.

[0235] As shown in Figure 6, formulations prepared in HEPES association buffer with 60%, 75% or 90%, in particular 60-75%, sucrose gave improved in vitro transfection efficiencies compared to untransfected controls and formulations prepared in association buffers with 0% or less than 60% sucrose. Of particular note is the significant increase in transfection efficiency of HEK cells seen with formulations prepared in 60% sucrose, with marked further increases in transfection efficiency of formulations prepared in 75% and 90% sucrose association buffers. Similar improved transfection efficiencies are seen in CHO cells, with a marked improvement with formulations prepared in 60% or 75% sucrose compared to formulations prepared in association buffers with 0% or less than 60% sucrose.

[0236] Similar results are observed in vivo (see Figure 7), with a markedly improved transfection efficiency of spleen cells by formulations prepared in 60% sucrose associated buffer compared to those prepared in association buffer comprising 0% or concentrations of sucrose other than 60%.

[0237] SEQUENCE LISTING

[0238] Throughout the following sequence listing: Xi represents Lys (K), Orn (O), Dab or Dap; X2 represents KA or AK; * indicates that the peptide continues from the side chain amino group and not from the a-amino group; O refers to ornithine; Dab refers to 2,4-diaminobutanoic acid; and Dap refers to 2,3-diaminopropionic acid.

[0239] SEQ ID NO: 1

[0240] AGYLLGOI N LKALAALAKAI L

[0241] SEQ ID NO: 2

[0242] AGYLLGX1INLKALAALAX2IL

[0243] SEQ ID NO: 3

[0244] AGYLLGX1 LKALAALAX2I L SEQ ID NO: 4

[0245] AGYLLGO*I N LKALAALAKAI L

[0246] SEQ ID NO: 5

[0247] AGYLLGK*I N LKALAALAKAI L

[0248] SEQ ID NO: 6

[0249] AGYLLGDab*INLKALAALAKAIL

[0250] SEQ ID NO: 7

[0251] AGYLLGDabl N LKALAALAKAI L

[0252] SEQ ID NO: 8

[0253] AGYLLGDap*INLKALAALAKAIL

[0254] SEQ ID NO: 9

[0255] AGYLLGO*I N LKALAALAAKI L

[0256] SEQ ID NO: 10

[0257] AGYLLGO*LKALAALAKAI L

[0258] SEQ ID NO: 11

[0259] AGYLLGDab*LKALAALAKAIL

[0260] SEQ ID NO: 12

[0261] AGYLLGDab*LKALAALAAKIL

[0262] SEQ ID NO: 13

[0263] HHYHHGO*ILLKALKALAKAIL

[0264] SEQ ID NO: 14

[0265] HHHHYHHGO*ILLKALKALAKAIL

[0266] SEQ ID NO: 15

[0267] HHHHHHGO*ILLKALKALAKAIL

[0268] SEQ ID NO: 16 HHHHHHYHHGO*ILLKALKALAKAIL

[0269] SEQ ID NO: 17

[0270] HYHHGO*ILLKALKALAKAIL

Claims

1. CLAIMS1. A method of preparing a formulation comprising a cell penetrating peptide and a nucleic acid molecule cargo, said method comprising the steps of:(i) providing a cell penetrating peptide in ethanol without an aprotic solvent;(ii) associating the cell penetrating peptide with a nucleic acid molecule cargo in an association buffer, thereby forming a cell penetrating peptide-cargo formulation, wherein the nucleic acid molecule cargo and association buffer are at a ratio amount of between 2 and 4 parts to 1 part cell penetrating peptide in ethanol and wherein the association buffer comprises between 30% and 90% sucrose; and(iii) storing the cell penetrating peptide-cargo formulation at -70°C or below, and optionally lyophilising the cell penetrating peptide-cargo formulation, wherein associating step (ii) is performed in a volume between 0.5x and 0.05x the final volume at which the formulation is prepared for administration.

2. The method of claim 1 , wherein the associated cell penetrating peptide-cargo formulation is in the form of a nanoparticle.

3. The method of claim 1 or claim 2, wherein the cell penetrating peptide comprises a fatty acid chain attached to the N-terminus, optionally wherein the fatty acid chain has 16 to 22 carbons atoms, such as 18 to 22 carbon atoms, and optionally wherein the fatty acid chain has 16, 18, 20 or 22 carbon atoms, such as 18, 20 or 22 carbon atoms, in particular 18 carbon atoms.

4. The method of any one of claims 1 to 3, wherein the cell penetrating peptide comprises the sequence of:AGYLLGOINLKALAALAKAIL (SEQ ID NO: 1), or the sequence of SEQ ID NO: 1 having one or more modifications selected from:(i) an N9L amino acid substitution;(ii) an A14K amino acid substitution;(iii) a deletion of the alanine residue at position 1 , or a deletion of the alanine 20 residue at position 1 and a deletion of the glycine residue at position 2;(iv) a substitution of the ornithine residue at position 7 with Dab or Dap; and(v) one or two amino acid substitutions at positions 9 to 21 of SEQ ID NO: 1 in addition to or instead of N9L and / or A14K, wherein the cell penetrating peptide comprises two or more histidine residues by substitution to the N-terminal part and / or addition to the N-terminus, andoptionally wherein the cell penetrating peptide is chemically modified at the C- terminus, such as by amidation and / or wherein the C-terminus is CONH2.

5. The method of any one of claims 1 to 3, wherein the cell penetrating peptide comprises the sequence of:AGYLLG Xi* INLKALAALA X2IL (SEQ ID NO: 2); or AGYLLG Xi* LKALAALA X2IL (SEQ ID NO: 3), wherein Xi represents Lys (K), Orn (O), Dab or Dap, wherein * indicates that the peptide optionally continues from the side chain amino group and not from the a-amino group, wherein X2represents KA or AK, and optionally wherein the cell penetrating amino acid sequence is chemically modified at the C-terminus, such as amidated, such as wherein the C-terminus is CONH2.

6. The method of claim 5, wherein Xi is K, Orn (O) or Dab, in particular Dab, and / or wherein the peptide continues from the side chain amino group and not from the a- amino group at the * position, and / or wherein X2is KA or X2is AK, and / or wherein the cell penetrating peptide comprises one or more further amino acid substitution at positions 9 to 21 of SEQ ID NO: 2 or at positions 9 to 19 of SEQ ID NO: 3.

7. The method of any one of claims 1 to 6, wherein the nucleic acid molecule cargo and association buffer are at a ratio amount of 2-4:1 to cell penetrating peptide in ethanol, in particular at a ratio amount of nucleic acid molecule cargo and association buffer to cell penetrating peptide in ethanol of 3:1.

8. The method of any one of claims 1 to 7, wherein the association buffer comprises between 50% and 90% sucrose and 10mM HEPES, in particular about 60% sucrose and 10mM HEPES.

9. The method of any one of claims 1 to 8, wherein associating step (ii) is performed in a volume between 0.5x and 0.1x the final volume at which the formulation is prepared for administration, in particular in a volume 0.1x the final volume at which the formulation is prepared for administration.

10. The method of any one of claims 1 to 9, wherein the method additionally comprises step (iv) of diluting the cell penetrating peptide-cargo formulation or resuspending thelyophilised formulation in a total volume between 2x and 20x the volume at which step (ii) is performed, such as a total volume between 2x and 10x, in particular in a total volume 10x the volume at which step (ii) is performed, optionally wherein diluting or resuspending is in a buffer comprising 10mM HEPES without sucrose.11 . The method of any one of claims 1 to 10, wherein step (iii) comprises storing the cell penetrating peptide-cargo formulation at -70°C or below without lyophilisation, or wherein step (iii) comprises lyophilising the cell penetrating peptide-cargo formulation.

12. The method of any one of claims 1 to 11 , wherein the nucleic acid molecule cargo is mRNA, plasmid DNA (pDNA), small-interfering RNA (siRNA) or micro RNA (miRNA), in particular mRNA, optionally wherein the mRNA encodes an antigenic protein or peptide, such as a virus or bacteria surface protein, in particular a capsid and / or viral envelope protein, such as a viral glycoprotein.

13. A cell penetrating peptide-cargo formulation obtainable and / or obtained by the method of any one of claims 1 to 12.

14. A pharmaceutical composition comprising the cell penetrating peptide-cargo formulation obtainable and / or obtained by the method of any one of claims 1 to 12, optionally further comprising one or more adjuvants.

15. The cell penetrating peptide-cargo formulation of claim 13 or pharmaceutical composition of claim 14 for use in a method of raising an immune response in a subject, or for use in the transport of the nucleic acid molecule cargo across a lipid membrane and subsequent delivery of said nucleic acid molecule cargo into a cell, optionally wherein the cell is in vivo, and / or wherein the cell is a cell of the spleen and / or lymph node, such as a dendritic cell (DC).

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