Ph-responsive synthetic peptide shuttle agents

Abstract

Described herein are pH-responsive synthetic peptide shuttle agent having increased cargo transduction activity at acidic pH than at neutral pH. The pH-responsive synthetic peptide shuttle agent generally comprises an amphipathic alpha-helical motif when in acidic pH, the amphipathic alpha-helical motif having solvent-exposed surface comprising a discrete hydrophilic cationic face and a discrete hydrophobic face. The pH-responsive synthetic peptide shuttle agent described herein may be further conjugated to a cargo for intracellular delivery, and / or a targeting ligand for tissue-specific delivery. Also described herein are lipid nanoparticles (LNPs) incorporating synthetic peptide shuttle agents or pH- responsive synthetic peptide shuttle agents for improved payload delivery to the cytosol.

Description

[0001] pH-RESPONSIVE SYNTHETIC PEPTIDE SHUTTLE AGENTS The present description relates to synthetic peptides that facilitate intracellular delivery and endosomal escape of cargoes. More specifically, the present description relates to pH-responsive synthetic peptide shuttle agents that exhibit increased cargo transduction activity at acidic pH compared to neutral pH, for example, suitable for encapsulation in lipid nanoparticles (LNPs). The present description refers to a number of documents, the contents of which are herein incorporated by reference in their entirety.

[0002] BACKGROUND

[0003] Synthetic peptides called shuttle agents represent a relatively new class of intracellular delivery agents having the ability to transduce many types of non-polyanionic cargos to the cytosolic / nuclear compartment of eukaryotic cells. Unlike traditional cell-penetrating peptide-based intracellular delivery strategies, synthetic peptide shuttle agent-mediated cargo transduction has been shown to occur with extremely rapid kinetics involving direct translocation across the cell membrane, bypassing the kinetically slower conventional cellular endocytic pathways (e.g., WO / 2016 / 161516; WO / 2017 / 175072;

[0004] WO / 2018 / 068135; WO / 2020 / 210916; WO / 2022 / 077121; WO / 2022 / 082315; WO / 2022 / 204806). One of the hallmarks of synthetic peptide shuttle agents is their ability to transduce cargoes in a variety of different eukaryotic cell types instantaneously upon contact. However, for some applications, some delay and / or control over the transduction activity of synthetic peptide shuttle agents would be desirable.

[0005] SUMMARY

[0006] In a first aspect, described herein is a pH-responsive synthetic peptide shuttle agent having increased cargo transduction activity at acidic pH than at neutral pH. The pH-responsive synthetic peptide shuttle agent generally comprises an amphipathic alpha-helical motif when in acidic pH, the amphipathic alpha-helical motif having solvent-exposed surface comprising a discrete hydrophilic cationic face and a discrete hydrophobic face. The discrete hydrophilic cationic face generally comprises at least one cationic residue (e.g., K or R) and at least one histidine residue that becomes protonated at acidic pH, resulting in the pH-responsive synthetic peptide shuttle agent having increased cargo transduction activity at acidic pH than at neutral pH.

[0007] In a further aspect, described herein is a composition comprising a pH-responsive synthetic peptide shuttle agent as described herein and a cargo for intracellular delivery.

[0008] In a further aspect, described herein is a lipid nanoparticle comprising a synthetic peptide shuttle agent and a cargo for cytosolic delivery. In some embodiments, the synthetic peptide shuttle agent may be a pH-responsive synthetic peptide shuttle agent as described herein.In a further aspect, described herein is a method for synthesizing a pH-responsive synthetic peptide shuttle agent as described herein.

[0009] General Definitions

[0010] Headings and other identifiers, e.g., (a), (b), (i), (ii), etc., are presented merely for ease of reading the specification and claims. The use of headings or other identifiers in the specification or claims does not necessarily require the steps or elements be performed in alphabetical or numerical order or the order in which they are presented.

[0011] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one” but it is also consistent with the meaning of “one or more”, “at least one”, and “one or more than one”.

[0012] The term “about”, when used herein, indicates that a value includes the standard deviation of error for the device or method being employed in order to determine the value. In general, the terminology “about” is meant to designate a possible variation of up to 10%. Therefore, a variation of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10% of a value is included in the term “about”. Unless indicated otherwise, use of the term “about” before a range applies to both ends of the range.

[0013] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0014] As used herein, “protein” or “polypeptide” or “peptide” means any peptide-linked chain of amino acids, which may or may not comprise any type of modification (e.g., chemical or post-translational modifications such as acetylation, phosphorylation, glycosylation, sulfatation, sumoylation, prenylation, ubiquitination, etc.). For further clarity, protein / polypeptide / peptide modifications are envisaged so long as the modification does not destroy the cargo transduction activity of the shuttle agents described herein, or the biological activity of the cargoes described herein. For example, shuttle agents described herein may be linear or circular, may be synthesized with one or more D- or L-amino acids. Shuttle agents described herein may also have at least one amino acid being replaced with a corresponding synthetic amino acid having a side chain of similar physiochemical properties (e.g., structure, hydrophobicity, or charge) as the amino acid being replaced.

[0015] As used herein, the term “synthetic” used in expressions such as “synthetic peptide” or “synthetic peptide shuttle agent” is intended to refer to non-naturally occurring molecules that can be produced in vitro (e.g., synthesized chemically and / or produced using recombinant DNA technology). The purities ofvarious synthetic preparations may be assessed by, for example, high-performance liquid chromatography analysis and mass spectroscopy. Chemical synthesis approaches may be advantageous over cellular expression systems (e.g., yeast or bacteria protein expression systems), as they may preclude the need for extensive recombinant protein purification steps (e.g., required for clinical use). In contrast, longer synthetic polypeptides may be more complicated and / or costly to produce via chemical synthesis approaches and such polypeptides may be more advantageously produced using cellular expression systems. In some embodiments, the peptides or shuttle agents of the present description may be chemically synthesized (e.g., solid- or liquid phase peptide synthesis), as opposed to expressed from a recombinant host cell. In some embodiments, the peptides or shuttle agent of the present description may lack an N-terminal methionine residue. A person of skill in the art may adapt a synthetic peptide or shuttle agent of the present description by using one or more modified amino acids (e.g., non-naturally-occurring amino acids), or by chemically modifying the synthetic peptide or shuttle agent of the present description, to suit particular needs of stability or other needs.

[0016] Other objects, advantages, and features of the present description will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.

[0017] BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In the appended drawings:

[0019] Fig. 1 shows a multiple sequence alignment of the amino acid sequences of the peptides FSD375D and FSD449D, with the position of a K to A substitution highlighted in black.

[0020] Fig. 2 shows a multiple sequence alignment between the peptide FSD449D and its variants (without their C-terminal cysteine residues): FSP001D - FSP017D, with the positions of the K, R, and H residues highlighted in black.

[0021] Fig. 3A shows flow cytometry analysis of the Cy5-labeled cargo (nls* peptide) delivery using 10 pM of pH-responsive shuttles at pH 5 and pH 7 in HeLa cells. Fig. 3B shows a flow cytometry analysis of the Cy5-labeled cargo (nls* peptide) delivery with different doses of pH-responsive shuttles at pH 5 and pH 7 in HeLa cells.

[0022] Fig. 4A-4S shows helical wheel projections of the “core” regions of the peptides FSD449D (Fig.

[0023] 4A), FSP001D to FSP017D (Figs. 4B-4R), and FSD375D (Fig. 4S) generated with an online helical wheel projection tool created by Don Armstrong and Raphael Zidovetzki (e.g., available at:

[0024] https: / / www.donarmstrong.com / cgi-bin / wheel.pl).Figs. 5A and 5B show the molar ellipticity of various pH-responsive shuttles in water at pH 5 and 10% TFE in water at pH 5 respectively. Fig. 5C shows the Helix % of the pH responsive shuttles of Fig. 5A and 5B.

[0025] Fig. 6A shows flow cytometry analysis of eGFP fluorescence intensity after (eGFP)-mRNA delivery using 1% of (Shuttle agent)-LNPs in HeLa cells. Fig. 6B shows firefly bioluminescence analysis of (FLuc)-mRNA delivery in HeLa cells using 1% (9.5 pM) (FSP003D)-LNP- vs control LNP lacking synthetic peptide shuttle agent (“no FS”). Fig. 6C shows firefly bioluminescence analysis of (FLuc)-mRNA delivery in HeLa cells using 1% (9.5 pM) (FSP016D)-LNP- and (FSP017D)-LNP- vs control LNP lacking synthetic peptide shuttle agent (“C-LNP”).

[0026] Fig. 7. shows synthetic peptide shuttle agent / mRNA complexation and characterization by electrophoretic mobility shift assay.

[0027] Fig. 8 shows firefly bioluminescence imaging following systemic administration of 1% (FSP003D)-LNP-(FLuc)-mRNA and LNP-(FLuc)-mRNA at 1 pg in C57B1 / 6 mice.

[0028] Fig. 9 shows 1% (FSP003D)-LNP delivery efficiency by organ bioluminescence quantification after systemic injection of 1 pg (FLuc)-mRNA.

[0029] Fig. 10 shows flow cytometry analysis of engineered HeLa cells 24 h after being contacted with LNPs loaded with PMO-(eGFP) cargo alone, or with PMO-(eGFP) cargo mixed with 7.5 pM of FSD10, in which successful PMO-(eGFP) cargo delivery to the cytosol directs a splicing correction of an (eGFP)-mRNA expressed by the engineered HeLa cells, resulting in GFP expression.

[0030] Fig. HA shows a flow cytometry analysis of (eGFP)-mRNA delivery to HeLa cells using (Shuttle agent)-LNPs (MC3) carrying 60 or 120 ng of (eGFP)-mRNA normalized to control LNPs without shuttle agents (C-LNP). Fig. 11B shows the cell viability of the Hela cells of Fig. HA.

[0031] Fig. 12A shows flow cytometry analysis of (eGFP)-mRNA delivery to Hela cells using FSP008D- LNPs prepared with the SM-102 LNP kit carrying 60 or 120 ng of (eGFP)-mRNA normalized to control LNPs without shuttle agents (C-LNP). Fig. 12B shows the cell viability of Hela cells of Fig.

[0032] 12A.

[0033] Fig. 13A shows a flow cytometry analysis of (eGFP)-mRNA delivery to HeLa cells using (Shuttle agent)-LNPs prepared with FSP008D, FSD10 and FSD375D, and their lipid conjugates carrying 60 or 120 ng of (eGFP)-mRNA, normalized to C-LNP (ratio = 1), 24 hours post-incubation in HeLa cells. Fig. 13B shows a flow cytometry analysis of (eGFP)-mRNA delivery to HeLa cells using (Shuttle agent)-LNPs prepared with FSP003D-, FSP005D- and FSD375D- lipid conjugates carrying 60 or 120 ng of (eGFP)-mRNA, normalized to C-LNP (ratio = 1), 24 hours post-incubation in HeLa cells.Fig. 14A and 14B shows firefly bioluminescence imaging and its quantification following systemic administration of 0.5 pg (FLuc)-mRNA with 1% FSD10-CLS, pH- responsive FSP003D-CLS LNPs and control LNP in BALB / c mice.

[0034] Figs. 15A - 15E show the quantified total radiance for each collected organ of the mice of Fig.

[0035] 14A and 14B.

[0036] Fig. 16A is a schematic representation of the time -dependent activation of the pH-responsive-cargo conjugates of the present technology when exposed to acidic pH in an endosome. Fig. 16B shows representative fluorescence microscopy images illustrating the time dependent activation of FSP005D-nls* in cells. Fig. 16C shows the quantification of the fluorescence signal in cell treated with FSD449, FSP005D and FSP008D conjugated to PMO as a cargo compared to the PMO alone at 5 min and 120 minutes post treatment. Fig. 16D are representative fluorescence microscopy images of the cells treated in Fig. 16C.

[0037] Figs. 17A and 17B show representative slides of the systemic biodistribution of pH-Responsive Shuttle-PMO conjugates in eGFP-CAG645 mice after systemic administration.

[0038] Fig. 18 shows representative slides of the systemic biodistribution of pH-Responsive Shuttle-PMO with and without GalNac conjugation in eGFP-CAG645 mice following systemic administration.

[0039] SEQUENCE LISTING

[0040] This application contains a Sequence Listing in computer-readable form created February 25, 2026, which is incorporated herein by reference in its entirety.

[0041] SEQ ID NO: Description

[0042] 1 FSD375D*

[0043] 2 FSD449D*

[0044] 3 FSP001D*

[0045] 4 FSP002D*

[0046] 5 FSP003D*

[0047] 6 FSP004D*

[0048] 7 FSP005D*

[0049] 8 FSP006D*

[0050] 9 FSP007D*

[0051] 10 FSP008D*

[0052] 11 FSD375D (core)

[0053] 12 FSD449D (core)

[0054] 13 FSP001D (core)

[0055] 14 FSP002D (core)

[0056] 15 FSP003D (core)

[0057] 16 FSP004D (core)

[0058] 17 FSP005D (core)

[0059] 18 FSP006D (core)

[0060] 19 FSP007D (core)

[0061]

[0062] 20 FSP008D (core)21 His-CM18-PTD4 (core)

[0063] 22 FSD18 (core)

[0064] 23 FSD250 (core)

[0065] 24 FSD174 (core)

[0066] 25 FSD333 (core)

[0067] 26 FSD395 (core)

[0068] 27 FSD396 (core)

[0069] 28 FSD397 (core)

[0070] 29 FSD398 (core)

[0071] 30 FSD399 (core)

[0072] 31 FSD400 (core)

[0073] 32-94 FSD375D (core) with different permutations of K / R to H substitutions 95-125 FSD449D (core) with different permutations of K / R to H substitutions

[0074] 126-156 His-CM18-PTD4 (core) with different permutations of K / R to H substitutions

[0075] 157-163 FSD18 (core) with different permutations of K / R to H substitutions

[0076] 164-194 FSD250 (core) with different permutations of K / R to H substitutions

[0077] 195-225 FSD174 (core) with different permutations of K / R to H substitutions

[0078] 226-288 FSD333 (core) with different permutations of K / R to H substitutions

[0079] 289-351 FSD395 (core) with different permutations of K / R to H substitutions

[0080] 352-414 FSD396 (core) with different permutations of K / R to H substitutions

[0081] 415-477 FSD397 (core) with different permutations of K / R to H substitutions

[0082] 478-604 FSD398 (core) with different permutations of K / R to H substitutions

[0083] 605-635 FSD399 (core) with different permutations of K / R to H substitutions

[0084] 636-698 FSD400 (core) with different permutations of K / R to H substitutions

[0085] 699 GGSGGGS linker domain

[0086] 700 FSP009D*

[0087] 701 FSP0010D*

[0088] 702 FSP011D*

[0089] 703 FSP012D*

[0090] 704 FSP013D*

[0091] 705 FSP014D*

[0092] 706 FSP015D*

[0093] 707 FSP009D (core)

[0094] 708 FSP010D (core)

[0095] 709 FSP011D (core)

[0096] 710 FSP012D (core)

[0097] 711 FSP013D (core)

[0098] 712 FSP014D (core)

[0099] 713 FSP015D (core)

[0100] 714 FSP016D*

[0101] 715 FSP017D*

[0102] 716 FSP016D (core)

[0103] 717 FSP017D (core)

[0104]

[0105] 718 FSD10

[0106] * Full-length but lacking C-terminal cysteine

[0107] DETAILED DESCRIPTION

[0108] In a first aspect, described herein is a composition comprising a cargo for intracellular delivery and a pH-responsive synthetic peptide shuttle agent. In some embodiments, the pH-responsive synthetic peptide shuttle agent may comprise or adopt the conformation of an amphipathic alpha-helical motif whenin acidic pH. In some embodiments, the pH-responsive synthetic peptide shuttle agent may comprise or adopt the conformation of an amphipathic alpha-helical motif both at neutral and acidic pH. In some embodiments, the amphipathic alpha-helical motif has a solvent-exposed surface comprising a discrete hydrophilic cationic face and a discrete hydrophobic face. The discrete hydrophilic cationic face generally comprises at least one positively-charged residue (e.g., K or R) and at least one histidine residue that becomes protonated at acidic pH, resulting in the pH-responsive synthetic peptide shuttle agent having increased cargo transduction activity at acidic pH than at neutral pH.

[0109] In some embodiments, the discrete hydrophilic cationic face described herein may comprise at least two, three, or four adjacent K, R, and / or H residues upon helical wheel projection, based on an alpha helix having an angle of rotation between consecutive amino acids of 100 degrees and / or an alpha-helix having 3.6 residues per turn. In some embodiments, the discrete hydrophilic cationic face described herein may comprise a segment of six adjacent residues comprising three to five K, R, and / or H residues upon helical wheel projection, based on an alpha helix having an angle of rotation between consecutive amino acids of 100 degrees and / or an alpha-helix having 3.6 residues per turn. Such helical wheel projections, as referred to herein, may be prepared using a variety of programs, such as the online helical wheel projection tool created by Don Armstrong and Raphael Zidovetzki (e.g., available at: https: / / www.donarmstrong.com / cgi-bin / wheel.pl ) or the online tool developed by Mol et al., 2024 (e.g., available at http: / / lbqp.unb.br / NetWheels / ). In some embodiments, the discrete hydrophilic cationic face described herein may comprise a total number of positively-charged residues (e.g., K and R residues) that is greater than the total number of positively-charged residues comprised in the discrete hydrophobic face, thereby creating an alpha-helix having a charge gradient or polarity. In some embodiments, the discrete hydrophilic cationic face described herein may lack negatively-charged amino acids (e.g., D or E) or may comprise no more than one or two negatively-charged amino acid residue.

[0110] In some embodiments, the amphipathic alpha-helical motif described herein may have a minimum length of 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 amino acids. In some embodiments, the amphipathic alpha-helical motif described herein may have a maximum length of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. In some embodiments, the amphipathic alpha-helical motif described herein may comprise the amino sequence of any one of SEQ ID NOs: 15-31, 707—713 and 716-717. In some embodiments, the amphipathic alpha-helical motif described herein may comprise the amino acid sequence of any one of SEQ ID NOs: 11, 12 or 21-31, wherein the at least one, two, or three positively-charged residues (e.g., K or R) comprised in the discrete cationic hydrophilic face is / are replaced with histidine. SEQ ID NOs: 21-29 correspond to the predicted amphipathic alpha-helical motifs of some synthetic peptide shuttle agents previously demonstrated to exhibit robust cargo transduction activity -i.e., His-CM18-PTD4, FSD18, FSD250, FSD174, FSD333, FSD395, FSD396, and FSD397, respectively(e.g., see Fig. 6 ofWO / 2020 / 210916). SEQ ID NOs: 32-698 correspond to the amphipathic alpha-helical motifs (“cores”) of SEQ ID NOs: 11, 12, and 21-31, except containing different permutations of K and / or R residues replaced with H residues. In some embodiments, the amphipathic alpha-helical motif described herein may have a hydrophobic moment (p) between a lower limit of 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, and an upper limit of 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, or 11.0. In some embodiments, the amphipathic alpha-helical motif described herein may have a net charge of at least -2 at neutral pH (e.g., pH 7) and at least +3 at acidic pH (e.g., pH 5).

[0111] In some embodiments, the discrete hydrophobic face described herein may comprise at least two or three adjacent residues selected from L, I, F, V, W, M, or any combination thereof, upon helical wheel projection, based on an alpha helix having angle of rotation between consecutive amino acids of 100 degrees and / or an alpha-helix having 3.6 residues per turn. In some embodiments, the discrete hydrophobic face described herein may comprise at least two or three adjacent L residues upon helical wheel projection, based on an alpha helix having an angle of rotation between consecutive amino acids of 100 degrees and / or an alpha-helix having 3.6 residues per turn. In some embodiments, the discrete hydrophobic face described herein may comprise a segment of eight, nine, or ten adjacent residues comprising at least five hydrophobic residues selected from: L, I, F, V, W, and M, upon helical wheel projection, based on an alpha helix having angle of rotation between consecutive amino acids of 100 degrees and / or an alpha-helix having 3.6 residues per turn. In some embodiments, the discrete hydrophobic face described herein may comprise a total number of positively-charged residues (e.g., K and R residues) that is less than the total number of positively-charged residues comprised in the discrete hydrophilic cationic face. In some embodiments, the discrete hydrophobic face described herein may lack negatively-charged amino acids (e.g., D or E). In other embodiments, the discrete hydrophobic face may comprise at least one or two negatively-charged residues. In some embodiment, the at least one or two negatively-charged residue may be incorporated in the discrete hydrophobic face by substituting a hydrophobic amino acid (e.g., A) with a negatively-charged amino acid (e.g., D or E).

[0112] In some embodiments, the pH-responsive synthetic peptide shuttle agent described herein may further comprise a flexible linker domain N-terminal or C-terminal with respect to the amphipathic alphahelical motif. In some embodiments, the pH-responsive synthetic peptide shuttle agent described herein may further comprise flexible linker domains flanking (e.g., N- and / or C-terminally) the amphipathic alpha-helical motif. In some embodiments, the linker domains described herein may comprise or consist of the sequence GGSGGGS (SEQ ID NO: 699). In some embodiments, the pH-responsive synthetic peptide shuttle agent may have a maximum length of 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 60, 65, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids. In some embodiments, the pH-responsive synthetic peptide shuttle agent described herein may have an isoelectric point (pl) of 6 to 13. In some embodiments, the pH-responsive synthetic peptide shuttle agent described herein may have a net charge of at least -2 at neutral pH (e.g., pH 7) and at least +3 at acidic pH (e.g., pH 5). In some embodiments, the pH-responsive synthetic peptide shuttle agent may have a hydrophobic moment (p) between a lower limit of 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, and an upper limit of 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, or 11.0. In some embodiments, the pH-responsive synthetic peptide shuttle agent described herein may comprise the amino acid sequence of any one of SEQ ID NOs: 5-10, 700-703, 705-706 and 716-717.

[0113] In some embodiments, the pH-responsive synthetic peptide shuttle agent described herein may lack a cell-penetrating domain and / or a histidine-rich domain. As used herein, the expression “cellpenetrating domain” refers to sequences derived from the class of conventional cell-penetrating peptides (CPPs) that generally mediate intracellular delivery with slower kinetics than that of synthetic peptide shuttle agents, and usually via conventional cellular endocytic pathways involving the formation of closed endosomes, endosomal maturation / acidification, and are therefore vulnerable to endosomal entrapment. Traditional CPPs require binding or covalent attachment to their cargoes for increased intracellular delivery, as opposite to synthetic peptide shuttle agents that possess increased cytosolic delivery when not covalently or non-covalently bound to their cargoes. As used herein, the expression “histidine-rich domain” refers to a segment of a polypeptide sequence that has a greater proportion or density of histidine residues based on the primary amino acid sequence of a polypeptide, without regard to the preponderance of the histidine residues within a three-dimensional shape or secondary structure adopted by the polypeptide. More specifically, as used herein, the expression “histidine-rich domain” comprises those as described in WO / 2016 / 161516, WO / 2017 / 175072, WO / 2018 / 068135, WO / 2020 / 210916, WO / 2022 / 077121, WO / 2022 / 082315, and WO / 2022 / 204806.

[0114] In some embodiments, the pH-responsive synthetic peptide shuttle agent described herein may further comprise at least one cysteine residue positioned at the N- and / or C-terminal portion, or at the N or C terminus, of the pH-responsive synthetic peptide shuttle agent. In some embodiments, the pH-responsive synthetic peptide shuttle agent described herein may be comprised in the composition described herein at a concentration that increases the cytosolic delivery of the cargo as compared to a corresponding composition lacking the pH-responsive synthetic peptide shuttle agent. In some embodiments, the concentration of the pH-responsive synthetic peptide shuttle agent in the composition described herein may be at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 pM. Insome embodiments, the pH-responsive synthetic peptide shuttle agent described herein may be covalently conjugated to a lipid (e.g., a cholesterol derivative, 1,2-dimyristoyl glycerol, a fatty acid, a PEGylated lipid, a phospholipid, an ionizable lipid). In some embodiments, the pH-responsive synthetic peptide shuttle agent described herein may be covalently conjugated to a biocompatible non-polyanionic hydrophilic polymer. As used herein, “non-polyanionic hydrophilic polymer” refers to water-soluble polymers that are not regularly negatively charged at physiological pH (e.g., in blood or other bodily fluids / secretions) or that do not contain sufficient negative charges at physiological pH to abrogate shuttle agent-mediated cargo transduction. In some embodiments, the biocompatible non-anionic hydrophilic polymer may have a linear, branched, hyper-branched, or dendritic structure. Branched, hyper-branched, or dendritic structures may be suitable for the synthesis of bioconjugates comprising shuttle agent multimers. In some embodiments, the biocompatible non-anionic hydrophilic polymer may be a polyether moiety, a polyester moiety, a polyoxazoline moiety, a polyvinylpyrrolidone moiety, a polyglycerol moiety, a polysaccharide moiety, a hydrophilic peptide or polypeptide linker moiety, a polysiloxane moiety, a polylysine moiety, a non-anionic polynucleotide analog moiety (e.g., a charge-neutral polynucleotide analog moiety having a phosphorodiamidate backbone, an amide (e.g., peptide) backbone, a methylphosphonate backbone, a neutral phosphotriester backbone, a sulfone backbone, or a triazole backbone; or a cationic polynucleotide analog moiety having an aminoalkylated phosphoramidate backbone, a guanidinium backbone, an S-methylthiourea backbone, or a nucleosyl amino acid (NAA) backbone), or any non-anionic derivative thereof, or any combination thereof. In some embodiments, the biocompatible non-anionic hydrophilic polymer may comprise a polyethylene glycol (PEG) moiety and / or a polyester moiety, or a non-anionic derivative thereof. In some embodiments, the biocompatible non-anionic hydrophilic polymer has a mass of at least 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14-, 15-, 16-, 17-, 18-, 19-, 20-, 21-, 22-, 23-, 24-, 25-, 26-, 27-, 28-, 29-, 30-, 31-, 32-, 33-, 34-, 35-, 36-, 37-, 38-, 39-, or 40-fold of the mass of the synthetic peptide shuttle agent. In some embodiments, the biocompatible non-anionic hydrophilic polymer has a mass of between 1-, 2-, 3-, 4-, 5-fold to 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14-, 15-, 16-, 17-, 18-, 19-, 20-, 21-, 22-, 23-, 24-, 25-, 26-, 27-, 28-, 29-, 30-, 31-, 32-, 33-, 34-, 35-, 36-, 37-, 38-, 39-, or 40-fold of the mass of the synthetic peptide shuttle agent. In some embodiments, the biocompatible non-anionic hydrophilic polymer has a mass of between about 1 to 80 kDa, 1 to 70 kDa, 1 to 60 kDa, 1 to 50 kDa, 1 to 40 kDa, 2 to 80 kDa, 2 to 70 kDa, 2 to 60 kDa, 2 to 50 kDa, 2 to 40 kDa, 3 to 80 kDa, 3 to 70 kDa, 3 to 60 kDa, 3 to 50 kDa, 3 to 40 kDa, 4 to 80 kDa, 4 to 70 kDa, 4 to 60 kDa, 4 to 50 kDa, 4 to 40 kDa, 5 to 80 kDa, 5 to 70 kDa, 5 to 60 kDa, 5 to 50 kDa, 5 to 40 kDa, 5 to 35 kDa, 10 to 35 kDa, 10 to 30 kDa, 10 to 25 kDa, or 10 to 20 kDa. In some embodiments, the non-anionic hydrophilic polymer has a size of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 kDa. As usedherein in the context of the sizes of biocompatible non-anionic hydrophilic polymers, the term “about” is intended to reflect the innate heterogeneity of polymer synthesis, wherein the size of the polymers generally refers to the average size or mass of the polymers in the preparation. Such variations are encompassed by the term “about” in such contexts.

[0115] In some embodiments, the pH-responsive synthetic peptide shuttle agent described herein may be comprised in a multimer comprising at least two pH-responsive synthetic peptide shuttle agents (i.e., shuttle agent monomers) tethered together (e.g., via said biocompatible non-anionic hydrophilic polymer). In some embodiments, the shuttle agent monomers are preferably tethered together at or towards their N-or C-terminal ends (e.g., via a branched or hyper-branched biocompatible non-anionic hydrophilic polymer) such that the N-terminal end of the shuttle agent’s cationic amphipathic core motif remains free or untethered. In some embodiments, compositions described herein may comprise a concentration of a pH-responsive synthetic peptide shuttle agent multimer, wherein the shuttle agent monomer concentration in the composition is at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1500, 2000, 2500, or 3000 pM. For example, a 25 pM concentration of a multimer tethering together four shuttle agent monomers would have a shuttle agent monomer concentration of 100 pM.

[0116] In some embodiments, the biocompatible non-anionic hydrophilic polymer may comprise a non-cleavable or a cleavable (e.g., degradable) linkage, wherein the cleavable linkage, can enable untethering of the pH-responsive synthetic peptide shuttle agents following administration. In some embodiments, the multimer may comprise a branched PEG, a hyper-branched PEG, a dendritic, and / or a polyester core. In some embodiments, a multimer comprising a polyester core may be degradable in vivo, enabling a gradual release or untethering of shuttle agent monomers following administration. In some embodiments, the biocompatible non-anionic hydrophilic polymer may be conjugated to the pH-responsive synthetic peptide shuttle agent via a cleavable linkage (e.g., a disulfide bond or a hydrolysable polyester bond). In some embodiments, the biocompatible non-anionic hydrophilic polymer may be conjugated to the pH-responsive synthetic peptide shuttle agent via a non-cleavable linkage (e.g., a maleimide bond).

[0117] Structure -activity relationship studies described previously and herein demonstrate that flexible linker domains are not responsible or necessary for the cargo transduction activity of synthetic peptide shuttle agents, as truncated shuttle agents lacking flexible linker domains have been shown to retain their cargo transduction activities (e.g., WO / 2022 / 082315). Structure -activity relationship studies describedpreviously and herein further show that the primary amino acid sequence of flexible linker domains is of lesser importance than the ability of the linker to adopt a random coil conformation. Nevertheless, flexible linker domains contribute to stability, overall solubility, ease of synthesis, efficacy, and / or versatility of synthetic peptide shuttle agents. In some embodiments, the pH-responsive synthetic peptide shuttle agent described herein may comprise a flexible linker domain that comprises uncharged hydrophilic residues that: adopt a random coil conformation; increase the stability of the amphipathic alpha-helical motif; increase the solubility of the pH-responsive synthetic peptide shuttle agent; or any combination thereof. In some embodiments, the flexible linker domain described herein may be comprised of uncharged hydrophilic residues (e.g., selected from glycine, serine, threonine, cysteine, tyrosine, asparagine, and / or glutamine). In some embodiments, the flexible linker domain described herein may be a glycine / serine-rich flexible linker domain.

[0118] In some embodiments, the pH-responsive synthetic peptide shuttle agent and the cargo are not covalently bound to one another.

[0119] In some embodiments, the pH-responsive synthetic peptide shuttle agent and the cargo are covalently bound to one another in a cleavable or non cleavable manner.

[0120] In some embodiments, the pH-responsive synthetic peptide shuttle agent and the cargo do not physically bind to one another. In some embodiments, the pH-responsive synthetic peptide shuttle agent and the cargo bind to one another sufficiently weakly to not inhibit cytosolic delivery of the cargo.

[0121] In some embodiments, the covalently bound pH-responsive synthetic peptide shuttle agent and the cargo may be cleaved or cleavable following cellular internalization and / or endosomal localization where the acidification and / or the intracellular disulfide bond cleavage leads to cargo release. In some such embodiments, the cargo may be an antisense oligonucleotide (e.g., a PMO) or a peptide.

[0122] In other embodiments, the pH-responsive synthetic peptide shuttle agent and the cargo may be bound to one another by any means described herewith and be further associated (e.g. covalently bound) with a targeting ligand. As used herein “targeting ligand” refers to any endogenous or exogenous molecule that results in the transport of the pH-responsive synthetic peptide shuttle agent and the cargo or the LNP in which they are incorporated in a specific cell or tissue. In some embodiments, such targeting ligands are selected from those known in the art. In some embodiments, the targeting ligand may be N-acetylgalactosamine (GalNac) which is a hepatocyte-targeting ligand that binds with high affinity to the asialoglycoprotein receptor (ASGPR), which is highly expressed on hepatocytes.

[0123] In some embodiments, cargo described herein may be a polynucleotide cargo (e.g., DNA and / or RNA cargo). As used herein, the term “polynucleotide cargo” also comprises synthetic polynucleotides that have the activity of natural polynucleotides (e.g., can participate in Watson-Crick base pairing). In some embodiments, the polynucleotide cargo may be mRNA, RNAi, shRNA, siRNA, microRNA,antisense RNA, IncRNA, saRNA, CircRNA, RNA or DNA aptamer, etc. In some embodiments, the cargo described herein may be an antisense oligonucleotide (ASO) cargo. In some embodiments, the ASO may be RNA or DNA. In some embodiments, the ASO may be a phosphorothioate (PS) ASO. In some embodiments, the cargo described herein may be a non-anionic polynucleotide analog, such as a chargeneutral or cationic antisense synthetic ASO. In some embodiments, the ASO may be a charge -neutral or cationic splice -switching oligonucleotide (SSO). In some embodiments, the polynucleotide cargo may be a charge-neutral polynucleotide analog cargo having a phosphorodiamidate backbone, an amide (e.g., peptide) backbone, a methylphosphonate backbone, a neutral phosphotriester backbone, a sulfone backbone, or a triazole backbone. In some embodiments, the polynucleotide cargo may be a cationic polynucleotide analog cargo having an aminoalkylated phosphoramidate backbone, a guanidinium backbone, an S-methylthiourea backbone, or anucleosyl amino acid (NAA) backbone. In some embodiments, the polynucleotide cargo may be a phosphorodiamidate morpholino oligomer (PMO), a peptide nucleic acid (PNA), a methylphosphonate oligomer, or a short interfering ribonucleic neutral oligonucleotide (siRNA). In some embodiments, the polynucleotide cargo may be a 5- to 50-mer, a 5-mer to 75-mer, or a 5-mer to 100-mer. In some embodiments, the polynucleotide cargo is not covalently linked to a cell-penetrating peptide, octa-guanidine dendrimer, or other intracellular delivery moiety. In some embodiments, the pH-responsive synthetic peptide shuttle agent described herein may reduce the amount or concentration of the cargo that is required to be administered to achieve its intended biological effect, as compared to administration of the cargo without the pH-responsive synthetic peptide shuttle agent.

[0124] In some embodiments, cargo described herein may be a protein or peptide cargo. In some embodiments, cargo described herein may be a small molecule cargo. In some embodiments, cargo described herein may be ribonucleoprotein cargo.

[0125] In a further aspect, described herein is a lipid nanoparticle comprising a synthetic peptide shuttle agent and a cargo for cytosolic delivery. In some embodiments, the synthetic peptide shuttle agent may be as described in WO / 2016 / 161516, WO / 2017 / 175072, WO / 2018 / 068135, WO / 2020 / 210916, WO / 2022 / 077121, WO / 2022 / 082315, and / or WO / 2022 / 204806. In some embodiments, the synthetic peptide shuttle agent may be covalently conjugated to a lipid (e.g., a cholesterol, a cholesterol derivative, 1,2 -dimyristoyl glycerol, a fatty acid, a PEGylated lipid, a phospholipid, an ionizable lipid) and / or to non-polyanionic hydrophilic polymer. In some embodiments, the synthetic peptide shuttle agent may be a pH-responsive synthetic peptide shuttle agent as described herein. In some embodiments, the cargo for cytosolic delivery may be a cargo as described herein.

[0126] In a further aspect, the pH-responsive synthetic peptide shuttle agent may be for use in increasing the cytosolic delivery of the cargo as compared to in the absence of the pH-responsive synthetic peptideshutle agent. In some embodiments, the synthetic peptide shutle agent (alone or conjugated to a lipid as disclosed herein) or the pH-responsive synthetic peptide shutle agent (alone or conjugated to a lipid as disclosed herein) may be for use in the manufacture of a lipid nanoparticle comprising a cargo as described herein for cytosolic delivery. In some embodiments, the pH-responsive synthetic peptide shutle agent may be for use in the manufacture of a composition as described herein.

[0127] In a further aspect, the composition described herein may be for use in the manufacture of a lipid nanoparticle (e.g., a lipid nanoparticle described herein). In a further aspect, the composition described herein may be for use in therapy, wherein the cargo is a therapeutic agent. In a further aspect, the composition described herein may be for use as a diagnostic agent, wherein the cargo is a detectable marker (e.g., radioactive marker, fluorescent marker, bioluminescent marker, contrast agent, a photoactivatable marker).

[0128] In a further aspect, the lipid nanoparticle described herein may be for use in therapy, wherein the cargo is a therapeutic agent. In a further aspect, the lipid nanoparticle described herein may be for use in the manufacture of a medicament for treating a disease or disorder ameliorated by intracellular delivery of the cargo. In a further aspect, the lipid nanoparticle described herein may be for use in the biomanufacture of non-replicating viral vectors (e.g., AAVs) or virus-like particles. In a further aspect, the lipid nanoparticle described herein may be for use as a diagnostic agent, wherein the cargo is a detectable marker (e.g., radioactive marker, fluorescent marker, bioluminescent marker, contrast agent, a photoactivatable marker). In a further aspect, the lipid nanoparticle described herein may be for use in the manufacture of a diagnostic agent, wherein the cargo is a detectable marker (e.g., radioactive marker, fluorescent marker, bioluminescent marker, a contrast agent, a photoactivatable marker).

[0129] EXAMPLES

[0130] Example 1: Materials and Methods

[0131] Materials

[0132] Acetonitrile (ACN) was purchased from Laboratoire Mat Inc (QC, CA). Formic Acid (FA), Aldrithiol-2 (DPDS), Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), Dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), 2,2,2-Trifluoroethanol (TFE), and boric acid were purchased from Millipore Sigma (MO, USA). Maleimid-PEG15-cholesterol (Mal-PEG15-CLS) was purchased from BroadPharm (CA, USA). Maleimid-PEG45-cholesterol (Mal-PEG45-CLS), Thiol-PEG45-cholesterol (SH-PEG45-CLS), Maleimid-PEG45-l,2-dimyristoyl-rac -glycerol (Mal-PEG45-DMG), Thiol-PEG45-1,2-dimyristoyl-rac -glycerol (Thiol-PEG45-DMG), Maleimid-PEG45-l,2-distearoyl-sn-glycero-3-phosphoethanolamine (Mal-PEG45-DSPE) and Maleimid-PEG45- l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (Mal-PEG45-DOPE) were purchased from Ruixibio Inc (SN, CN). DBCO-amine was purchased from Vector Laboratories (CA, USA). Anhydrous Ethanol 100% was purchased from Greenfield Global (ON, CA). Water Dnase Rnase Protease free, Tris, EDTA disodium dihydrate (Na2EDTA), sucrose and agarose were purchased from BioShop Canada Inc. (ON, CA). TBE 10X is homemade following this recipe: for 1 L, dissolve 108 g Tris base, 55 g boric acid, and 40 mL of 0.5 M EDTA (pH 8.0) in deionized water. EDTA solution is homemade following this recipe: for IL, add 186.12 g Na2EDTA in deionized water, pH adjusted to 8 with NaOH. GelRed 10 000X was purchased from Cedarlane Laboratories (ON, CA). Phosphate-Buffered Saline 10X (PBS) (pH 7.4, RNase-free for LNPs), sodium Acetate (3 M), pH 5.5, RNase-free, and Quant-it™ RiboGreen RNA Quantification Kit, Slide-A-Lyzer™ MINI Dialysis Devices and Pierce™ Microdialysis Plates, Hoechst 33342 was purchased from Invitrogen. Nunc™ Lab-Tek™ II Chambered Coverglass, CellLight™ Early Endosomes-GFP and CellTracker Orange were purchased from Thermofisher Scientific (MA, USA). Triton X-100, Sodium hydroxide and Hydrochloric acid (HC1) were purchased from VWR (NJ, USA). Sulfo-Cy5-Maleimid (sCy5-Mal) was purchased from Lumiprobe (MD, USA). All peptides were purchased from Expeptise (QC, CA) or GL Biochem (SH, CN) or Wuxi (CN). PMOs, including PMO-Cys, PMO-N3, and HS-PMO-(DBCO), were obtained from Wuxi (CN). RNAs were obtained from Genscript (NJ, USA), or TriLink BioTechnologies (CA, USA), or RNA Technologies and Therapeutic (QC, CA). LipidLaunch™ LNP-MC3 and LNP-0315 kits were purchased from Cayman Chemical (MI, USA). Dulbecco’s Modified Eagle’s Medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640 media, Opti-mem, Penicillin (50 U / mL), Streptomycin (50 pg / mL), L-Glutamine (PSG), and Trypsin-EDTA 0.05-0.5% were purchased from Fisher Scientific (Coming), ON, CA). Fetal Bovine Serum (FBS) was purchased from Wisent (QC, CA). Puromycin was purchased from Invivogen (CA, USA). Microfluidic Mixer MIX-4 Chip, gasket sheets, and individual reservoir connectors were purchased from Precigenome (CA, USA). Lipofectamine 2000, Neon™ NxT Electroporator, Electrolyte Buffer El 00, and Resuspension T Buffer were purchased from Invitrogen (MA, USA). PBS solution IX was prepared internally using a standard protocol for cell culture only. Human cervical carcinoma (HeLa) cells were purchased from the American Type Culture Collection (Ref. CCL-2). Monodisperse PEGs (SDPD-dPEGn-NHSester, 3 -Azidopropylamine, and amine-DBCO were obtained from Vector Laboratories (CA, USA). Sulfo-Cy5-Mal (sCy5-Mal), was purchased from Lumiprobe (MD, USA).

[0133] Ultra-Performance Liquid Chromatography (UPLC)

[0134] All reactions were monitored using a UPLC system that consisted of an Acquity UPLC binary solvent manager equipped with an Acquity automatic sample manager and a Photodiode Array (PDA) detector from Waters (Waters Inc., Bedford, MA, USA). The solvent system was composed of Milli-Q water (H2O) containing 0.1% formic acid (solvent A) and acetonitrile containing 0.08% formic acid(solvent B). Separation was achieved by reversed-phase with the following gradient: 0 - 0.40 min (98% A), 0.40 - 1.20 min (72% A), 2.20 - 2.40 min (30% A) 2.40 - 3.10 (10 % A) and 3.10 - 3.21 min (98% A) with a flow rate of 0.5 mL / min through an Acquity UPLC BEH Phenyl column (2.1 x 50 mm, particles 1.7 pm) kept at room temperature. The detector wavelength was set to 214, 229, 254, and 280 nm, and the injection volume was between 1 and 10 pL, depending on sample concentration.

[0135] Preparative HPLC

[0136] Purification was performed using a Waters 2487 high-performance liquid chromatography (HPLC) with a dual absorbance detector, equipped with a Waters 600 controller. The injection loop is 30 pL, and the column is an Xbridge Prep 19 mm x 150 mm, phenyl 5 pm. Solvent A is composed of Milli-Q water containing 0.1% formic acid (FA), and solvent B is composed of acetonitrile containing 0.08% FA. Purification was performed at 10 mL / min.

[0137] LCMS

[0138] Samples were analyzed on an ACQUITY UPLC (Waters Inc., Bedford, MA, USA) equipped with an ACQUITY UPLC BEH Phenyl column (130 A, 1.7 pm, 2.1 x 50 mm, Waters, Inc., Bedford, MA, USA). The gradient program was 2-28 % B in 0.80 min, 28-70 % B in 1.20 min, 70-90 % B in 0.7 min, and return at 2 % B in 0.11 min with mobile phase A (water with 0.1%FA) and mobile phase B (acetonitrile with 0.1 % FA) and was applied at a flow rate of 0.5 mL / min. The peptide identity was verified by mass spectrometry on a Xevo-TQD mass spectrometer (Waters Inc., Bedford, MA, USA) equipped with an ESI source and calibrated with the Xevo TQ-S micro installation std kit (Waters Inc., Bedford, MA, USA) as calibration solution. Source temperature was set to 150 °C. Data was acquired by scanning over the m / z range 50-2000 with a scan time of 0.3 sec. Analysis was set in the positive-ion mode, and the ESI-MS mass spectra were deconvoluted with the Waters MassLynx analysis software (Waters Inc., Bedford, MA, USA).

[0139] Alternatively, a Quadrupole Time-of-Flight mass spectrometry was used to characterize the peptides and conjugates. For that, samples were analyzed on an ACQUITY H-Class (Waters Inc., Milford, MA, USA) equipped with an ACQUITY UPLC BEH Phenyl column (1.7 pm 2.1 x 75 mm, Waters, Inc., Milford, MA, USA). The gradient program was 5% B for 1 min of 5-95% B in 3 min, hold 95% B for 0.5 min, 95-5% B in 0.1 min, 5% B for 1.4 min with mobile phase A (water with 0.1%FA) and mobile phase B (acetonitrile with 0.1 % FA) and was applied at a flow rate of 0.5 mL / min. The identity of the conjugated peptides was verified by mass spectrometry on an Xevo-G2-XS mass spectrometer (Waters Inc., Milford, MA, USA) equipped with an ESI source and calibrated with 0.5 mM of sodium format in 90: 10 IPA:Water as a calibration solution. Source temperature was set to 150 °C. Data was acquired by scanning over the m / z range 500-2000 with a scan time of 0.1 sec. Analysis was set in the positive-ion mode, and the ESI-MS mass spectra were deconvoluted using the Unifi v3.1 analysis software (Waters Inc., Milford, MA, USA).

[0140] Synthesis of peptides-lipid / sterol

[0141] The conjugation of peptides to a lipid / sterol was performed via a thiol-maleimide chemistry reaction. All reactions were carried out by adding the peptide and the lipid / sterol to a reaction vessel, stirred following conditions, quantity, and solvent as indicated in the following table. The reaction was monitored by UPLC. Crude products were purified by preparative HPLC. Pure fractions were pooled and lyophilized. The final compounds were characterized by LC / MS (ESI).

[0142] Table 1: Reaction conditions for the synthesis of peptides-lipid conjugates

[0143] Synthesis name Peptide Lipid Solvent Duration and Yield Quantity Quantity Temp.

[0144] FSP008D-SMal-PEGi9- FSP008D Mal-PEG19-CLS H2O / DMF Ih 61% CLS 0.73pmol 1.09pl(1.5eq) 80 / 20 1ml RT

[0145] FSP005D-SMal-PEGi9- FSP005D- Mal-PEG19-CLS H2O / DMF 2h30 28% CLS Cys 2.69pmol(1.5eq) 80 / 20 1ml RT

[0146] 1.8 pmol

[0147] FSP003-SMal-PEGi9 CLS FSP003-Cys Mal-PEGw-CLS H2O / DMF 2h30 36% 0.72 pmol 1,09 pmol (1.5 80 / 20 1ml RT

[0148] eq)

[0149] FSD375-SMal-PEGi9-CLS FSD375-Cys Mal-PEGw-CLS H2O / DMF 3hRT 5%

[0150] 3.52 pmol 8.80 pmol (2.5eq) 80 / 202mL

[0151] FSD10-SMal-PEGi9-CLS FSDIO-Cys Mal-PEGw-CLS H2O / DMF 15h 8%

[0152] 3.84 pmol 5.37 pmol (1.5 80 / 20 ImL

[0153] eq)

[0154] FSP008D-SMal-PEG4s- FSP008D- Mal-PEG45-DMG H2O / DMSO 3h 23% DMG Cys 1.09 (1.5 ea) 70 / 30 ImL RT

[0155] 0.73 pmolSynthesis of FSP008D-SMal-PEG19-CLS

[0156]

[0157] The conjugation of FSP008D to sterol was performed via a thiol-maleimide chemistry reaction. To a reaction vessel containing 0.73 pmol of FSP008D-Cys in H2O (500 pL), 1.09 pmol (1.5 eq) ofmal-PEGig-CLS in DMF (500 pL) was added. The mixture was stirred at room temperature for Ih, and the reaction was monitored by UPLC. Crude FSP008D-SMal-PEGi9-CLS was purified by preparative HPLC. Pure fractions were pooled and lyophilized, yielding about 61%. The final compound was characterized by LC / MS (ESI).

[0158] 838.16 1001 769.33.914.23

[0159] 146.99 1645.56 0 '' •|“| ~i' i ' i i,r.

[0160] 200 400 600 800 1000 1200 1400 1600 1800

[0161] Synthesis of FSP003D-SMal-PEG19-CLS

[0162]

[0163] The conjugation of FSP003D to a cholesterol was performed via a thiol-maleimide chemistry reaction. To a reaction vessel containing 0.72 pmol of FSP003D-Cys in H2O (800 pL), 1.09 pmol (1.5 eq) of mal-PEG19-CLS in DMF (200 pL) was added. The mixture was stirred at room temperature for 2h30 and the reaction was monitored by UPLC. Crude FSP003D-SMal-PEGi9-CLS was purified by preparative HPLC.Pure fractions were pooled and lyophilized, yielding about 36.16 %. The final compound was characterized by LC / MS (ESI).

[0164] Synthesis of FSP005D-SMal-PEG19-CLS

[0165]

[0166] The conjugation of FSP005D to a cholesterol was performed via a thiol-maleimide chemistry reaction. To a reaction vessel containing 1.80 pmol of FSP005D-Cys in H2O (800 pL), 2.69 pmol (1.5 eq) of mal-PEG19-CLS in DMF (200 pL) was added. The mixture was stirred at room temperature for 2h30 and the reaction was monitored by UPLC. Crude FSP005D-SMal-PEGi9-CLS was purified by preparative HPLC. Pure fractions were pooled and lyophilized, yielding about 27.51 %. The final compound was characterized by LC / MS (ESI).

[0167]

[0168] Synthesis of FSD10-SMal-PEG19-CLS

[0169]

[0170] The conjugation of FSD10 to a cholesterol was performed via a thiol-maleimide chemistry reaction. To a reaction vessel containing 3.84 pmol of FSDIO-Cys in H2O (800 pL), 5.37 pmol (1.5 eq) of mal-PEGig-CLS in DMF (200 pL) was added. The mixture was stirred at room temperature for 15h and the reaction was monitored by UPLC. Crude FSD 10-SMal-PEGi -CLS was purified by preparative HPLC. Pure fractions were pooled and lyophilized, yielding about 8.25 %. The final compound was characterized by LC / MS (ESI).

[0171]

[0172] FSD375L -SMal-PEG19-CLS

[0173]

[0174] The conjugation of FSD375L to a cholesterol was performed via a thiol-maleimide chemistry reaction. To a reaction vessel containing 3.52 pmol of FSD375L-Cys in H2O (1.6mL), 8.8 pmol (2.5 eq) of mal-PEG19-CLS in DMF (400pL) was added. The mixture was stirred at room temperature for 3h, and the reaction was monitored by UPLC. Crude FSD375L-SMal-PEGi9-CLS was purified by preparative HPLC. Pure fractions were pooled and lyophilized, yielding about 5.3 %. The final compound was characterized by LC / MS (ESI).36957

[0175] 100-

[0176] 766.49

[0177] 274.50

[0178] 110.91

[0179]

[0180] 200 1800

[0181] Synthesis of FSP008D-SMal-PEG45-DMG

[0182]

[0183] The conjugation of FSP008D to DMG was performed via a thiol-maleimide chemistry reaction. To a reaction vessel containing 0.73 pmol of FSP008D-Cys in H2O (700 pL), 1.09 pmol (1.5 eq) of mal-PEG45-DMG in DMSO (300 pL) was added. The mixture was stirred at room temperature for 3h, and the reaction was monitored by UPLC. Crude FSP008D-SMal-PEG45-DMG was purified by preparative HPLC. Pure fractions were pooled and lyophilized, yielding about 23%. The final compound was characterized by LC / MS (ESI).

[0184]

[0185] Circular Dichroism

[0186] Circular dichroism studies at pH 5 were performed using a Jasco J-815 spectropolarimeter (Easton, Maryland, USA). Samples were dissolved in H2O, or in 10%trifluoroethanol (TFE) in H2Oto give a final concentration of 250 pM, the pH of the buffers was adjusted using a solution of HC1 (IM) and spectra were recorded using a 0.2 mm path length quartz cells. For each measurement, ten scans were collected from 190 to 250 nm with a scanning speed of 100 nm / min at 37 °C. All spectra were converted to molar ellipticity (deg.cm2.dmol-1) after subtraction of the spectra buffer in the absence of peptide. Conversion to molar ellipticity was performed using the following equation:

[0187] Ml _ _ 9 X P _

[0188] L

[0189]

[0190] J10 x n x C P™CP x I

[0191] Where: [0] (mdeg.cnU.dmol1) is the molar ellipticity; 0 (mdeg) is the recorded signal; Mp(g.mol1) is the peptide molecular weight; n is the number of amino acids; C™pis the (g. L1) peptide weight concentration and 1 (cm) is the optical path of the cell.

[0192] The percentage of a-helix (helix %) content was determined as [0] / [0]a, where [0] is the molar ellipticity at 222 nm and [0]athe molar ellipticity of a fully structured peptide calculated using the following equation:

[0193] 7 2,5\

[0194] [£?]„ = -4000 - ( 1 - \ n )Where: - 40000 is the estimated ellipticity of a fully structured, infinitely long helix, and n is the number of amino acid residues in the peptide.

[0195] Electrophoretic Mobility Shift or Gel Retardation Assay

[0196] Gel retardation was performed by preparing peptide / mRNA complexes in a 40-pL solution of 50 mM sodium acetate buffer at pH 5. For this, 2 pg of (eGFP)-mRNA was mixed with 1% of peptide (9.6 pM) and incubated for two hours at room temperature. The complexes were then loaded in a pre-stained 1% agarose gel with 10 000X Gel Red in RNAse-free TBE IX buffer. The gel was run under 125V voltage for 45 minutes in RNAse-free TBE IX, then imaged using Gel Doc XR (Bio-rad, CA, USA) and Gel documentation system (Image Lab software, Bio-rad, CA, USA).

[0197] Cell Culture

[0198] Human cervical carcinoma HeLa cells (ATCC™ CCL-2) were cultured according to the manufacturer’s instructions in complete DMEM, consisting of DMEM supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U / mL penicillin, and 100 pg / mL streptomycin.. For peptide cargo (DIR-NLS) delivery experiments in all cell lines, plain RPMI 1640 medium (pH 5 or 7) was used, whereas for lipid nanoparticle (LNP) delivery experiments, complete DMEM was used for all cell lines.

[0199] In Vitro Transduction of DRI-NLS647(nls*) at pH 5 / 7

[0200] HeLa cells were seeded (15 000 cells / well) in a 96-well plate one day prior to transduction. Acidic delivery buffer was prepared by adding a few drops of hydrochloric acid [1 M] (HC1) to RPMI 1640 media to reach a pH of 5 using a pH meter (Accumet Basic AB 15 pH meter, Fisher Scientific, Hampton, NH, USA), the media was then filtered and used to prepare the delivery mixtures. Peptides at the indicated concentrations and 10 pM of Cy5 -labeled D-retro-inverso NLS peptide (DRI-NLS647or nls*) were mixed in 100 pL of RPMI at pH 5 or 7. Cells were washed once with sterile PBS IX and incubated for five minutes with the delivery mixtures or the cargo alone as a control (50 pL / well).

[0201] Following the five-minute incubation, 100 pL of complete DMEM was added to each well to dilute and inactivate the shuttle’s peptide activity, the total volume was replaced with fresh complete DMEM media. Cells were then incubated for 1 h at 37 °C in 5% CO2 and transduction efficiency was characterized by fluorescence microscopy imaging (Revolve, Echo; San Diego, CA, USA) and flow cytometry (Cytoflex™, Beckman Coulter; Indianapolis, IN, USA).Microscopy Imaging

[0202] Fluorescence microscopy imaging of HeLa cells was performed directly on the 96-well plate using a Revolve R4 hybrid inverted fluorescence microscope in the upright position at 20x magnification. The focus was manually performed, and the fluorescence intensity was adjusted automatically for each well.

[0203] Flow Cytometry

[0204] For flow cytometry sample preparation, media was first removed from each well, and cells were washed with PBS IX, then trypsinized for 10 minutes at 37 °C in 5% CO2 using 50 pL of 0.05% trypsin per well to detach all cells. Cells were then resuspended in 100 pL of complete DMEM, to inactivate the trypsin. A total of 150 pL per cell suspension was transferred to a transparent round bottom 96-well plate for flow cytometry analysis to determine the percentage of positive fluorescent cells (FITC fdter for (eGFP)-mRNA, APC fdter for DRI-NLS647) and their emitted signal intensity (Cytoflex™, Beckman Coulter; Indianapolis, IN, USA). Briefly, singlet cells were selected (FSC-H vs FSC-A), and the size and granularity measures of the cells allowed us to choose viable cells (FSC-A vs SSC-A). Then, from the viable cells, the percentage of cells with a fluorescence signal greater than the maximum fluorescence of untreated cells was used to identify positive fluorescent cells and determine the delivery efficiency.

[0205] Furthermore, cellular toxicity for all cargos was assessed using cytometric measurements of cell size (FSC) and granularity (SSC). The percentage of cell viability was calculated by comparing the events / pL in the viable population of each delivered cell condition to its untreated control (FSC-A vs SSC-A).

[0206] LNP Preparation

[0207] LNPs were prepared using the LipidLaunch™ LNP- MC3, or SM-102 kit according to the manufacturer’s instructions unless otherwise specified. LNP-MC3 consists of the lipid / sterol Dlin-MC3-DMA, 1,2, DSPC, cholesterol, and DMG-PEG45 at a molar ratio of 50:10:38.5: 1.5% respectively. LNP-SM-102 consists of the lipid / sterol SM-102, 1,2, DSPC, cholesterol, and DMG-PEG45 at molar ratios of 50: 10:38.5: 1.5% respectively. As readout, two types of mRNAs were used: an enhanced green fluorescent protein (eGFP)-mRNA and a firefly luciferase (FLuc)-mRNA. Typically, the mRNAs were dissolved in a 50 mM sodium acetate solution (pH = 4, RNase-free H2O) to achieve final concentrations ranging from 25 to 35 ng / pL in a 200 pL LNP final volume. The incorporation of synthetic peptide shuttles or peptide-lipid / sterol conjugates was performed during the LNP synthesis by replacing 1-2 % of the cholesterol needed for LNP synthesis with an equimolar amount of peptide or peptide conjugate. Peptides or peptide-lipid / sterol conjugates were dissolved in RNAse-free water to obtain a final concentration in the LNP mixture ranging from 1-20 pM. The peptide solution was added as a solution or lyophilized and mixedwith the lipids / sterol already dissolved in ethanol. The LNP formation can be prepared by pipet-mixing or using a microfluidic system. For pipet-mixing, the mRNA solution and peptides / lipids / sterol are pipet-mixed for 15 seconds and incubated at room temperature for ten minutes without agitation to prepare the LNPs. For the microfluidic device, the Flex-S system from Precigenome (CA, USA) was used to generate the LNPs. Briefly, the mRNA solution was loaded into the aqueous reservoir, while peptides / lipids / sterol were added to the organic solvent reservoir, and both streams were processed at a flow rate of 3 mL / min before being immediately collected in the collection tank. The newly synthesized LNPs were purified by dialysis at room temperature using IX PBS 10% sucrose to increase particle stability, using either Slide-A-Lyzer™ MINI Dialysis Devices or Pierce™ Microdialysis Plates with a 20kDa cutt-off Encapsulation efficiency and size distribution / polydispersity were respectively determined by Quant-iT RiboGreen RNA assay and DLS as described herein.

[0208] Physical Characterization of Lipid Nanoparticles

[0209] Size distribution, polydispersity index (PDI), and zeta potential of the LNPs were measured by dynamic light scattering (DLS) using a Zetasizer NanoZS from Malvern (Malvern Panalytical Ltd, UK). Measurements were performed at 22 °C in triplicates using a disposable plastic micro cuvette (ZEN0040) for size or a folded capillary zeta cell (DTS1070) for zeta potential. Particle size (Z -average) and zeta potential were presented in (%) intensity and millivolt (mV), respectively.

[0210] mRNA Encapsulation Efficiency

[0211] The concentration of encapsulated mRNA was measured using a Quant-iT™ RiboGreen™ RNA assay (ThermoFisher Scientific). Briefly, 5-20 pL (minimum 25 ng mRNA / well) of each sample to be quantified was loaded in a microplate, and the volume was completed to 100 pL with IX TE buffer (RNAse free) for non-encapsulated mRNA quantification, or 1% Triton X-100 in IX TE buffer (RNAase free) for total mRNA quantification. The samples were loaded along with dilutions of the provided ribosomal RNA (100 pg / mL) from 0 to 2000 ng / mL in the appropriate buffer to generate a standard curve. The Quant-iT RiboGreen RNA Reagent was diluted 1 / 200 in TE buffer, and 100 pL of this solution was added to each well. The plate was incubated for 2-5 min at room temperature, protected from light, and fluorescence was measured using a fluorescence microplate reader (excitation ~480 nm, emission ~520 nm). The same protocol was performed in the presence of Shuttle agents in the LNP formulations. Encapsulation efficiency percentage was calculated using the following equation:

[0212] (Total mRNA — Free mRNA)

[0213] EE% = x 100%

[0214] Total mRNALNP-Mediated Delivery of mRNAs

[0215] HeLa cells were cultured in complete DMEM and were used to investigate the delivery efficiency of mRNAs. Prior to delivery, adherent cells were seeded in a 96-well plate (10000 - 15 000 cells / well) and allowed to attach for at least 4 h at 37 °C in 5% CO2 before testing. Media was removed and replaced with 100 pL of LNP-mRNA or Shuttle-incorporated LNP-mRNA [(Shuttle agent)-LNP-mRNA] in complete DMEM containing 15, 30, 60, or 120 ng of mRNA per well. Suspension cells were seeded in a 96-well plate (50 000 cells / well in lOpL volume) with fresh media. LNP-mRNA or Shuttle-incorporated LNP-mRNA [(Shuttle agent) -LNP-mRNA] in lOOpL complete DMEM containing 15, 30, 60, or 120 ng of mRNA per well were added. Cells were incubated for 16 h at 37 °C in 5% CO2. The percentage of eGFP-positive cells was determined by flow cytometry. Ratios of mean fluorescence intensity were calculated by normalizing the mean fluorescence intensities of the shuttle LNP candidates to the control C-LNP without peptide agent for each delivered mRNA concentration. For (FLuc)-mRNA, cells were visualized by microscopy with transillumination for shape morphology. Luminescence was quantified with the One-step luciferase assay kit using a LUMIstar Omega luminometer microplate reader (BMG LABTECH, DE) following the manufacturer’s protocol (BPS Bioscience, CA, USA).

[0216] Bioluminescent Firefly Luciferase Assay

[0217] At 16 h post-delivery of the (FLuc)-mRNA, a One-Glo Reagent (ONE-Glo™ + Tox Luciferase Reporter and Cell Viability Assay (Promega, WI, USA) was made by combining the ONE-Glo™ Luciferase Assay Buffer with the ONE-Glo™ Luciferase Assay Substrate, provided with this system, and directly added to each well containing the culture media, prepared according to the manufacturer’s instructions, using a 1 / 1 One-Glo Reagent to cell culture media ratio, then incubated 3 minutes with agitation at room temperature. The content of each well was transferred into a white 96-well plate compatible with the luminometer microplate reader (LUMIstar Omega), and the firefly luminescence was measured for each condition with the gain adjusted to avoid signal saturation.

[0218] In Vivo Delivery of (FLuc)-mRNA Mediated by (Peptide)-LNPs

[0219] Animals. The TransBIOTech animal care facility is accredited by the Canadian Council on Animal Care (CCAC). This study was approved by the Cegep de Levis Animal Care Committee and complied with CACC standards and regulations governing the use of animals for research. Female C57Bl / 6 mice (Charles River, Rayleigh, USA) aged 6-8 weeks at delivery were used for this study. Following arrival in the animal facility, all animals were subjected to a general health evaluation. An acclimation period of 5-7 days is allowed before the beginning of the study. The animals were housedunder standardized environmental conditions. The mice were housed in auto-ventilated cages, 3-5 per cage. Each cage was equipped with a manual water distribution system. A standard certified commercial rodent diet and water were provided ad libitum for the acclimation phase. It is considered that there are no known contaminants in the diet and water that would interfere with the objectives of the study. Each cage was identified for the corresponding group, indicating the treatment and the identity of the animals housed in the cage. The animal room was maintained at a controlled temperature of 21.0 ± 1°C and a relative humidity of 40 ± 10%. A controlled lighting system guaranteed 12 hours of light and 12 hours of dark per day for all animals. Adequate ventilation of 18-20 air changes per hour was maintained.

[0220] Sample Preparation and Injection. LNP (FLuc)-mRNA formulations with or without peptide were prepared as described above. Samples were sent ready to use and were stored at 4 °C until use at the animal care facility. Products were left to equilibrate at room temperature before being well mixed by inversion prior to use for injection. The lower part of the back, as well as both thighs of each mouse, were shaved to maximize the visualization of any leakage prior to injection. Two hundred microliters of each LNP formulation containing 1 and 5 pg (FLuc)-mRNA were injected systemically. Injections were performed using a 29G syringe in the tail vein of each mouse.

[0221] In-Life Observations. Weight and health were recorded for each date so that each observation is associated with a particular date. General health observations were evaluated daily as well.

[0222] In Vivo Imaging. Twenty -four hours (24 h) after administration, animals were injected with freshly prepared D-luciferin solution intraperitoneally (30 mg / mL x 5 mL / kg for 150 mg / kg, GoldBio, MO, USA,). Ten minutes post-D-luciferin injection, mice were imaged for luminescence under isoflurane-mediated anesthesia on their back within the imaging chamber of an IVIS Lumina XR (Perkin Elmer, MA, USA) in luminescence mode. Total flux in the mouse abdomen was determined for each mouse using areas of interest.

[0223] Euthanasia. Mice were euthanized after the in vivo imaging session at 24 h post-injection. All organs were collected and placed in a Petri dish covered with D-luciferin at 0.3 mg / mL prior to imaging. Total flux in these tissues was determined using the area of interest and used to draw histograms using the organ weight as a normalizer. For bone marrow, the left femur was collected, and both extremities were cut with scissors. Then, the femur was centrifuged for one minute in an Eppendorf tube to extract the bone marrow, which was suspended in 0.3 mg / mL D-luciferin prior to imaging the tube along with organs.

[0224] PMO Encapsulation in LNPs

[0225] The formulation of LNP-0315 was adapted from the manufacturer’s protocol with slight modifications. For this formulation, a Splicing Switch Oligonucleotide PMO (eGFP) was used as apayload. The SSO directs a splicing correction of an (eGFP)-mRNA that contains a human beta-globin intron with an aberrant 654-splicing mutation. Typically, 0.6 mg of PMO (eGFP) was dissolved in a 50 mM sodium acetate solution (pH = 5; RNase-free H2O), then lipids and sterols dissolved in ethanol were added, and the solution was pipet-mixed for 15 seconds. The mixture was incubated at room temperature for 10 min without agitation. After incubation, the mixture was purified by centrifugal dialysis at 4 °C with IX PBS (Sterile filtered, RNAse / Dnase free) using Amicons with a 30 kDa cut-off.

[0226] PMO Delivery in HeLa eGFP Cells

[0227] HeLa eGFP cells were cultured in DMEM, supplemented with 10% FBS and 1% PSG (penicillin, streptavidin, and L-glutamine) and 500 ng / pL of puromycin, and were used to investigate the delivery efficiency of (eGFP)-PMO. Prior to delivery, cells were seeded in a 96-well plate (15 000 cells / well) and allowed to attach for at least 4 h at 37 °C in 5% CO2 before testing. Media was removed and replaced with 100 pL of LNP-(eGFP)-PMO or (eGFP)-PMO alone in plain RPMI containing 2.5, 5, 10, 20, 40 pg of (eGFP)-PMO per well. Cells were incubated for 24 h at 37 °C in 5% CO2. The percentage of eGFP+ cells was determined by flow cytometry.

[0228] Conjugation of pH-responsive synthetic peptide shuttle agents to functional cargos

[0229] Synthesis of nls-SMal-sCy5 (nls*)

[0230] OVNH2nls RT, 2h ^NX_. SH+. H ACN / H2O

[0231]

[0232] nls-Cys sCy5-Mal nls-SMal-sCy5 (nls*) Florescence labelling of nls-Cys was carried out as follows. To a 7.6 pmol of nls-Cys, was added 15.2 pmol of sCy5-Mal dissolved in 122 pl ACN. The final volume of the mixture was adjusted to 1 ml with ACN / H2O 50% (v / v). The reaction was stirred for 2 h at room temperature and monitored by UPLC-UV at 280 nm. Once the reaction was completed, the mixture was purified by preparative HPLC.

[0233] Fractions corresponding to the final compound were pooled and lyophilized. The reaction yield was about 80%. The purity of the fraction pool was confirmed by mass spectrometry. LC / MS (ESI) calculated for nls* C148H222N39O47S3' [M+H]+m / z 3396.82, found 3396.89.Synthesis of nls-SMal-sCy5-N3ACN / H2O 50 / 50 RT, 2h

[0234]

[0235] nls-Cys-N3sCy5-Mal nls-SMal-sCy5-N3

[0236] The nls(Cys)-N3 was labeled via thiol-maleimide click chemistry reaction. To 5.35 pmol of nls(Cys)-N3was added 30.7 pmol (2eq) of sCy5-Mal. The final volume was adjusted to 2 ml of ACN / H2O 50% (v / v), The mixture was stirred 2 h at room temperature and the reaction was monitored by UPLC-UV 280 nm. Once the reaction is completed, the final product was purified by preparative HPLC, giving 90% yield. Pure fractions were pooled, lyophilized, and characterized by mass spectrometry. LC / MS (ESI) calculated for nls-SMal-sCy5-N3C160H243N44O51S3- [M+5H]5+m / z 3695.14, found 3695.77.Synthesis of OPSS-PEG12-DBCO

[0237]

[0238] OPSS-PEG12-NHS ester Amine-DBCO OPSS-PEG12-DBCO

[0239] To synthesize OPSS-PEG12-DBCO, 10.96 pmol of OPSS-PEG12-NHS ester was added to 16.45 pmol (1.5eq) of the amine-DBCO in 5.5 ml ACN / H2O 50% (v / v). The mixture was stirred for 2 h at room temperature. The reaction was monitored by UPLC and purified by HPLC, giving 54% yield. Pure fractions were pooled, lyophilized, and characterized by mass spectrometry. LC / MS (ESI) calculated for OPSS-PEG12-DBCO C53H76N4O15S2 [M+2H]2+m / z 1073.32, found 1072.58.

[0240]

[0241] Synthesis of OPSS-PEGi2-trz-nls-SMal-sCy5

[0242] ACN / H2O

[0243] 50 / 50

[0244] nls RT, 2h nls-SMal-sCy5-N3OPSS-REG12-N3

[0245]

[0246] OPSS-PEG12-trz-nls-SMal-sCy5

[0247] Synthesis of OPSS-PEGi2-trz-nls-SMal-sCy5 was carried out as follows. To a 3.79 pmol of nls-SMal-sCy5-Ns, was added 4.55 pmol (1.2eq) of OPSS-PEG12-DBCO. The final volume of the mixture was adjusted to 700 pl with ACN / H2O 50% (v / v). The reaction was stirred for 2h at room temperature and monitored by UPLC-UV at 280 nm. Once the reaction was completed, the mixture was purified by preparative HPLC. Fractions corresponding to the final compound were pooled and lyophilized. Thereaction yield was about 80%. The purity of the fraction pool was confirmed by mass spectrometry.

[0248] LC / MS (ESI) calculated for nls* C148H222N39O47S3- [M+H]+m / z 3396.82, found 3396.89.

[0249] Synthesis of FSP005D-nls*

[0250]

[0251] OPSS-PEG12-trz-nls-SMal-sCy5 FSP005D-PEG12-trz-nls-SMal-sCy5 Synthesis of FSP005D-nls* was carried out as follows. To a 0.92 pmol of OPSS-PEGn-trz-nls-SMal-sCy5, was added 1.15 pmol (1.25eq) of FSP005-Cys. The final volume of the mixture was adjusted to 460 pl with ACN / H2O 50% (v / v). The reaction was stirred for 2h at room temperature and monitored by UPLC-UV at 214 nm. Once the reaction was completed, the mixture was purified by preparative HPLC. Fractions corresponding to the final compound were pooled and lyophilized. The reaction yield was about 80%. The purity of the fraction pool was confirmed by mass spectrometry. LC / MS (ESI) calculated for nls* C326H49SNS90IOIS5_[M+H]+m / z 7440.39 found 7439.11.

[0252] 20251 104_SCAN_FCL-376_20250CT20_FD10_2UL_SUB MS2 ES+

[0253]

[0254] Synthesis of FSD449D-nls*

[0255]

[0256] OPSS-PEG12-trz-nls-SMal-sCyS FSD449D-PEG12-trz-nls-SMal-sCy5 Synthesis of FSD449D-nls* was carried out as follows. To a 1.04 pmol of OPSS-PEGn-trz-nls- SMal-sCy5, was added 1.04 pmol (leq) of FSD449-Cys. The final volume of the mixture was adjusted to 1 ml with ACN / H2O 50% (v / v). The reaction was stirred for 2h at room temperature and monitored by UPLC-UV at 280 nm. Once the reaction was completed, the mixture was purified by preparative HPLC. Fractions corresponding to the final compound were pooled and lyophilized. The reaction yield was about 80%. The purity of the fraction pool was confirmed by mass spectrometry. LC / MS (ESI) calculated for nls* C326H5O8N87OIOI S5- [M+H]+m / z 7422.46 found 7419.99.

[0257] Intens. J

[0258] [mAU]

[0259] 60-

[0260] 40-

[0261] 20- X

[0262] 0- - > - v,..

[0263]

[0264] 0.0 0.5 1.0 1 5 2.0 2.5 Time [min]Synthesis of FSP005D-PMO FSP005D ACN H.’O RNAse free PMO %;50'50 H’ l-,

[0265] FSP005D O'" 'OH RT 6h

[0266]

[0267] PMO Cys F SP005D OPSS F SP005D SS PMO Synthesis of FSP005D-PMO was carried out as follows. To a 1.18 pmol of FSP005D-OPSS, was added 1.41 pmol (1.2eq) ofPMO-Cys. The final volume of the mixture was adjusted to 1.18 ml with ACN / H2O RNAses free 50% (v / v). The reaction was stirred for 6h at room temperature and monitored by UPLC-UV at 254 nm. Once the reaction was completed, the mixture was purified by preparative HPLC. Fractions corresponding to the final compound were pooled and lyophilized. The reaction yield was about 25%. The purity of the fraction pool was confirmed by mass spectrometry. LC / MS (ESI) calculated for

[0268]

[0269] Synthesis of OPSS-PEG12-N3

[0270] ACN / HjO RNAse free 50 / 50 RT, 1h

[0271]

[0272] OPSS-PEG12-N3PMO-DBCO OPSS-PEG12-PMO To synthesize OPSS-PEG12-N3, 43.86 pmol of OPSS-PEG12-NHS ester and 52.63 pmol (E2eq) of 3-azidropropylamine were dissolved in 4 ml of ACN / H2O 50% (v / v). The reaction was stirred at room temperature for 1 h and monitored by UPLC-UV at 229 nm. Once the reaction was completed, the final product was purified by preparative HPLC, giving 63% yield. Pure fractions were pooled, lyophilized.Inters.

[0273] [mAU]

[0274] 1000

[0275] 800

[0276] 600

[0277] 400

[0278] 200

[0279] 0

[0280]

[0281] 00 0.5 1 0 1 5 2.0 2.5 Time [mm] Synthesis of OPSS-PEGi2-trz-PMO

[0282] o ACN / H2O RNAse free 50 / 50 RT, 1h

[0283]

[0284] OPSS-REG12-N3 PMO-DBCO OPSS-PEG12-PMO PEGylation of the PMO was performed by reacting 3.23 pmol of PMO(EGFP)-DBCO and 4.84 pmol of OPSS-PEGlk-Ns ( 1.5eq) via the strain-promoted azide-alkyne cycloaddition (SPAAC). The mixture was dissolved in 1.6 ml of ACN / H2O RNAses free 50% (v / v). The reaction was stirred at room temperature for 1 h and monitored by UPLC-UV at 254 nm. Once the reaction is completed, the final product was purified by preparative HPLC, giving 90% yield. Pure fractions were pooled, lyophilized, and characterized by mass spectrometry. LC / MS (ESI) calculated for OPSS-PEGn-trz-PMO

[0285] C268H41 oN 107O862S2 [M+H]+m / z 7097.56, found 7097.40.

[0286] Intens.

[0287] [mAU] 2500 2000 1500 1000 500 0 0.0 0.5 1.0 1.5 2.0 2.5 Time [min]

[0288]

[0289] | IA FCL-323 pool 2025MAY26 1-B,3 01 27171.d: UV Chromatogram, 254 nm |

[0290]

[0291] OPSS-PEG12-PMO FSP008D-Cys FSP008D-SS-PEG12-PMO Conjugation of the PEGylated PMO to FSP008D via a disulfide bond was performed by reacting 0.70 pmol of the OPSS-PEGn-trz-PMO and 1.41 pmol (2eq) of FSP008D-Cys. The reactants were dissolved in 1.4 ml of ACN / H2O (DNAse / RNAse free) 50% (v / v). The reaction was stirred at room temperature for 5 h and monitored by UPLC-UV at 214 nm. The final product was purified by preparative HPLC, giving 38% yield, and characterized by mass spectrometry. LC / MS (ESI) calculated for FSP008D-SS-PEGi2-trz-PMO (FPSOO8D-SS-PEG12-PMO) C418H643N154O125P17S2 [M+H]+m / z 10416,37 found 10417.00.Intens (mAU] ' 800 ' 600 • 400 - 200 • 0 • -200 • 00 05 1 0 1 5 20 25 Time [min]

[0292] .... A ■,

[0293] Synthesis of FSD449D-SS-PEG ^trz-PMO

[0294]

[0295] OPSS-PEG12-PMO FSD449-Cys FSD449D-SS-PEG12-PMO Conjugation of the PEGylated PMO to FSD449D via a disulfide bond was performed by reacting 0.70 pmol of the OPSS-PEGn-trz-PMO and 1.41 pmol (2eq) of FSP449D-Cys. The reactants were dissolved in 1.4 ml of ACN / H2O (DNAse / RNAse free) 50% (v / v). The reaction was stirred at room temperature for 6 h and monitored by UPLC-UV at 254 nm. The final product was purified by preparative HPLC, giving 21% yield, and characterized by mass spectrometry. LC / MS (ESI) calculated for FSD449D-SS-PEGi2-trz-PMO (FSD449D-SS-PEGI2-PMO) C379H593N146O121P17S2 [M+H]+m / z 9721.49, found 9752.5.Intens.

[0296] [mAU]

[0297] 2500

[0298] 2000

[0299] 1500

[0300] 1000

[0301] 500

[0302] 0

[0303]

[0304] 0.0 0.5 1 0 1.5 2.0 2.5 Time [min]

[0305] Synthesis of FSPOOSD-SS-PEG^trz-PMO c> PMO o FSP003D FSP003D o

[0306]

[0307] OPSS-PEG12-PMO FSP003D-Cys FSP003D-SS-PEG12-PMO Conjugation of the PEGylated PMO to FSP003D via a disulfide bond was performed by reacting 1.41 pmol of the OPSS-PEGn-trz-PMO and 2.82 pmol (2eq) of FSP003D-Cys. The reactants were dissolved in 1 ml of ACN / H2O (DNAse / RNAse free) 50% (v / v). The reaction was stirred at room temperature for 4 h 30 and monitored by UPLC-UV at 214 nm. The final product was purified by preparative HPLC, giving 38% yield, and characterized by mass spectrometry. LC / MS (ESI) calculatedfor FSP008D-SS-PEGi2-trz-PMO (FPS003D-SS-PEGI2-PMO) C551H855N117O111P19S2 [M+H]+m / z 9750.44, found 9751.5.

[0308]

[0309]

[0310] Generation of an SSO-inducible HeLa EGFP-654 cell line

[0311] To study the delivery of the functional PMO(EGFP), an SSO-inducible HeLa EGFP-654 cell line was generated by nucleofection (Lonza 4D Nucleofector) of an EGFP-654 expression cassette at the AAVS1 locus (PMID: 12426578). Briefly, wild-type HeLa cells were co-transfected via electroporation with a plasmid coding for Cas9 and a guide RNA targeting the AAVS 1 locus, along with a donor plasmid containing a puromycin-resistant gene, an EGFP-654 reporter cassette, and homology arms to the AAVS1 locus sequence (OriGene Technologies, CAT#: GE100023 and customized GE100024). Cells were selected for puromycin resistance, and single clones were isolated, validated, and used for in vitro functional delivery of PMO studies. To ensure stability of the HeLa EGFP-654 line, cells were maintained in DMEM supplemented with 10% FBS, 1% PSG, and puromycin (500 ng / mL) at 37 °C and 5% CO2.

[0312] Cell delivery assays for pH-responsive synthetic peptide shuttle agents conjugated to functional cargos One day before delivery, the SSO-inducible HeLa EGFP-654 cells were seeded in a 96-well plate (20,000 cells / well) and allowed to attach for at least 16 h at 37 °C in 5% CO2 before testing. A nuclear stain was performed using 0.2 mg / mL of Hoechst 33342 per well. Cells, protected from light, were incubated with the nuclear stain for 30 min at room temperature before treatment with the conjugates. Cells were then washed with sterile PBS IX and incubated for different timepoints at room temperature or at 37 °C (for timepoints longer than 5 minutes) with delivery mixtures containing pH-Responsive-DRI-NLS-647 or PMO conjugates (50 pL per well) or cargo alone prepared in RPMI media at specific concentrations. At the end of the incubation time, 100 pL of complete DMEM was added to each well to dilute and inactivate the conjugate’s activity. Cells were finally washed with PBS IX and incubated withfresh complete DMEM and incubated for 1 h (fluorescent cargo) or overnight (functional PMO) at 37 °C in 5% CO2 before flow cytometry and microscopy analyses.

[0313] Cell viability and cytotoxicity assays

[0314] One day prior to treatment, cells were seeded in a 96-well plate at a density of 15,000 cells / well and allowed to adhere for at least 16 h at 37 °C in 5% CO2. Cells were then incubated with peptides at a final concentration of 20, 40, or 60 pM, according to each experimental condition. Incubation times were 5 or 30 min for the FSD449 peptide and 5 min or 2h for all pH-responsive shuttles. Untreated cells incubated with complete DMEM served as negative controls, while positive cytotoxicity controls were generated by adding 1 pL of lysis buffer from the CytoTox-ONE™ Homogeneous Membrane Integrity Assay kit 5 min before analysis. Following incubation, culture media were collected from each well and transferred to a black, clear-bottom 96-well plate. An equal volume of CytoTox-ONE™ reagent was added to each well and incubated at room temperature for 10 min to allow reaction with released LDH. The reaction was then terminated by adding 25 pL of Stop Solution per well. Fluorescence was measured using a plate reader at an excitation wavelength of 560 nm and an emission wavelength of 590 nm. Cytotoxicity (%) was calculated as 100 x (Experimental signal - Background) / (Positive control -Background). Cell viability was assessed in parallel using a resazurin-based metabolic activity assay. Immediately after media removal for LDH analysis, 100 pL of plain RPMI medium was added to the remaining cells, followed by 20 pL of resazurin at a 0.15 mg / mL concentration. Cells were incubated for approximately 1 h at 37 °C and 5% CO2, ensuring that fluorescence signals remained below saturation. Supernatants were then transferred to a new black, clear-bottom 96-well plate, and fluorescence was measured at an excitation wavelength of 560 nm and an emission wavelength of 590 nm. Cell viability (%) was calculated using the same formula described above.

[0315] Measurement of cargo uptake by flow cytometry

[0316] In vitro delivery efficiency analysis was performed 1 h post-delivery for all DRI-NLS conjugates in HeLa cells and 24 h after delivery for the EGFP expression in SSO-inducible HeLa EGFP-654 cells. For flow cytometry sample preparation, media was first removed from each well, and cells were washed with PBS IX, then trypsinized for 10 min at 37°C in 5%CO2 using 50 pL 0.05% trypsin per well to detach all cells. Cells were then resuspended in 100 pL of complete DMEM, to inactivate the trypsin. A total of 150 pL per cell suspension was transferred to a transparent U-bottom 96-well plate for flow cytometry analysis to determine the percentage of positive fluorescent cells and their emitted signal intensity (Cytoflex, Beckman Coulter). Briefly, singlet cells were selected (FSC-H vs FSC-A), and the size and granularity measures of the cells allowed us to choose viable cells (FSC-A vs SSC-A). Then,from the viable cells, the percentage of cells with a fluorescence signal greater than the maximum fluorescence of untreated cells was used to identify positive fluorescent cells and determine the delivery efficiency. Furthermore, cellular toxicity for all cargos was assessed using cytometric measurements of cell size (FSC) and granularity (SSC). The percentage of cell viability was calculated by comparing the events / pL in the viable population of each delivered cell condition to its untreated control (FSC-A vs SSC-A).

[0317] Microscopy imaging

[0318] For in vitro studies, fluorescence live microscopy images of cells were captured directly on the 96 well plate using a Revolve R4 hybrid inverted fluorescence microscope in the upright position (Echo, BICO company) at 20X magnification. The focus was manually performed, and the fluorescence intensity was adjusted automatically for each well. The Hoechst 33342, EGFP+, and sCy5+cells were analyzed using the DAPI, fluorescein isothiocyanate, and Cy5 filters, respectively.

[0319] In vivo delivery of pH-responsive-PMO Conjugates by intravenous administration

[0320] Animals. FVB CAG-EGFP*lRkol / RjulJ mice (Strain #027617) were obtained from Jackson Laboratory and bred homozygously. These mice express the EGFP protein coding sequence interrupted by the intron of human beta-globin encoding the 654-splicing mutation. The mutant EGFP RNA is expressed in all cell types but is nonfunctional. Upon splicing correction with an ASO, functional EGFP is expressed. Both male and female mice aged 6-8 weeks were used for the studies. This study was approved by the Cegep de Levis Animal Care Committee (#003-23) and complied with CACC standards and regulations governing the use of animals for research. Five microns filtered, activated-charcoal treated, reverse-osmosis-treated tap water was provided to the animals. A standard certified commercial rodent diet (Envigo 2018) was also provided ad libitum. Test articles were administered to the animals by intravenous injection of the tail vein at a volume of 0.2mL. The mice body weight was measured daily and clinical signs were evaluated 0.083h, 0.5h, Ih, 2h, 4h, 24h, 48h and 72h after the administration. The clinical sign evaluation was based on the animal’s gait, grimace, mobility, response to stimuli, hair coat condition, respiration, hydration status, and any other indicators of distress or pain.

[0321] Euthanasia. The mice were euthanized 72h after administration by cardiac puncture under isoflurane anesthesia. At euthanasia, targeted organs were collected for eGFP imaging.

[0322] Microscopy Imaging. Organs were fixed with 4% PFA overnight at 4°C and immersed in a 30% sucrose solution for 24 h at 4°C. Tissues were transferred to a 20% sucrose: OCT (1:1) solution, cut into 7-pm -thick sections with a cryostat, and mounted on glass slides with a coverslip using a Prolong GlassNucBlue (Invitrogen). Images were taken with an automated slide scanner (PANNORAMIC MIDI II, 3DHistech Utd.) within 1-4 days after mounting.

[0323] Statistical Analysis

[0324] Statistical analysis and graphing were performed using GraphPad Prism (GraphPad Software, MA, USA). For in vitro studies, all represented data indicate a minimum experiment replicate of n = 3. Statistical significance for in vitro and in vivo studies, when applicable, was determined using a two-way ANOVA and a multiple comparisons test. The test type is indicated in each figure (p < 0.05). The in vivo data represents a single experiment with n = 3 per group.

[0325] Example 2: Synthetic peptide shuttle agents

[0326] Synthetic peptides called shuttle agents represent a relatively new class of intracellular delivery agents having the ability to transduce many types of non-polyanionic cargos to the cytosolic / nuclear compartment of eukaryotic cells. Unlike traditional cell-penetrating peptide-based intracellular delivery strategies, synthetic peptide shuttle agent-mediated cargo transduction has been shown to occur with extremely rapid kinetics involving direct translocation across the cell membrane, bypassing the kinetically slower conventional cellular endocytic pathways (e.g., WO / 2016 / 161516; WO / 2017 / 175072;

[0327] WO / 2018 / 068135; WO / 2020 / 210916; WO / 2022 / 077121; WO / 2022 / 082315; WO / 2022 / 204806). Also, unlike traditional cell-penetrating peptides, synthetic peptide shuttle agents have been shown to be highly effective when not covalently linked or not electrostatically complexed with their cargos during transduction. Although synthetic peptide shuttle agents were initially developed and optimized for transducing protein cargos, subsequent studies demonstrated the versatility of the platform to transduce different types of non-polyanionic cargos into even some of the most difficult-to-transduce / transfect cells (e.g., NK cells, Del’Guidice et al., 2018, Mac Donald et al., 2023) and tissues (e.g., airway epithelial cells, Krishnamurthy et al., 2019, Kulhankova et al., 2023, Uno et al., 2023, Kulhankova et al., 2024; depilated skin of mice, WO / 2020 / 210916), thereby underscoring the robustness of the platform.

[0328] One of the hallmarks of synthetic peptide shuttle agents is their ability to transduce cargoes in a variety of different eukaryotic cell types virtually instantaneously upon contact. However, for some applications, some delay and / or control over the transduction activity of synthetic peptide shuttle agents would be desirable. The work presented herein relates to the development of a new subclass of pH-responsive synthetic peptide shuttle agents having increased transduction activity at acidic pH as compared to at neutral or basic pH. Such pH-responsive synthetic peptide shuttle agents could prove particularly useful for more controlled transduction of cargoes, for example, in acidic tumormicroenvironments, or in facilitating the release of endosomally-trapped cargoes upon endosomal acidification.

[0329] Although different subclasses of synthetic peptide shuttle agents have emerged, a common structural feature shared by nearly all synthetic peptide shuttle agents that exhibit a significant degree of cargo transduction activity is their secondary structure: namely, the presence of a “core” segment of at least 12 to 15 amino acids long having an amphipathic alpha-helical structure with a discrete cationic hydrophilic face and a discrete hydrophobic face (WO / 2022 / 082315). Truncation studies showed that synthetic peptide shuttle agents consisting of this “core” region alone, or the “core” region flanked on one or both sides by flexible glycine / serine-rich segments, were sufficient for cargo transduction activity (WO / 2022 / 082315), with the latter subclass of synthetic peptide shuttle agents generally showing a greater ability to transduce Cas9-RNP complexes than other synthetic peptide shuttle agents. The synthetic peptide shuttle agent FSD375 described in WO / 2022 / 082315 proved to be particularly versatile, as it was even able to deliver functional genome-editing Cas9-RNP and base-editing ABE-Cas9-RNP complexes in the notoriously refractory Human Bronchial Epithelial cell line, CFF-16HBEge (Figs. 7 and 8 of WO / 2022 / 082315). FSD375D was therefore employed as a starting point for the development of a new subclass of pH-responsive synthetic peptide shuttle agents, with the amino acid sequence of FSD375D being identical to that of FSD375 except that all the amino acids of FSD375D are D-amino acids. Synthetic peptide shuttle agents were previously shown to be active regardless of whether they are composed of amino acids in their L or D conformations (e.g., WO / 2020 / 210916). The use of peptides comprised entirely of D-amino acids are generally more resistant to degradation (e.g., by proteases) and / or may have a longer half-life than a corresponding peptide comprised of only L-amino acids.

[0330] Example 3: Development of pH-responsive synthetic peptide shuttle agents Structure -function studies we performed on the FSD375D peptide (SEQ ID NO: 1) revealed that a single lysine to alanine substitution at position 21 (i.e., K21A) resulted in an increase in baseline transduction activity over FSD375 (data not shown) while reducing the peptide’s C-terminal positive charge density. The peptide FSD449D (SEQ ID NO: 2) was synthesized containing this substitution and further included a C-terminal cysteine residue (to provide a convenient thiol functional group to facilitate potential future conjugations). An alignment of the amino acid sequences of FSD375D and FSD449D is shown in Fig. 1, with the “core” segment in bold and position 21 being highlighted in black.

[0331] A strategy we explored to obtain pH-responsive synthetic peptide shuttle agents was to make use of the amino acid histidine. The imidazole side chain of histidine has a pKa of about 6, resulting in it being predominantly uncharged (unprotonated) at physiological pH and predominantly cationic (protonated) at acidic pH. A series of variant peptides were then synthesized in which one or more of thepositively-charged lysine (K) or arginine (R) residues of FSD449D were replaced with histidine. A

[0332] summary of the variant peptides synthesized and tested in this Example is shown in Table 2, along with their total number of histidine residues, hydrophobic moment (p), and predicted relative net charge at both neutral pH 7 and acidic pH 5. An alignment of the different variant peptides synthesized based on FSD449D is shown in Fig. 2, in which the K, R, and H residues are highlighted in black.

[0333] Table 2: Name, structure, and physical properties of studied peptides

[0334] Average

[0335] Rel. charge Hydrophilicity Peptide Sequence lacking C-terminal Cys # His pH7 pH5 pH pl FSD10 KWKLARAFARAIKKLGGSGGGSYARALRRQARTG 0 10 10 7.02 0.34 12.82 FSD375D GGSGGGSKWKLARAFARAIKKLGGSGGGS 0 6 6 4.4 0.20 12.55 FSD449D GGSGGGSKWKLARAFARAIKALGGSGGGS 0 5.02 5.02 3.72 0.08 12.54 FSP001D GGSGGGSHWHLAHAFAHAIHALGGSGGGS 5 0.47 4.53 4.16 -0.52 8.15 FSP002D GGSGGGSKWHLAHAFAHAIKALGGSGGGS 3 2.27 4.72 4.14 -0.28 10.66

[0336]

[0337] FSP003D GGSGGGSHWKLAHAFARAIHALGGSGGGS 3 2.25 4.72 4.04 -0.28 11.56 FSP004D GGSGGGSHWHLAHAFARAIKALGGSGGGS 3 2.29 4.74 4.02 -0.28 11.56 FSP005D GGSGGGSHWHLAHAFARAIKALGGSGGGS 2 3.16 4.83 3.91 -0.16 12.50 FSP006D GGSGGGSKWHLAHAFAHAIKALGGSGGGS 2 3.20 4.83 4.03 -0.16 11.74 FSP009D GGSGGGSHWKLAHAFARAIHALGGSGGGS 2 3.20 4.83 3.73 -0.16 12.50 FSP010D GGSGGGSHWKLARAFAHAIHALGGSGGGS 3 2.27 4.74 3.88 -0.28 11.56 FSP011D GGSGGGSHWKLAHAFARAIHALGGSGGGS 2 3.20 4.83 3.86 -0.16 11.74 FSP012D GGSGGGSKWKLAHAFARAIKALGGSGGGS 1 4.11 4.92 3.83 -0.04 11.84 FSP013D GGSGGGSHWKLARAFAHAIHALGGSGGGS 4 1.34 4.64 3.85 -0.40 11.06 FSP014D GGSGGGSHWKLLHLFLRLIHLLGGSGGGS 3 2.27 4.79 3.76 -0.50 11.56 FSP015D GGSGGGSHWKLAHLFLRAIHALGGSGGGS 3 2.29 4.74 3.61 -0.37 11.56 FSP008D GGSGGGSKWHLLKIWSHLIKIWRHLGGSGGGS 3 4.29 6.75 7.51 -0.34 11.84 FSP007D GGSGGGSHWHLLHLWSRLLKLWGGSGGGS 3 2.22 4.75 6.03 -0.52 11.56 FSP016D GGSGGGSHWHLAHEFAHEIHALGGSGGGS 5 -1.81 3 6.03 -1.39 6.26 FSP017D GGSGGGSHWHLAHEFEHAIHALGGSGGGS 5 -1.81 3 4.75 -1.39 6.26

[0338] The ability of the peptides in Table 2 to transduce a fluorescently-labeled (D) retro-inverso NLS peptide (nls*) cargo (Auger et al., 2024) in cultured HeLa cells in vitro was tested at both pH 7 and pH 5 as described in Example 1. Results are shown in Table 3 and Fig. 3A and 3B.Table 3: Cargo transduction activity of peptides at pH 7 and pH 5 in HeLa cells Transduction efficiency

[0339] Peptide (10 pM) pH7 pH5 Change*

[0340] FSD449 50.7% 52.3% +1.6

[0341] FSP001D 2.8% 13.4% +10.6

[0342] FSP002D 4.4% 10.2% +5.8

[0343] FSP003D 12.4% 57.4% +45

[0344] FSP004D 11.1% 38.4% +27.3

[0345] FSP005D 45.7% 62.4% +16.7

[0346] FSP006D 19.8% 37.8% +18

[0347] FSP009D 11.4% 20.8% +9,4

[0348] FSP010D 3.7% 16.8% +13,1

[0349] FSP011D 8.5% 26.2% +17,7

[0350] FSP012D 11.9% 29.9% +18,0

[0351] FSP013D 4.8% 15.5% +10,7

[0352] FSP014D 15.6% 68.7% +53,1

[0353] FSP015D 40.5% 56.1% +15,6

[0354] FSP008D 41.9% 61.9% +20

[0355] FSP007D 15.1% 34.2% +19.1

[0356] FSP016D 2.4% 4.6% +2.2

[0357]

[0358] FSP017D 2% 5.4% +3.4

[0359] * Change in cargo transduction efficiency from pH7 to pH5

[0360] As mentioned in Example 2, a common structural feature shared by nearly all synthetic peptide shuttle agents that exhibit a significant degree of cargo transduction activity is the presence of a “core” segment of at least 12 to 15 amino acids long having an amphipathic alpha-helical structure with a discrete cationic hydrophilic face and a discrete hydrophobic face (Fig. 4A - 4S). This structure can be readily seen upon a helical wheel projection of the core segment of FSD449D, as shown in Fig. 4A in which the hydrophobic face is indicated in a dotted line and cationic R / K residues are circled.

[0361] In the FSP001D peptide, all the cationic K and R residues of FSD449D are replaced with histidine residues (Fig. 4B) and this completely abolished cargo transduction activity at pH 7 and yielded only low transduction activity at pH 5 (Table 3 and Fig. 3). For FSP002D (Fig. 4C), which showed no cargo transduction activity at pH 7 and vey low activity at pH 5, the two positively-charged residues were split, one K resided in the hydrophobic face and one K in the cationic face (Fig. 4C). FSP013D (Fig. 4N), in which 4 / 5 of the cationic K and R residues were replaced with histidine residues and thus comprises one R in its cationic face, also showed the virtually no cargo transduction activity at pH 7 and low activity at pH 5.

[0362] FSP016D and FSP017D also derived from FSD449D, in which in addition to having all cationic K and R residues replaced by histidine, were also designed to introduce negatively charged amino acidsby substituting alanine residues with glutamic acid residues (Fig. 2 and Table 2), did not show any cargo transduction activity at neither pH 7 nor pH5.

[0363] Strikingly, many Shuttle peptide variants tested showed activity at pH 5, with FSP003D (Fig. 4D), FSP004D (Fig. 4E), FSP006D (Fig. 4G), FSP007D (Fig. 4H), FSP005D (Fig. 4F), and FSP014D (Fig. 40) showing the most significant activation of their cargo transduction ability at pH 5 compared to their activity at pH 7. Each of these Shuttle peptide variants retained two to three K / R residues that were originally in the cationic face of FSD449D (Fig. 4A), indicating that the retention of at least two K / R residues on the cationic face of the core from the original sequence results in a significant increase in shuttle activity at pH 5, although at least one K / R was sufficient in some instances.

[0364] FSP009D (Fig. 4 J), and FSP010D (Fig. 4K) both retained two to three K / R residues on their cationic face and showed some activity at pH 5. However, their hydrophobic moments (pH) were lower compared to most other pH-responsive Shuttles containing two to three K / R residues from the original sequence (Table 2). The pH value is a measure of the segregation of hydrophilic and hydrophobic amino acid residues along the helical axis of a peptide. It quantifies the amphiphilicity of a helix, which is related to interactions with a lipid bilayer. A high pH suggests a strong tendency for the peptide to interact with lipid membranes and is often correlated with the ability of the peptide to penetrate or permeabilize cell membranes. This is supported for example by FSP008D (Fig. 41), which contains 3 histidine residues and has a very high pH (Table 2). This peptide showed an acid-activation and a transduction ability at pH 5 that is comparable to the most efficient pH-responsive Shuttles (Fig. 3A). Surprisingly, FSP014D which is among the most efficient pH-responsive shuttles, has a low pH (Table 2). However, this Shuttle is highly hydrophobic with the lowest average hydrophilicity (about -0.50) among all active Shuttle peptides (Table 2), suggesting that peptide hydrophobicity has an impact on activity and, to some extent, can compensate for a low pH.

[0365] As seen, in Figs. 5A-5C, circular dichroism studies performed at pH 5 in H2O and in 10%TFE in H2O revealed that low cargo transduction activity peptides FSP001 and FSP013 generally adopted a random coil conformation at pH 5 (2% helical content), while the peptides FSD449, FSP012, FSP007 and FSP008 exhibiting robust cargo transduction activity generally had the highest helical content (5%). FSP008 reached over 15% helical content in 10% TFE. Data also show that increasing the number of histidine that were in the cationic face of FSD449 generally decreased the ability to form helical structures in H2O and in 10% TFE.

[0366] Taken together, the structure -activity relationship studies in this Example strongly suggest that synthetic peptide shuttle agents can be engineered to exhibit greater cargo transduction activity at acidic pH compared to neutral or slightly basic pH. This can be achieved by incorporating histidine residues into the cationic face of the amphipathic alpha-helix. In general, the data also show that it is beneficial to keepat least one of the positively charged residues, such as lysine or arginine, on the cationic face of the core from the original sequence. Moreover, having a greater number of positively charged residues in the cationic face than in the hydrophobic face was also seen to be beneficial.

[0367] However, further studies conducted to evaluate the delivery efficiency of pH-responsive Shuttles with reduced positive charge at pH 7, such as FSP013D, showed that delivery of the cargo can still be achieved at higher concentrations despite the reduced positive charge. As seen in Fig. 3B, a dose response study was performed using one representative shuttle from each charge group. The results show that the delivery efficiency of pH-responsive shuttles with reduced initial charge (2+) and (1+) increased in a concentration-dependent manner. These shuttles remained well tolerated at concentrations up to 60 pM, with only a slight decrease in viability observed for FSP013D at 100 pM (data not shown). In contrast, the delivery efficiency of the fully histidine-based FSP001D, having no positively charged residues on its cationic face (Fig. 4B) was not improved, even at the highest tested concentration (100 pM).)

[0368] These results also demonstrated, somewhat unexpectedly, that replacement of some K / R residues in FSD449D with histidine residues (e.g., FSP003D, FSP005D, FSP008D and FSP014D) achieves cargo transduction activities at pH 5 that even exceeded the maximum transduction activity of FSD449D at pH 7, suggesting the intriguing combination of pH-responsive synthetic peptide shuttle agents and an acidic delivery medium to achieve enhanced cargo transduction. Additionally, pH of the sequence can be tuned to further improve cargo transduction of the pH-responsive Shuttle peptide. Hence, parameters such as the number and position of histidine residues, hydrophobicity of the sequence, and amphiphilicity of the helix are all parameters that can be fine-tuned to design an acid-responsive peptide with a cargo transduction ability that exceeds the original Shuttle.

[0369] Example 4: Characterization of LNPs incorporating pH-responsive synthetic peptide shuttle agents Lipid nanoparticles (LNPs) are one of the most commonly used non-viral nanocarriers employed for delivering nucleic acids and other payloads into cells. LNPs are typically formulated using cationic or ionizable lipids, helper lipids, PEG-lipids, and / or sterol lipids in well-defined ratios. In particular, ionizable lipids have been employed in Modema’s and Pfizer / BioNTech’s mRNA LNP-based COVID-19 vaccines and are currently being investigated to deliver mRNAs to infections, cancer, and genetic disorders. Despite such efforts, studies revealed that only <15% of the encapsulated RNA reaches the cytoplasm (Zhang et al., 2022; Sabnis et al., 2018; Maugeri et al., 2019; and Muller et al., 2024). The rest of the internalized RNA mainly accumulates in lysosomes for degradation (Paramasivam et al., 2022; Paramasivam et al., 2013; Wittrup et al., 2015; Zeng et al., 2023; and Chatterjee et al., 2024). Thus, facilitating endosomal / lysosomal escape of LNPs would improve the efficacy and potentially lower LNP / mRNA dosing requirements. While some attempts have been made to exploit cell-penetratingpeptides (CPPs) or fusogenic peptides to enhance cytosolic delivery, such approaches generally involve attaching the peptides to a lipid, resulting in LNPs “displaying” peptides on their surface, known as peptide-decorated LNPs (Khalil et al., 2014; Aschmann et al., 2024; Zeng et al., 2023; Tateshita et al., 2019; and Abd Elwakil et al., 2019). However, such a design results in cationic LNPs that can randomly interact with proteins and cells in vivo, which could potentially be detrimental, and endosomal entrapment remains a major hurdle.

[0370] We explored whether physically incorporating pH-responsive synthetic peptide shuttle agents into LNPs (e.g., LNP-MC3) could facilitate endosomal escape and cytosolic delivery of mRNA payloads. Experimentally, pH-responsive synthetic peptide shuttle agents were pipet-mixed with the mRNA and the lipid / sterol to form the LNPs, as described in Example 1. After dialysis in PBS, the resulting (Shuttle agent)-LNPs were characterized by dynamic light scattering (DLS) to determine particle size, polydispersity (PDI), and zeta potential (Z). The results are summarized in Table 4.

[0371] Table 4: Physical characterization of LNPs by dynamic light scattering Peptide (1%) Size (nm) PDI Z (mV) Encap. Eff.

[0372]

[0373] 84.5 0.08 -2.750 100%

[0374] FSD449D 91.7 0.12 -6.807 97%

[0375] FSP001D 220.6 0.16 -2.467 72%

[0376] FSP003D 156.8 0.087 -3.420 85%

[0377] FSP004D 144 0.208 -2.913 90%

[0378] FSP005D 150.5 0.206 -1.737 90%

[0379] FSP006D 175.4 0.16 -1.827 86%

[0380] FSP008D 127.9 0.181 - 73%

[0381] FSP014D 192,7 0.09 - 36.1%

[0382] FSP015D 143.9 0.17 - 76.5%

[0383] FSP016D 90.1 0.18 - 96%

[0384] FSP017D 68.5 0.25 - 97%

[0385] Although DLS characterization showed that mRNA-LNPs that incorporated a peptide tended to have larger diameters, nearly all (Shuttle agent)-LNPs had size ranges within those generally considered ideal for LNPs (e.g., 20-200 nm). The encapsulating efficiency of the (Shuttle agent)-LNPs obtained with the various pH responsive synthetic peptide shuttle agents ranged from about 37% to about 97%.

[0386] Example 5: In vitro evaluation of mRNA-LNPs incorporating pH-responsive synthetic peptide shuttle agents

[0387] We next investigated the ability of different (Shuttle agent)-LNPs to deliver an mRNA payload encoding eGFP or Firefly luciferase (Flue) to the cytosol and result in the expression of a functionalprotein, as described in Example 1. Briefly, delivery efficiency was evaluated by incubating freshly prepared (Shuttle agent)-LNPs or control LNP formulations lacking the Shuttle agent, prepared by pipet mixing, in HeLa cells (60 ng of mRNA; 1% = 9.5 pM of peptide). The results in Fig. 6A showed that the peptides FSP003D, FSP004D, FSP005D, and FSP006D all strikingly increased eGFP expression as compared to the control LNP formulation by more than two-fold. All of these peptides had also shown robust intracellular delivery of the nls* peptides in HeLa cells at pH 5 (Table 3 and Fig. 3), suggesting that their ability to mediate cargo transduction across the plasma membrane also translated to their ability to mediate mRNA escape from acidified endosomes / lysosomes. The experiment was repeated with the peptide FSP003D with firefly luciferase (FLuc) -encoding mRNA, with the readout being bioluminescence (instead of fluorescence). The results in Fig. 6B show that incorporating the FSP003D peptide in the LNP formulation resulted in an increase in luciferase expression as compared to the control LNP formulations lack any Shuttle agent, in line with the results in Fig. 6A. In contrast, the FSP001D peptide, which exhibited minimal intracellular delivery in HeLa cells at pH 5 (Table 3 and Fig. 3A), did not result in an increase in eGFP expression as compared to the control LNP formulation (Fig. 5A).

[0388] Interestingly, the peptides FSD449D and FSP008D both did not result in an increase in eGFP expression as compared to the control LNP formulation despite both peptides exhibiting high intracellular delivery in HeLa cells at pH 5 and pH 7 (Table 3 and Fig. 3A). These peptides were suspected to form overly tight polyplexes with mRNA due to them having the highest overall relative charges at pH 7 (Table 2) compared to the other peptides. To investigate this hypothesis, we examined the strength of electrostatic interactions between the peptides and the mRNA by an electrophoretic mobility shift assay, as described in Example 1. The results shown in Fig. 7 reveal that the peptide / mRNA electrostatic interactions for the peptides FSD449 and FSP008D when LNPs were prepared by pipet mixing were sufficiently strong to prevent the release and migration of mRNA, which was not the case for the other peptides. These results suggest that overly strong interactions between the peptide and mRNA may interfere with the ability of the mRNA to reach the cytosol and / or its translation. Conversely, synthetic peptide shuttle agents that have weaker interactions with the mRNA cargo (e.g., by having reduced overall charge at neutral pH) are advantageous to employ in the (Shuttle agent)-LNP formulations prepared by pipet mixing.

[0389] FSP016D and FSP017D exhibiting a relative charge of -1.81 at neutral pH and +3 at acidic pH (Table 1) were additionally tested to determine their ability to deliver an mRNA payload when physically incorporated into LNPs. While FSP017D led to a low improvement of mRNA delivery at a delivery dose of 120 ng of mRNA / well, FSP016D achieved approximately 8- and 17-fold higher (FLuc)-mRNA delivery and expression at both delivered doses of 60 and 120 ng mRNA / well, respectively, relative to the control LNP (Fig.6C).Example 6: In vivo evaluation of mRNA-LNPs incorporating pH-responsive synthetic peptide shuttle agents

[0390] LNPs were formulated with mRNA encoding firefly luciferase ((FLuc)-mRNA) with or without incorporation of the pH-responsive synthetic peptide shuttle agent FSP003D and were administered in mice, as described in Example 1. Briefly, LNPs containing 1 or 5 pg of mRNA / mouse were injected via the tail vein, and whole-body bioluminescence was measured 24 h post administration. Emitted photons from live animals were quantified with an exposure time of 1 min. The region of interest was quantified as average radiance (represented by color bars) as shown in Fig. 8. Visual assessment of the images suggested that, at both doses, mRNA-LNPs formulated with FSP003D resulted in a higher luminescence than mRNA-LNPs lacking the peptide, suggesting a beneficial role of the incorporation of pH-responsive synthetic peptide shuttle agents in LNPs for RNA delivery to the cytosol.

[0391] To evaluate transduction levels and biodistribution, mice were sacrificed 24 h post administration, and the liver, spleen, kidney, pancreas, lungs, and heart were collected and exposed to D-luciferin at 0.3 mg / mL for luminescence imaging and quantification. The results in Fig. 9 show (FLuc)-mRNA was delivered in all the collected organs with both LNP formulations, indicating no change in biodistribution due to the presence of FSP003D in the LNPs. This suggests that the FSP003D peptide is either not exposed at the surface of the LNPs, or that its exposition is too low to impact the normal biodistribution of the LNPs, which is advantageous for FSP003D as a drop-in ingredient in existing LNP formulations. Strikingly, at 1 pg of (FLuc)-mRNA per mouse, the level of luminescence was consistently higher in all six organs collected for LNPs formulation with FSP003D. Similar trends were observed for administration at 5 pg of (FLuc)-mRNA per mouse (data not shown).

[0392] Example 7: Synthetic peptide shuttle agents facilitate endosomal escape of LNP-encapsulated PMOs

[0393] The results shown in Example 5 suggested that synthetic peptide shuttle agents having higher overall relative charges at pH 7 (e.g., FSD449D and FSP008D) formed overly tight polyplexes with anionic mRNA cargo when LNPs were prepared by pipet mixing, thereby interfering with the mRNA’s ability to reach the cytosol and / or undergo translation. To evaluate whether synthetic peptide shuttle agents having higher overall relative charges would improve endosomal escape of non-anionic cargoes, we encapsulated an electrically neutral phosphorodiamidate morpholino oligo (PMO) cargo in LNPs by pipet mixing as described in Example 1, with or without a synthetic peptide shuttle agent. The synthetic peptide shuttle agent selected was one previously demonstrated to exhibit robust cargo transduction activity - i.e., FSD10, first described in WO / 2018 / 068135. Of note, FSD10 is a 33-residue synthetic peptide shuttle agent having a net charge of +10 at pH 7 due to the presence of ten K / R residues. ThePMO cargo is a Splicing Switch Oligonucleotide PMO (eGFP) that directs a splicing correction of an (eGFP)-mRNA in an engineered HeLa cell line (WO / 2022 / 077121), resulting in GFP expression upon successful PMO delivery to the cytosol. The results in Fig. 10 show that the incorporation of FSD10 in the LNP formulation significantly increased PMO endosomal escape and splicing correction, resulting in increased GFP fluorescence. These results point to a role of synthetic peptide shuttle agents (pH-responsive or otherwise) in enhancing the endosomal escape of LNP -encapsulated payloads other than mRNA.

[0394] Example 8: In vitro evaluation of microfluidic mRNA-LNPs incorporating pH-responsive synthetic peptide shuttle agents

[0395] State-of-the-art fabrication of LNPs is now largely performed using microfluidic devices. Thus, we carried out this approach to prepare LNPs incorporating Shuttle peptides. Microfluidic mixing is a technology wherein an organic phase comprising one or more lipids is rapidly mixed with the aqueous phase comprising a nucleic acid cargo (e.g., mRNA). The microfluidic system enables controlled and continuous mixing under laminar flow conditions, facilitating the spontaneous self-assembly of LNPs with uniform size distribution, high encapsulation efficiency, and reproducible physicochemical properties. We reproduced (eGFP)-mRNA-loaded (Shuttle agent)-LNPs using microfluidic mixing and evaluated their efficiency in HeLa cells. Consistent with the pipet mixing procedure, incorporation of FSP003D within LNPs using the Flex S microfluidic device led to an increase in eGFP expression compared to the control LNP (C-LNP) without Shuttle (Fig. 11 A) which were not due to differences in cell viability following treatment, as shown in Fig. 11B. Notably, the FSP008D candidate, previously ineffective when produced via pipet-mixing, exhibited the highest delivery efficiency when using the Flex S, indicating that the mixing procedure can have an impact on Shuttle incorporation on the LNPs, as well as its ability to associate with and deliver the cargo.

[0396] As seen in Fig. 11 A, at a dose of 60 ng of mRNA, LNPs incorporating FSP003D and FSP008D yielded approximately 9-fold and 15-fold increases in eGFP expression, respectively, compared to the control C-LNP. An increase in eGFP expression was also observed with LNPs incorporating FSP003D and FSP008D as compared to those incorporating the original FSD449D synthetic peptide shuttle agent. While increasing the mRNA dose to 120 ng did not show any improvement in protein expression with LNPs incorporating FSP003, LNPs containing FSP008 showed a further increase in protein expression, approximately 25-fold relative to C-LNP.

[0397] Incorporation of FSD449D, as well as the remaining pH responsive FSP004D, FSP006D, FSP007D, FSP005D (Fig. 11 A), showed a moderate increase in protein expression when compared to C-LNP. With FSP008D being the most amphiphilic pH-responsive Shuttle (pH = 7.51), these results suggestthat a high pH may be beneficial and can improve LNP-mediated mRNA delivery. Taken together, these results indicate that incorporation of the original synthetic peptide shuttle agents as well as the pH responsive Shuttle peptides in LNPs promotes higher mRNA delivery in the intracellular space.

[0398] In addition, LNPs incorporating FSP008D and (eGFP)-mRNA-loaded were also prepared with the SM-102 ionizable lipid LNP kit using microfluidic mixing. Their ability to deliver (eGFP)-mRNA was also evaluated in HeLa cells as described above, the results of which are shown in Fig. 12A. As seen in Fig. 12A, similar results were obtained with the SM-102 ionizable lipid LNP kit, wherein the incorporation of the pH-responsive Shuttle FSP008D in the LNP resulted in a significant increase in the delivery and expression efficiency of the mRNA(eGFP) compared to C- LNPs (control LNP without pH responsive Shuttles), which was not due to differences in cell viability between the different treatments (Fig. 12B).

[0399] Example 9: Characterization of LNPs covalently conjugated to pH-responsive synthetic peptide shuttle agents

[0400] In Examples 5-7, synthetic peptide shuttle agents were incorporated into LNPs by physical entrapment. In the present Example and as detailed in Example 1, we explored a different approach: covalently conjugating synthetic peptide shuttle agents to individual LNP components, such as the cholesterol (CLS), and dimyristoylglycerol (DMG), that are commonly used in LNP formulations.

[0401] Conjugation was performed through a PEG linker (PEG, or PEG45) to extend the distance between the lipid and the Shuttle peptide and increase the solubility of the resulting conjugates. Without being bound by theory, it is believed that this strategy may enforce the incorporation of the Shuttle peptide within the lipid layer of the LNP by hydrophobic interactions during particle formation. For these studies, the pH responsive shuttles FSP003D, FSP005D and FSP008D were used for lipid / sterol conjugation. In addition, the original synthetic peptide shuttle agents, FSD375 and FSD10, were also conjugated to cholesterol. The resulting conjugates were incorporated in LNPs by replacing a percentage of the cholesterol used for LNP, and the LNPs were prepared using a microfluidic device, as detailed in Examples 1 and 8. Particle properties of the LNPs were assessed by dynamic light scattering. The results are shown in Table 5 and demonstrate that incorporation of the conjugates in LNPs resulted in particles with hydrodynamic diameters that ranged within those generally considered as ideal for LNPs (e.g., 20-200 nm), except for FSPOO8D-PEG19-CLS, which led to premature precipitation and resulted in particles with a larger hydrodynamic diameters (248.9 nm). Strikingly, incorporation of the original synthetic peptide shuttle agents conjugates (FSD10-mal-PEG19-CLS and FSD375-mal-PEG19-CLS) within the LNPs led to particles with similar properties as the C-LNP (without the pH-responsive or non-responsive peptide shuttles), whereas incorporation of the pH-responsive shuttle peptide conjugates led to generally largerparticles, some of which (e.g., those made with FSP008D-mal-PEG45-DMG and FSP008D-mal-PEGi9-CLS) had somewhat lower encapsulation efficiencies.

[0402] Table 5: Physical characterization LNPs eGFP (mRNA) by dynamic light scattering Lipid Conjugate Size (nm) PDI Encap. Eff.

[0403] C-LNP 85.4 0.09 95% FSD10-mal-PEG19-CLS 116.2 0.12 93% FSD375-mal-PEG19-CLS 80.8 0.06 97% MC3 FSP008D-mal-PEG45-DMG 185.4 0.09 74%

[0404] FSP008D-mal-PEG19-CLS 248.9 0.15 78% FSP003-mal-PEG19-CLS 175.5 0.27 100%

[0405]

[0406] FSP005-mal-PEG19-CLS 155.1 0.46 99%

[0407] Following particle characterization, formulations were assessed for mRNA delivery in HeLa cells. Overall, incorporation of synthetic peptide shuttle agents (original or pH-responsive) and their conjugates in LNPs leads to particles that enable mRNA delivery with an efficiency that is similar to or above the delivery efficiency of the C-LNP. As seen in Fig. 13A, conjugation of FSP008D with a lipid or cholesterol did not improve mRNA delivery compared to FSP008D alone. On the contrary, LNPs formulated with FSPOO8D-PEG45-DMG resulted in a slightly lower mRNA delivery compared to FSP008D alone, and LNPs formulated with FSPOO8D-PEG19-CLS showed even lower mRNA delivery than C-LNPs when 60 ng of mRNA was loaded onto the LNP, which was likely due to the precipitation of the particles, as detailed above. Without being bound by theory, it is believed that with the high hydrophobicity (average hydrophilicity = -0.34) and pH (7.51) of FSP008D, conjugation to hydrophobic molecules such as DMG and CLS may decrease its water solubility and can potentially lead to precipitation, thus reducing its incorporation in the lipid layer of the LNPs.

[0408] On the other hand, the LNPs formulated with the original synthetic peptide shuttle agent FSD10, were less efficient compared to LNPs containing FSP008D alone (Fig. 13A) but more efficient than C-LNPs. FSD10, like FSP008D, has a high pH (7.02), but is hydrophilic (average hydrophilicity = 0.34). Without being bound by theory, it is believed that the hydrophilicity of FSD 10 may prevent the peptide from entering the lipid layer of the LNPs, which can explain the low impact on the delivery efficiency of these particles. However, conjugation to cholesterol, seemed to promote its insertion in the lipid layer of LNPs, which resulted in a delivery efficiency that is similar to LNPs incorporating FSP008D.

[0409] Similar results were observed with the synthetic peptide shuttle agent FSD375, having a pH of 4.4, which is lower than the pH of FSD 10, and is also hydrophilic (average hydrophilicity = 0.20). LNPs incorporating both FSD375D and FSD375-PEG19-CLS demonstrated an increase in mRNA delivery compared to C-LNP. However, as seen in Fig. 13A, conjugation to cholesterol was most beneficial when120 ng of mRNA was loaded into the LNP. Nonetheless, the delivery efficiency of these particles was lower compared to LNPs containing FSP008D or FSD10-PEG19-CLS.

[0410] Overall, these results demonstrate that conjugation of the original synthetic peptide shuttle agents to lipids or sterols can further improve LNP efficiency compared to unconjugated Shuttle.

[0411] LNPs generated with FSP003-mal-PEG19-CLS and FSP005-mal-PEG19-CLS were also tested for their delivery efficiency of (eGFP)-mRNA in HeLa cells. As seen in Fig. 13B, an increase in eGFP expression compared to C-LNPs, was observed with LNPs generated with non-pH responsive shuttle agents and with pH-responsive shuttle agents conjugated to cholesterol, suggesting that ensuring shuttle insertion within LNPs through cholesterol conjugation is favorable to improve the delivery efficiency of LNPs. Of note, none of the differences between the efficiency of mRNA delivery seen in Fig. 13A and 13B were associated with differences in cell cytotoxicity (data not shown).

[0412] Example 10: In vivo evaluation of mRNA-LNPs incorporating synthetic peptide shuttle agents conjugated to cholesterol

[0413] The performance of LNPs incorporating FSD10-Cholesterol or FSP003D-Cholesterol was further evaluated in mice. In this study, 0.5 pg of LNPs containing 10 pM FSD10-CLS or FSP003D-CLS, along with a control LNP formulation, were administered systemically via the tail vein. Four hours post injection, animals were injected with D-luciferin and imaged for luminescence with a IVIS Lumina XR imager. Corresponding images for each condition are represented in Fig. 14A.

[0414] Visual evaluation of the whole-body bioluminescence intensity, together with quantitative analysis of total radiance (Fig. 14B), revealed higher luminescence when mice were treated with LNPs incorporating FSD10-Cholesterol or FSP003D-Cholesterol compared to control LNPs. These Shuttle-modified LNPs exhibited bioluminescence levels up to five times higher than the control LNP.

[0415] The liver, spleen, pancreas, kidneys and lungs were further collected from these mice and incubated with D-luciferin for ex vivo bioluminescence imaging and quantification. As seen in Fig. ISA-ISE, all LNPs showed similar biodistribution, indicating that incorporation of FSD10-CLS or FSP003D-CLS had minimal impact on LNP distribution in mice. However, compared to mice treated with control LNPs, systemic administration of shuttle-modified LNPs showed a higher total radiance in most organs, with a higher luminescence observed in the liver (18-fold increase) and spleen (20-fold increase) (Fig. 15A and 15C).

[0416] Example 11: Conjugating the pH-responsive shuttle peptides to a peptide or an ASO cargo Having established that pH-responsive shuttles exhibit acid-dependent endosomal activation (e.g., Fig.3), we next examined their activity when covalently attached to cargo molecules. While co-incubationstrategies are suitable for in vitro peptide characterization, systemic in vivo applications may benefit from stable peptide-cargo constructs to ensure the presence of both entities in biodistributed tissues. We, therefore, determined whether pH-dependent activation, endosomal escape, and functional intracellular delivery are preserved when the pH responsive shuttles are conjugated to a cargo. To address this, we selected FSP005D and FSP008D as representative pH-responsive shuttles and conjugated them to either the nls* peptide and / or the splice-switching PMO(EGFP) as provided in Example 1. The delivery efficiency of the resulting conjugates was evaluated in vitro.

[0417] The proposed activation mechanism of pH-responsive-cargo conjugates is illustrated schematically in Fig. 16A and involves peptide activation driven by endosomal acidification following cellular internalization, combined with intracellular disulfide bond cleavage leading to cargo release. To assess whetherthis activation occurs in vitro, the intracellular behavior of FSP005D-nls* conjugate was examined by fluorescence microscopy over increasing incubation times (5 min, 30 min, 1 h, and 2 h). The cationic parent FSD449D-nls* conjugate was included as a pH-independent positive control to enable direct comparison of intracellular trafficking and delivery kinetics (Fig. 16B).

[0418] As seen in Fig. 16B, treatment with the parent FSD449D-nls* conjugate resulted in rapid membrane-destabilizing activity, resulting in efficient intracellular nls* delivery, with nuclear accumulation observed within 5 min of incubation. In contrast, the FSP005D-nls* conjugate exhibited no detectable delivery at the 5 -min time point, consistent with its inactive state at neutral pH. After 30 min of incubation, endosomal entrapment of the nls* was observed. Nuclear localization of the nls* cargo, confirmed by colocalization with the Hoechst-labeled nucleus, became evident only after 1 h of incubation and was further enhanced after 2 h. This time -dependent intracellular delivery suggests that FSP005D becomes active upon endosomal acidification, enabling cytoplasmic delivery and subsequent nuclear accumulation of the nls* cargo.

[0419] To determine whether the pH-responsive activation profile of such conjugates is maintained when the pH-responsive peptide shuttle is conjugated to other cargoes, such as biologically antisense oligonucleotide cargos, the delivery efficiency of FSP005D and FSP008D conjugated to a PMO(EGFP) were evaluated in HeLa EGFP-654 reporter cells, in which restoration of EGFP expression provides a functional readout of nuclear delivery and splice correction. As a reference for pH-independent delivery, the parent FSD449D shuttle peptide conjugated with PMO (FSD449D-PMO) was included as a positive control. Flow cytometry analysis showed that a short 5-min incubation with the parent FSD449D-PMO resulted in rapid and efficient splice correction, with approximately 60% of cells restoring EGFP fluorescence, consistent with an efficient cargo delivery under physiological conditions (Fig. 16C and 16D). In contrast, the conjugate with the pH-responsive shuttle, FSP005D-PMO, displayed minimal activity at the 5 min time point, with fewer than 20% EGFP+cells, consistent with low residual activity atneutral pH (Fig. 16C and 16D). Extending the incubation time to 2 h led to a significant increase in FSP005D-PMO-mediated splice correction, with EGFP restoration approaching -80% of cells. Under the same conditions, PMO alone produced a negligible EGFP signal, confirming that efficient splice correction requires cytoplasmic delivery (Fig. 16C).

[0420] The FSP008D conjugate had a different delivery profile than the FSP005D conjugate. As seen in Fig. 16C, this conjugate had a high residual activity after 5 minutes incubation, which can be explained by its cationicity (+4) that is close to the fully cationic parent FSD449D peptide conjugate (FSD449D-PMO). Nevertheless, this conjugate showed an increase in cargo delivery after 2h of incubation, suggesting its activation over time in endosomes. Fluorescence microscopy (Fig. 16D) also corroborates these findings. Together, these results demonstrate that the activation profile of the pH-responsive peptides depend on their initial charge and sequence and is fully retained upon covalent conjugation to a functional antisense cargo, enabling efficient intracellular delivery upon peptide activation.

[0421] Example 12: the PMO-based pH-responsive shuttle conjugates enable safe splice correction in vivo In previous studies, Feldan explored the potential of FSD10 shuttle conjugated to a cargo for biological applications (Auger et al., 2024). Both in vitro and in vivo studies for lung delivery demonstrated that linking the shuttle to the cargo through a cleavable or non-cleavable bond preserved shuttle activity and enabled efficient delivery of the fluorescently labeled DRI-NLS647peptide. Notably, cleavable conjugates showed superior cytosolic delivery in vitro. This conjugation strategy also proved highly promising for systemic administration, as it maintains proximity between the two entities, thereby minimizing the dilution effects that may occur when the non-conjugated components are administered. Systemic administration of highly cationic shuttle peptides, however, often led to cellular cytotoxicity (Cardozo AK, et al., 2007; Kawaguchi, Yoshimasa et al., 2024). In particular, highly cationic molecules have been shown to increase nonspecific membrane perturbation and induce cellular swelling associated with disrupted ionic homeostasis, resulting in dose-dependent toxicity in vivo (Vedadghavami et al., 2020; Frohlich, 2012). In this context, our pH-responsive conjugates, which display reduced cationic character and have demonstrated good viability profiles at higher concentrations and longer incubation times, represented promising alternatives for systemic administration. To test this, FSP008D-PMO and FSP005D-PMO conjugates, were administered systemically through the tail vein at 40 mg / kg in eGFP-CAG645 mice, and eGFP fluorescence was imaged 72 hours post injection.

[0422] Microscopy analysis showed that a single dose of 40 mg / kg was sufficient to achieve efficient delivery of the PMO to multiple organs, including kidney, liver and muscles (Fig. 17A). As shown in Fig.

[0423] 17A, different biodistribution profiles were observed between the FSP005D-PMO and the highly hydrophobic FSP008D-PMO conjugates. For the latter, a low number of eGFP+cells were observed in thekidney. However, the systemic administration of this FSP008D-PMO conjugate resulted in an efficient splicing correction in the liver, as evidenced by the presence of numerous eGFP+ hepatocytes. In contrast, the FSP005D-PMO predominantly accumulated in the kidney and to a lesser extent in the liver (Fig.

[0424] 17A), and in comparison, with the FSP008D-PMO, seemed to favor delivery to skeletal muscles.

[0425] FSP008D-PMO is more cationic than FSP005D-PMO at physiological pH. Without being bound by theory, it is believed that FSP005D-PMO accumulates mostly in the kidney because of clearance due to its low cationicity, suggesting that biodistribution of the pH responsive peptide shuttle conjugates may be sequence dependent.

[0426] We also observed that, administration of FSP003D-PMO conjugate also required higher doses of systemic administration to achieve a delivery comparable to FSP005D and FSP008D conjugates (Fig. 17B) indicating that delivery efficiency of the conjugates may also be dependent on the peptide sequence. Additionally, Fig. 17B also demonstrated that these conjugates could be administered at doses up to 80 mg / kg in mice which further outlined the safety of the pH-responsive conjugates. In contrast, peptide-PMO (PPMO) which are CPP-PPMO conjugates have been reported to induce a dose limiting toxicity at higher concentrations, including an acute renal toxicity in mice when injected at comparable or lower doses. These toxic effects have been largely attributed to the high permanent cationicity of the CPP and their nonspecific interactions with cellular membranes and serum proteins. For instance, conventional PPMO, including RXR and Pip-based PMO conjugates have been reported to induce renal or systemic toxicity at doses above 10-15 mg / kg. Thus, the absence of overt toxicity observed with the FSP003D-PMO conjugate at doses up to 80 mg / kg, and with 40mg / kg of the FSP008D- and FSP005D-PMO conjugates, highlights the improved safety profile of these pH-responsive peptides, which likely results from their lower initial cationicity at physiological pH.

[0427] Taken together, these findings indicate that the biodistribution of pH-responsive conjugates is strongly influenced by their initial charge and amino acid sequence and underscore their potential for the safe systemic delivery of ASOs.

[0428] Example 13: The PMO-based pH-responsive conjugates conjugated to ligands for tissue / cellular targeting

[0429] To evaluate whether the pH-responsive conjugates could be redirected toward a specific organ, a N-acetylgalactosamine (GalNAc) ligand was conjugated to FSP004D-PMO and FSP005D-PMO. GalNAc is a well-established hepatocyte -targeting ligand that binds with high affinity to the asialoglycoprotein receptor (ASGPR), which is highly expressed on hepatocytes.

[0430] FSP004D-PMO-GalNac and FSP005D-PMO-GalNac conjugates, at 20 mg / kg, were systemically administered through the tail vein of eGFP-CAG645 mice, and eGFP fluorescence was evaluated 72hours post injection. As seen in Fig. 18, conjugation of the GalNAc ligand into the FSP004D-PMO and FSP005D-PMO conjugates markedly directed biodistribution of the conjugates to the liver compared to the FSP004D-PMO and FSP005D-PMO alone. Compared to the pH-responsive-PMO conjugates alone, the GalNAc -modified conjugates showed a drastic reduction in the number of eGFP -positive cells in the kidney, resulting in a substantial increase in eGFP -positive hepatocytes in the liver. In these experiments the PMO conjugated to GalNac at a dose of 30 mg / kg was administered through the tail vein and used as a position control. These results demonstrate that the delivery ability of the pH-responsive shuttle platform can be rationally modulated through ligand conjugation, supporting its potential for targeted delivery of biotherapeutics.

[0431] REFERENCES

[0432] Abd Elwakil et al., “Lung-Endothelium-Targeted Nanoparticles Based on a pH-Sensitive Lipid and the GALA Peptide Enable Robust Gene Silencing and the Regression of Metastatic Lung Cancer.” Adv Funct Mater 29, 1807677. https: / / doi.org / 10.1002 / ADFM.201807677.

[0433] Aschmann et al. “Lipid-Based Nanoparticle Functionalization with Coiled-Coil Peptides for In Vitro and In Vivo Drug Delivery.” Accounts of chemical research vol. 57,8 (2024): 1098-1110. doi: 10.1021 / acs.accounts.3c00769

[0434] Auger et al., “Enhancing peptide and PMO delivery to mouse airway epithelia by chemical conjugation with the amphiphilic peptide S10.” Molecular therapy. Nucleic acids vol. 35,3 102290. 31 Jul.

[0435] 2024, doi: 10.1016 / j.omtn.2024.102290

[0436] Cardozo et al., “Cell-permeable peptides induce dose- and length-dependent cytotoxic effects.” Biochimica et Biophysica Acta (BBA) - Biomembranes. 2007 Sep;1768(9):2222-2234. doi: 10.1016 / j.bbamem.2007.06.003.

[0437] Chatterjee et al. “Endosomal escape: A bottleneck for LNP-mediated therapeutics.” Proceedings of the National Academy of Sciences of the United States of America vol. 121,11 (2024): e2307800120. doi: 10.1073 / pnas.2307800120

[0438] DelGuidice et al., “Membrane permeabilizing amphiphilic peptide delivers recombinant transcription factor and CRISPR-Cas9 / Cpfl ribonucleoproteins in hard-to-modify cells.” PloS one vol. 13,4 e0195558. 4 Apr. 2018, doi: 10.1371 / journal.pone.0195558

[0439] Frohlich E., “The role of surface charge in cellular uptake and cytotoxicity of medical nanoparticles. Int J Nanomedicine. 2012;7:5577-91. doi: 10.2147 / IJN. S36111.

[0440] Gilleron et al. “Image-based analysis of lipid nanoparticle-mediated siRNA delivery, intracellular trafficking and endosomal escape.” Nature biotechnology vol. 31,7 (2013): 638-46.

[0441] doi:10.1038 / nbt.2612Kawaguchi, Yoshimasa et al. “E3MPH16: An efficient endosomolytic peptide for intracellular protein delivery.” Journal of controlled release: official journal of the Controlled Release Society vol.

[0442] 367 (2024): 877-891. doi: 10.1016 / j.jconrel.2024.01.067

[0443] Khalil et al., “Octaarginine- and pH sensitive fusogenic peptide-modified nanoparticles for liver gene delivery.” Journal of controlled release: official journal of the Controlled Release Society vol.

[0444] 156,3 (2011): 374-80. doi:10.1016 / j.jconrel.2011.08.012

[0445] Krishnamurthy et al., “Engineered amphiphilic peptides enable delivery of proteins and CRISPR- associated nucleases to airway epithelia.” Nature communications vol. 10,1 4906. 28 Oct. 2019, doi: 10.1038 / s41467-019- 12922-yKulhankova et al., 2023

[0446] Kulhankova et al., “Amphiphilic shuttle peptide delivers base editor ribonucleoprotein to correct the CFTR R553X mutation in well-differentiated airway epithelial cells.” Nucleic acids research vol.

[0447] 52,19 (2024): 11911-11925. doi:10.1093 / nar / gkae819

[0448] Luo et al., “Genome Editing in Ferret Airway Epithelia Mediated by CRISPR / Nucleases Delivered with Amphiphilic Shuttle Peptides.” Human gene therapy vol. 34,15-16 (2023): 705-718. doi:10.1089 / hum.2023.016

[0449] Mac Donald et al., " KLR 1 knockout overcomes HLA-E-mediated inhibition and improves NK cell antitumor activity against solid tumors.” Frontiers in immunology vol. 14 1231916. 21 Aug. 2023, doi: 10.3389 / fimmu.2023.1231916

[0450] Maugeri et al., “Linkage between endosomal escape of LNP-mRNA and loading into EVs for transport to other cells.” Nature communications vol. 10,1 4333. 24 Sep. 2019, doi:10.1038 / s41467-019- 12275-6

[0451] Mol et al., “NetWheels: A Web Application to Create High Quality Peptide Helical Wheel and Net Projections.” Journal of Bioinformatics and Systems Biology. 7 (2024): 98-100.

[0452] Muller et al., “Kinetics of RNA-LNP delivery and protein expression.” European journal of pharmaceutics and biopharmaceutics: official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e. Evol. 197 (2024): 114222.

[0453] doi: 10.1016 / j.ejpb.2024.114222

[0454] Paramasivam et al. “Endosomal escape of delivered mRNA from endosomal recycling tubules visualized at the nanoscale.” The Journal of cell biology vol. 221,2 (2022): e202110137.

[0455] doi: 10.1083 / jcb.202110137

[0456] Sabnis et al., “A Novel Amino Lipid Series for mRNA Delivery: Improved Endosomal Escape and Sustained Pharmacology and Safety in Non-human Primates.” Molecular therapy: the journal of the American Society of Gene Therapy vol. 26,6 (2018): 1509-1519.

[0457] doi:10.1016 / j.ymthe.2018.03.010Tateshita et al., “Development of a lipoplex-type mRNA carrier composed of an ionizable lipid with a vitamin E scaffold and the KALA peptide for use as an ex vivo dendritic cell-based cancer vaccine.” Journal of controlled release: official journal of the Controlled Release Society vol.

[0458] 310 (2019): 36-46. doi:10.1016 / j.jconrel.2019.08.002

[0459] Vedadghavami et al., “Overcoming negatively charged tissue barriers: Drug delivery using cationic peptides and proteins”. Nano Today. 2020 Oct;34: 100898. doi: 10.1016 / j.nantod.2020.100898. Wittrup et al. “Visualizing lipid-formulated siRNA release from endosomes and target gene knockdown.” Nature biotechnology vol. 33,8 (2015): 870-6. doi:10.1038 / nbt.3298

[0460] Zeng et al. “Efficient mRNA delivery using lipid nanoparticles modified with fusogenic coiled-coil peptides.” Nanoscale vol. 15,37 15206-15218. 29 Sep. 2023, doi: 10.1039 / d3nr02175k Zhang et al., “Together is Better: mRNA Co-Encapsulation in Lipoplexes is Required to Obtain Ratiometric Co-Delivery and Protein Expression on the Single Cell Level.” Advanced science (Weinheim, Baden-Wurttemberg, Germany) vol. 9,4 (2022): e2102072.

[0461] doi: 10.1002 / advs.202102072

[0462] WO / 2016 / 161516

[0463] WO / 2017 / 175072

[0464] WO / 2018 / 068135

[0465] WO / 2020 / 210916

[0466] WO / 2022 / 077121

[0467] WO / 2022 / 082315

[0468] WO / 2022 / 204806

Claims

CLAIMS1. A composition comprising a cargo for intracellular delivery and a pH-responsive synthetic peptide shuttle agent, the pH-responsive synthetic peptide shuttle agent comprising an amphipathic alphahelical motif when in acidic pH, the amphipathic alpha-helical motif having a solvent-exposed surface comprising a discrete hydrophilic cationic face and a discrete hydrophobic face, wherein the discrete hydrophilic cationic face comprises at least one positively-charged residue (e.g., K or R) and at least one histidine residue that becomes protonated at acidic pH, resulting in the pH-responsive synthetic peptide shuttle agent having increased cargo transduction activity at acidic pH than at neutral pH.

2. The composition of claim 1, wherein the discrete hydrophilic cationic face:(a) comprises at least two, three, or four adjacent K, R, and / or H residues upon helical wheel projection, based on an alpha helix having angle of rotation between consecutive amino acids of 100 degrees and / or an alpha-helix having 3.6 residues per turn;(b) comprises a segment of six adjacent residues comprising three to five K, R and / or H residues upon helical wheel projection, based on an alpha helix having angle of rotation between consecutive amino acids of 100 degrees and / or an alpha-helix having 3.6 residues per turn; (c) comprises a total number of positively-charged residues (e.g., K and R residues) that is greater than the total number of positively-charged residues comprised in the discrete hydrophobic face;(d) lacks negatively-charged amino acids (e.g., D or E) or contains no more than one or two negatively- charged amino acids; or(e) any combination of (a) to (d).

3. The composition of claim 1 or 2, wherein discrete hydrophobic face:(a) comprises at least two or three adjacent hydrophobic residues selected from L, 1, F, V, W, M, or any combination thereof, upon helical wheel projection, based on an alpha helix having angle of rotation between consecutive amino acids of 100 degrees and / or an alphahelix having 3.6 residues per turn;(b) comprises a segment of eight, nine, or ten adjacent residues comprising at least five hydrophobic residues selected from: L, 1, F, V, W, and M, upon helical wheel projection, based on an alpha helix having angle of rotation between consecutive amino acids of 100 degrees and / or an alpha-helix having 3.6 residues per turn;(c) comprises a total number of positively-charged residues (e.g., K and R residues) that is less than the total number of positively-charged residues comprised in the discrete hydrophilic cationic face; or(d) any combination of (a) to (c).

4. The composition of any one of claims 1 to 3, wherein the amphipathic alpha-helical motif:(a) has a minimum length of 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 amino acids;(b) has a maximum length of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids;(c) comprises the amino acid sequence of any one of SEQ ID NOs: 15-20, 707-713, and 716- 717;(d) comprises the amino acid sequence of any one of SEQ ID NOs: 11, 12, or 21-31, wherein the at least one, two, or three positively-charged residues (e.g., K or R) comprised in the discrete cationic hydrophilic face is replaced with histidine;(e) has a hydrophobic moment (p) between a lower limit of 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, and an upper limit of 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, or 11.0;(f) has a net charge of at least -2 at neutral pH (e.g., pH 7) and at least +3 at acidic pH (e.g., pH 5); or(g) any combination of (a) to (f).

5. The composition of any one of claims 1 to 4, wherein the pH-responsive synthetic peptide shuttle agent:(a) further comprises a flexible linker domain N -terminal or C-terminal of the amphipathic alpha-helical motif, or comprises flexible linker domains flanking (e.g., N- and / or C- terminally) the amphipathic alpha-helical motif;(b) has a maximum length of 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 60, 65, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids;(c) has an isoelectric point (pl) of 6 to 13;(d) has a net charge of at least -2 at neutral pH (e.g., pH 7) and at least +3 at acidic pH (e.g., pH 5);(e) has a hydrophobic moment (p) between a lower limit of 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3,6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, and an upper limit of 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, or 11.0;(f) lacks a cell-penetrating domain and / or a histidine -rich domain;(g) comprises at least one cysteine residue positioned at the N- and / or C-terminal portion, or at the N or C terminus, of the pH-responsive synthetic peptide shuttle agent;(h) is comprised in the composition at a concentration that increases the cytosolic delivery of the cargo as compared to a corresponding composition lacking the pH-responsive synthetic peptide shuttle agent;(i) is covalently conjugated to a lipid (e.g., cholesterol, 1,2-dimyristoyl glycerol, a PEGylated lipid, a phospholipid, an ionizable lipid) and / or to non-polyanionic hydrophilic polymer; or (j) any combination of (a) to (i).

6. The composition of claim 5, wherein the pH-responsive synthetic peptide shuttle agent comprises a flexible linker domain that:(a) comprises uncharged hydrophilic residues that: adopt a random coil conformation; increase the stability of the amphipathic alpha-helical motif; increase the solubility of the pH- responsive synthetic peptide shuttle agent; or any combination thereof;(b) is comprised of uncharged hydrophilic residues (e.g., selected from glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine);(c) is a gly cine / serine -rich flexible linker domain; or(d) any combination of (a) to (c).

7. The composition of any one of claims 1 to 6, wherein the pH-responsive synthetic peptide shuttle agent and the cargo:(a) are not covalently bound to one another;(b) are covalently bound to one another in a cleavable or noncleavable manner;(c) do not physically bind to one another; or(d) bind to one another sufficiently weakly to not inhibit cytosolic delivery of the cargo.

8. The composition of any one of claims 1 to 7, wherein the cargo:(a) is a polynucleotide cargo (e.g., DNA and / or RNA cargo);(b) an antisense oligonucleotide (ASO) cargo;(c) a protein or peptide cargo;(d) a small molecule cargo;(e) a ribonucleoprotein cargo; or(f) any combination thereof (a) to (e).

9. The composition of any one of claims 1 to 8, wherein the pH-responsive synthetic peptide shuttle agent and the cargo are bound to one another and are further associated with (e.g. covalently conjugated to) a targeting ligand (e.g. N-acetylgalactosamine (GalNac)).

10. A composition comprising the pH-responsive synthetic peptide shuttle agent as defined in any one of claims 1 to 8 and the cargo for intracellular delivery as defined in claim 7 or 8 dissolved in an acidic buffer or solvent.

11. The composition of claim 10, wherein the acidic buffer or solvent has a pH of less than 7.0, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, 6.0, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1, 5.0, 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, or 3.0.

12. A lipid nanoparticle comprising a synthetic peptide shuttle agent and a cargo for cytosolic delivery.

13. The lipid nanoparticle of claim 12, wherein the synthetic peptide shuttle agent is covalently conjugated to a lipid (e.g., cholesterol, 1,2-dimyristoyl glycerol, a PEGylated lipid, a phospholipid, an ionizable lipid) and / or to non-polyanionic hydrophilic polymer.

14. The lipid nanoparticle of claim 12 or 13, wherein the cargo is as defined in claim 8.

15. The lipid nanoparticle of claim 12 or 13, wherein the synthetic peptide shuttle agent is the pH-responsive synthetic peptide shuttle agent as defined in any one of claims 1 to 7, and the cargo is as defined in claim 7 or 8.

16. A pH-responsive synthetic peptide shuttle agent as defined in any one of claims 1 to 9.

17. A synthetic peptide shuttle agent as defined in claim 13.

18. The composition as defined in any one of claims 1 to 11 for use:(a) in the manufacture of a lipid nanoparticle (e.g., the lipid nanoparticle as defined in any one of claims 12 to 15), a polymeric nanoparticle, liposomes, or other intracellular delivery vehicle);(b) in therapy, wherein the cargo is a therapeutic agent;(c) as a diagnostic agent, wherein the cargo is a detectable marker (e.g., radioactive marker, fluorescent marker, bio luminescent marker, a contrast agent, a photoactivatable marker); or (d) in biomanufacturing of a cell transfection agent.

19. The pH-responsive synthetic peptide shuttle agent of claim 16 for use:(a) in increasing the intracellular delivery of the cargo as compared to in the absence of the pH- responsive synthetic peptide shuttle agent;(b) in the manufacture of a lipid nanoparticle comprising a cargo for intracellular delivery (e.g., a cargo as defined in claim 7 or 8); or(c) in the manufacture of composition as defined in any one of claims 1 to 11.

20. The synthetic peptide shuttle agent as defined in claim 17 for use in the manufacture of a lipid nanoparticle comprising a cargo for intracellular delivery (e.g. a cargo as defined in claim 8).

21. The lipid nanoparticle as defined in any one of claims 12 to 15 for use:(a) in therapy, wherein the cargo is a therapeutic agent; or(b) in the manufacture of a medicament for treating a disease or disorder ameliorated by intracellular delivery of the cargo;(c) as a diagnostic agent, wherein the cargo is a detectable marker (e.g., radioactive marker, fluorescent marker, bio luminescent marker, a contrast agent, a photoactivatable marker); or (d) in the manufacture of a diagnostic agent, wherein the cargo is a detectable marker (e.g., radioactive marker, fluorescent marker, bioluminescent marker, a contrast agent, a photoactivatable marker); or(e) in biomanufacturing of a cell transfection agent.

22. A method for synthesizing a pH-responsive synthetic peptide shuttle agent, the method comprising: (a) designing a candidate pH-responsive synthetic peptide shuttle agent as defined in any one of claims 1 to 8; and (b) chemically synthetizing the candidate, wherein the candidate pH-responsive synthetic peptide shuttle agent is a pH-responsive synthetic peptide shuttle agent when the candidate pH-responsive synthetic peptide shuttle agent has increased cargo transduction activity at acidic pH than at neutral pH.

Images