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WO2025207027A1 - Compositions de coacervat - Google Patents

Compositions de coacervat

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Publication number
WO2025207027A1
WO2025207027A1 PCT/SG2025/050203 SG2025050203W WO2025207027A1 WO 2025207027 A1 WO2025207027 A1 WO 2025207027A1 SG 2025050203 W SG2025050203 W SG 2025050203W WO 2025207027 A1 WO2025207027 A1 WO 2025207027A1
Authority
WO
WIPO (PCT)
Prior art keywords
peptide
coacervate
coacervates
peptides
composition
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/SG2025/050203
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English (en)
Inventor
Yue Sun
Ali Gilles Tchenguise MISEREZ
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nanyang Technological University
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Nanyang Technological University
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Filing date
Publication date
Application filed by Nanyang Technological University filed Critical Nanyang Technological University
Publication of WO2025207027A1 publication Critical patent/WO2025207027A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein

Definitions

  • the present invention lies in the field of targeted delivery of active agents using peptide coacervates including isolated peptides, methods of peptide coacervate formation, and active agent recruitment and delivery using the peptide coacervates.
  • Such coacervates may be used in diagnostic or therapeutic approaches.
  • Coacervate compositions are able to cross the cell membrane, are not trapped inside endosomal vesicles, and so can directly deliver the biomacromolecule into the cell.
  • Coacervates can thus be employed to deliver biomacromolecules (e.g. active agents or payloads) into cells.
  • the Humboldt squid (Dosidicus gigas) beak includes a hard biomolecular composite made of chitin and proteins.
  • the squid beak proteins were recently isolated and sequenced and two families of proteins, Dosidicus gigas chitin binding beak proteins (DgCBPs) and Dosidicus gigas histidine-rich beak proteins (DgHBPs) were discovered within the beak.
  • DgCBPs Dosidicus gigas chitin binding beak proteins
  • DgHBPs Dosidicus gigas histidine-rich beak proteins
  • DgCBPs likely bind to chitin to form a chitin-DgCBPs scaffold, while DgHBPs exhibit self-coacervation ability, a liquidliquid phase separation (LLPS) process resulting in the formation of highly concentrated protein microdroplets.
  • DgHBP coacervates have been hypothesized to infiltrate the chitin-DgCBPs scaffold (Tan et al. (2015) Nat. Chem. Biol. 11 (7), 488-95) followed by interchain covalent crosslinking during maturation, with the very high cross-link density imparting the beak with its impressive mechanical properties (Mirobo et al. (2007) Acta Biomater. 3 (1), 139-49; Mirobo et al. (2010) J.
  • the DgHBPs identified have been sequenced and have been found to exhibit a two-domain organisation.
  • the N-terminal domains contain non- repetitive, long stretches of Alanine (Ala) and Histidine (His)-rich regions, whereas the C-terminal domains includes tandem His- and Gly-rich penta-repeat motifs.
  • the C-terminal domain motifs were found to be responsible for DgHBPs self-coacervation properties (Cai et al. (2017) Soft Matter 13 (42), 7740-7752). Coacervates can be generated from these histidine-rich beak protein peptides (HBpep).
  • HBpep coacervates have the ability to recruit various biomacromolecules with high efficiency of above 95%, and exhibit low toxicity (Lim, Z.W. et al., Bioconjugate Chem., 2018, 29, 2176). HBpep coacervates were also demonstrated to be able to cross the cell membrane via an endocytosis-free pathway (Lim, Z.W. et al., Acta Biomat, 2020, 110, 221). It has therefore been suggested that self- coacervating HBpeps may be potential candidates for intracellular delivery of therapeutics. Preliminary attempts to use HBpep coacervates to recruit and deliver proteins resulted in successful transmembrane delivery. For example, the inventors observed that HBpep coacervates successfully recruited biomacromolecules such as insulin and doxorubicin, and delivered said coacervates intracellularly (US 2019/0388357).
  • biomacromolecules such as insulin and doxorubicin
  • WO2021246961 discloses peptides derived from DgHB protein sequence capable of forming coacervates and recruiting therapeutic macromolecules within the peptide coacervates at pH of about 6.5. Such peptides are based on the tandem repeat GHGXY (SEQ ID NO: 25, where X could be valine (V), proline (P), or leucine (L) [HBpepVPL]). However, such peptide forming coacervates are unsuitable for recruiting macromolecules that can only be solubilised at higher alkali pH.
  • the inventors have found that the previously existing drawbacks of delivery platforms based on HBpepVPL coacervates could be overcome by using modified peptides, as described herein, for coacervate formation.
  • the present invention is based on the inventors' finding that peptide coacervates formed from the (modified) isolated peptides described herein can be used for the recruitment of therapeutic macromolecules that are soluble at pH above 6.5, e g. in the pH range of about 7.0- 9.0.
  • the isolated peptide coacervates formed may co-recruit one, two or more active agents to be applicable and effective in the management and/or treatment of diseases or disorders, such as cancer, or for diagnostic applications.
  • the inventors’ findings provide general guidelines and concepts for designing isolated peptide coacervates with LLPS ability for direct cytosolic release of the active agents which may be applicable in various applications, including bio-inspired protocells and smart drug-delivery systems.
  • the present invention provides, inter alia, coacervate forming peptides, methods of preparing coacervate compositions, that may comprise one or more biomacromolecules (payloads), the coacervate compositions per se, and their uses, e g. in methods of treatment or diagnosis.
  • coacervate compositions can be used to deliver active agents into the cell and release it directly in the cytosol.
  • payloads/active agents may be useful in various applications, including bioinspired protocells and smart drug-delivery systems.
  • an isolated peptide comprising the amino acid sequence of Formula (I):
  • Z is tryptophan (W) or is absent
  • O is GHGX 1 Y (SEQ ID NO: 1)
  • not all of the X 1 residues are proline (P).
  • the lysine residue is located at the 1 st, 6th, 11th, 16th, 21 st, or 26 th position from the N-terminus.
  • B is GHGPY (SEQ ID NO: 2)
  • G is glycine (G)
  • H is histidine (H)
  • Y is tyrosine (Y)
  • P is proline (P);
  • each X 1 is independently selected from alanine (A), glycine (G), serine (S), asparagine (N), histidine (H), arginine (R), aspartic acid (D) and tyrosine (Y);
  • K is lysine (K) optionally modified with a self-immolative moiety
  • the isolated peptide comprises or consists of an amino acid sequence, such as but not limited to:
  • lysine residue (K) is modified at an epsilon (s)-amino group with a self- immolative moiety, such as (SP) group.
  • compositions for delivery of an active agent comprising a peptide coacervate, wherein the peptide coacervate comprises:
  • the active agent is selected from the group comprising: proteins, (poly)peptides, carbohydrates, nucleic acids, lipids, (small) chemical compounds, nanoparticles, and combinations thereof.
  • a method for the recruitment of an active agent in a peptide coacervate comprising:
  • the pH of (a) the aqueous solution of the coacervate-forming peptides; and/or (b) the aqueous solution of an active agent; and/or (c) the aqueous solution of the aqueous solution of the coacervate-forming peptides mixed with the aqueous solution of an active agent is above 6.5, such as above 7.0, such as above 7.5, above 8.0, above 8.5, or in the range 7.0-9.0, including 7.0-8.0 and 8.0-9.0.
  • the aqueous solution of the active agent is buffered such that the combination of the aqueous solution of the active agent with the aqueous solution of the coacervate-forming peptides has a pH of > about 5.0 and ⁇ about 9.0, such as > about 6.5 and ⁇ about 9.0, or >about 7.0 and ⁇ about 8.0.
  • a method for the delivery of an active agent comprising:
  • compositions including a peptide coacervate wherein the peptide coacervate comprises: a. one or more isolated peptides of the first and/or eighth aspects of the invention, optionally wherein the lysine residue (K) is modified at an epsilon (s)-amino group with a self-immolative moiety; b. an active agent, wherein the active agent is recruited in the peptide coacervate; and
  • a method for treating or diagnosing a condition or disease in a subject in need thereof comprising:
  • the lysine residue (K) in the coacervate-forming peptide is modified at an epsilon (E)- amino group with a self-immolative moiety.
  • an isolated peptide of the first and/or eighth aspect of the invention for preparing a coacervate composition.
  • an isolated peptide of the first aspect of the invention in conjunction with an active agent for preparing a coacervate composition comprising the active agent.
  • Z is tryptophan (W) or is absent
  • O is GHGX 1 Y (SEQ ID NO: 1)
  • G is glycine (G)
  • H is histidine (H)
  • Y is tyrosine (Y)
  • each X 1 is independently selected from alanine (A), glycine (G), serine (S), asparagine (N), histidine (H), arginine (R), aspartic acid (D), tyrosine (Y), proline (P), lysine (K) and glutamine (E).
  • n is 0 - 5 m is 0 - 5 n+m is 3, 4, 5, 6, 7 or 8, optionally wherein at least two of the X 1 residues are different to each other.
  • the peptide of Formula (IV) is a peptide that comprises the amino acid sequence of Formula (V):
  • O is GHGX 1 Y (SEQ ID NO: 1)
  • Figure 2 Optical microscopic images of HBpep(AP)-SP and HBpep(GP)-SP at various pHs.
  • Figure 5 Fluorescence microscopy images of HeLa cells treated with EGFP-loaded HBpep- SP variant coacervates for 10 minutes, 4 hours and 24 hours.
  • Figure 6 Fluorescence microscopy images of HeLa cells treated with R-PE-loaded HBpep- SP variant coacervates for 24 hours.
  • Figure 7 Fluorescence microscopy images of HeLa cells treated with EGFP-loaded HBpep(HP)-SP and HBpep(RP)-SP coacervates for 10 minutes and 4 hours.
  • Figure 9 Fluorescence microscopy images of HeLa cells treated with EGFP-loaded HBpep(NP)-SP coacervates for 4 hours and 24 hours.
  • Figure 10 Fluorescence microscopy images of HeLa cells treated with EGFP-loaded HBpep(YP)-SP coacervates for 4 hours and 24 hours.
  • Figure 11 Fluorescence microscopy images of HeLa cells treated with EGFP-loaded HBpep(RPY)-SP coacervates for 10 minutes and 4 hours.
  • Figure 12 Fluorescence microscopy images and FACS of HeLa cells treat with EGFP- loaded HBpep(VPL)-SP, HBpep(HP)-SP HBpep(RPY)-SP coacervates for 2 and 4 hours.
  • Figure 13 Fluorescence microscopy images of HeLa cells treated with R-PE-loaded HBpep- SP variant coacervates for 24 hours.
  • Figure 14 Fluorescence microscopy images of HeLa cells treated with AF-IgG-loaded HBpep-SP variant coacervates for 24 hours.
  • Figure 15 Relative cell viabilities of HeLa cells treated with saporin-loaded HBpep-SP variant coacervates at various saporin concentrations for 24 hours.
  • Figure 16 Fluorescence microscopy images of HeLa cells treated with EGFP-loaded HBpep(DPY)-SP coacervates for 4 hours and 24 hours.
  • Figure 17 Fluorescence microscopy images of primary human fibroblast cells treated with EGFP-loaded HBpep(VPL)-SP (left), HBpep(HP)-SP (middle), and HBpep(RPY)- SP (right) coacervates for 24 hours.
  • FIG. 19 Fluorescence microscopy images of HeLa cells treated with EGFP-loaded HBpep(VPL)-SP (left), HBpep(HP)-SP (middle), and HBpep(RPY)-SP (right) coacervates in the presence of 10% FBS for 4 hours.
  • Figure 20 Fluorescence microscopy images and FACS of HeLa cells treated with FAM-Smac- loaded HBpep-SP variant coacervates for 4 hours.
  • Figure 22 Fluorescence microscopy images and FACS of HeLa cells transfected with EGFP- encoded pDNA, EGFP-encoded mRNA and FAM-siRNA mediated by HBpep-SP variant coacervates for 24 hours.
  • Figure 23 Analysis of indel frequency at the HBB locus in HeLa cells treated with (a) HBB targeted pDNA loaded HBpep-SP variant coacervates compared to Lipofectamine 2000, (b) HBB targeted mRNA/sgRNA loaded HBpep-SP variant coacervates compared to Lipofectamine 3000, and (c) HBB targeted Cas9 RNP loaded HBpep- SP variant coacervates compared to Lipofectamine CRISPRMAX.
  • Figure 24 Cell viability of HBpep-SP variant coacervates at the concentrations of 0.1 mg/mL in HeLa cells.
  • FIG. 25 Fluorescence microscopy images of RAW264.7 (immortalized macrophage) cells treated with EGFP-loaded HBpep(VPL)-SP (left), HBpep(HP)-SP (middle), and HBpep(RPY)-SP (right) coacervates for 4 hours.
  • Figure 37 The responsivity of cation-n stabilized complex coacervates to different concentrations of proteins
  • Figure 38 Macrophage engineering mediated by RP-K sp /YP complex coacervates.
  • compositions for delivery of an active agent may include a peptide coacervate, said peptide coacervate comprising peptides derived from histidine-rich proteins and comprising the amino acid sequence of Formula I II, III, IV or V disclosed herein, and said active agent, wherein the active agent is encapsulated in the coacervate, as well as methods of manufacture thereof and methods of use thereof.
  • the peptides may be produced by genetic engineering techniques as known to those skilled in the art.
  • the peptides thus artificially produced may represent amino acid stretches of the proteins they are derived from but do not encompass the full native protein sequence.
  • the derived peptides are N- and/or C-terminally truncated fragments of the respective histidine-rich protein.
  • the peptides may also comprise amino acid substitutions, deletions or insertions relative to the protein sequence they are derived from.
  • the peptides include fragments and variants of histidine-rich proteins that do not occur in nature and have typically been artificially produced
  • the peptides are, in various embodiments, artificial peptides, such as those created by genetic engineering techniques. Suitable synthesis methods are well-known to those skilled in the art and may be selected using their routine knowledge.
  • Coacervate has the meaning as commonly understood in the art.
  • Coacervates are two-phase liquid compositions, i.e. exhibiting liquid-liquid phase separation (LLPS), comprising or consisting of a concentrated macromolecule-rich (or coacervate) phase and a dilute macromolecule-depleted phase.
  • the two phases of the peptide coacervates are one peptide-rich coacervate phase and one dilute peptide-depleted phase.
  • the peptide-rich coacervate phase is also referred to herein as “peptide coacervate (micro)droplets”.
  • a self-coacervating peptide is one that is capable of exhibiting coacervation (or LLPS).
  • LPS liquid-liquid phase separation
  • macromolecules e.g. peptides
  • the two or more peptides possess opposite net charges to facilitate assembly into dense microdroplets driven by weak molecular interactions.
  • the peptides capable of forming coacervates for use in the present invention are derived from histidine-rich proteins, in particular derived from the histidine-rich proteins of the beak of a squid, in particularthe Humboldt squid (Dosidicus gigas).
  • HBpep(VPL) is the nomenclature of the coacervate forming peptide having the following sequence: GHGVY-GHGVY-GHGPY-K-GHGPY-GHGLY-W (SEQ ID NO: 13).
  • HBpep(VPL)-SP is the nomenclature of the coacervate forming peptide having the following sequence: GHGVY-GHGVY-GHGPY-K(SP)-GHGPY-GHGLY-W (SEQ ID NO: 14).
  • HBpep(VPL) and HBpep(VPL)-SP can be used as reference or control coacervate forming peptides.
  • HBpep(VPL)-SP may be the preferred control or reference peptide when comparing to a variant peptide that also has an SP attached to the lysine residue.
  • coacervates may be referred to herein based on the substitutions in the HBpep peptide; for example HBpep(RPY)-SP coacervates may be referred to as RPY coacervates or RPY variant.
  • aqueous solution means that the dilute phase is mainly water, i.e. comprises at least 50 vol.% water.
  • the composition may use water as the only solvent, i.e. no additional organic solvents, such as alcohols, are present.
  • the composition is an aqueous composition that additionally contains one or more solvents other than water, with water however being the major constituent, i.e. being present in an amount of at least 50, at least 60, at least 70, at least 80, at least 90, at least 95 or 99 vol.%.
  • Encapsulate as used herein in relation to the active agent, means that the active agent is entrapped in the peptide coacervate phase, for example the coacervate droplets formed by the peptides. Said entrapment may be such that the active agent is completely surrounded by peptides forming the coacervate phase but also includes embodiments, where the active agent is at least partially exposed on the surface of the respective coacervate phase, for example by being tethered to the colloidal phase via a certain group or moiety.
  • protein relates to polypeptides, i.e. polymers of amino acids connected by peptide bonds, including proteins that comprise multiple polypeptide chains.
  • a polypeptide typically comprises more than 50, for example, 100 amino acids or more.
  • peptide relates to polymers of amino acids, typically short strings of amino acids.
  • the peptides may include only amino acids selected from the 20 proteinogenic amino acids encoded by the genetic code, namely, glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, serine, threonine, asparagine, glutamine, tyrosine, tryptophan, histidine, arginine, lysine, aspartic acid, glutamic acid, cysteine, and methionine.
  • amino acids are also designated herein by their three or one letter code (as above).
  • peptides may be dipeptides, tripeptides or oligopeptides of at least 4 amino acids in length.
  • the typical length for the peptides of the invention may range from at least about 16 amino acids to 150, preferably to 80, 70, 60 or 50 amino acids in length, for example, at least 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length.
  • the upper limit for example, being 50, 40 or 35 amino acids.
  • peptide(s) refers to a unique polymer of amino acids, in accordance with various embodiments. Unless indicated otherwise, the standard single letter amino acid code is used herein; with “X” being used to allow for any amino acid.
  • isolated peptide we mean one that has been isolated or purified from its natural environment or produced artificially (e.g. on a peptide synthesiser).
  • a method reciting “a peptide” also covers the method on a population of peptides (typically the same type).
  • SR self-immolative
  • SR self-immolative
  • the molecule autocatalytically cleaves itself to release the functional group, typically in form of a harmless by-product, such that the unmodified side chain amino group of the lysine residue (K) is reformed.
  • the self-immolative (SR) moiety comprises or includes a disulfide bond (-S-S-), i.e.
  • SA refers to the immolative moiety that possesses an acetyl group at the extremity of the self-immolative moiety (generically referred to as HBpep-SA)
  • SP refers to the immolative moiety that possesses a phenyl group at the extremity of the self-immolative moiety (generically referred to as HBpep- SP).
  • HBpep-SA and HBpep-SP are collectively referred to as HBpep-SR.
  • isolated relates to the fact that the referenced peptide is at least partially separated from other components it may (naturally or non-natu rally) associate with, for example other molecules, cellular components and cellular debris. Said isolation may be achieved by purification protocols for proteins and peptides well known to those skilled in the art.
  • isolated peptides also applies to non-natural peptides which are synthesised or produced synthetically (e.g. in vitro).
  • an isolated peptide comprising the amino acid sequence of Formula (I):
  • Z is tryptophan (W) or is absent
  • O is GHGX 1 Y (SEQ ID NO: 1)
  • G is glycine (G)
  • H is histidine (H)
  • Y is tyrosine (Y)
  • each X 1 is independently selected from alanine (A), glycine (G), serine (S), asparagine (N), histidine (H), arginine (R), aspartic acid (D), tyrosine (Y), and proline (P)
  • n is 0 - 5 m is 0 - 5 n+m is 3, 4, 5, 6, 7 or 8, optionally wherein at least two of the X 1 residues are different to each other.
  • Each O may be referred to herein as a penta-peptide motif.
  • not all of the X 1 residues are proline (P).
  • the lysine residue is located at the 1 st, 6th, 11th, 16th, 21 st, or 26 th position from the N-terminus.
  • the lysine residue is at the 16 th position from the N- terminus (of the peptide).
  • n 3 and m is 2.
  • the peptide of Formula I is a peptide that comprises the amino acid sequence of Formula (II):
  • O is GHGX 1 Y (SEQ ID NO: 27)
  • G is glycine (G)
  • H is histidine (H)
  • Y is tyrosine (Y)
  • P is proline (P);
  • each X 1 is independently selected from alanine (A), glycine (G), serine (S), asparagine (N), histidine (H), arginine (R), aspartic acid (D) and tyrosine (Y);
  • the sum of a+b+c+d+e is 5.
  • the peptide of Formula I is a peptide that comprises the amino acid sequence of Formula (III):
  • O is GHGX 1 Y (SEQ ID NO: 1)
  • K is lysine (K) optionally modified at an epsilon (E)- amino group with a self-immolative moiety; and z -is tryptophan (W) or is absent, n is 1 to 4; optionally wherein at least two of the X 1 residues are different to each other.
  • any peptide of the first aspect of the invention including any peptide that comprises the amino acid sequence of Formula I, II or III.
  • the isolated peptide according to the first or eighth aspects of the invention is capable of forming coacervates at pH 7.0.
  • the isolated peptide according to the first or eighth aspects of the invention is capable of forming coacervates at pH 7.5.
  • the isolated peptide according to the first or eighth aspects of the invention is capable of forming coacervates at pH 8.0.
  • the isolated peptide according to the first or eighth aspects of the invention is capable of forming coacervates at pH 8.5. In particular embodiments, the isolated peptide according to the first or eighth aspects of the invention is capable of forming coacervates at pH 9.0.
  • the isolated peptide according to the first or eighth aspects of the invention is capable of forming coacervates in the pH range 7.0 - 9.0.
  • all the residues at the X 1 positions are the same and are selected from: A, G, and H.
  • Examples of such peptides are the HBpep(AP)-SP, HBpep(GP)-SP and HBpep(HP)-SP variants.
  • Such peptides form coacervates with enhanced cellular uptake compared to HBpep(VPL)-SP.
  • Such peptides form coacervates capable of enhanced gene transfection compared to HBpep(VPL)-SP.
  • the amino acid residue at each X 1 position is different to the residue at the other X 1 positions.
  • the two N-terminal X 1 amino acid residues are the same as each other and the C terminal X 1 amino acid residue is different to the two N-terminal X 1 amino acids.
  • Examples of such peptides include the HBpep(RPY)-SP and HBpep(DPY)-SP variants.
  • the isolated peptide comprises the Z tryptophan. In other embodiments, the isolated peptide lacks the Z tryptophan.
  • the peptides of the invention are capable of LLPS formation in a wide range of alkaline pH.
  • the pH of the aqueous solution comprising the peptide of the first aspect of the invention may be below 9, for example below 8.5, or below 8.0, or below 7.5, or below 7.0.
  • the isolated peptide of the first or eighth aspects of the invention is able to form coacervates at a pH between about 6.5 and 9.0, such as between about 7.0 and 9.0 or 7.0 and 8.0.
  • the peptide is capable of undergoing liquid-liquid phase separation (LLPS) at a pH of 8.0.
  • LLPS liquid-liquid phase separation
  • the peptide is capable of undergoing liquid-liquid phase separation (LLPS) at a pH of 9.0.
  • LLPS liquid-liquid phase separation
  • the peptide is capable of undergoing liquid-liquid phase separation (LLPS) in the pH range of about 7.0 to about 8.0.
  • LLPS liquid-liquid phase separation
  • the peptide is capable of undergoing liquid-liquid phase separation (LLPS) in the pH range of about 8.0 to about 9.0.
  • LLPS liquid-liquid phase separation
  • the isolated peptide of the first or eighth aspects of the invention is able to form coacervates that are more efficient at coacervate disassembly when in the cell cytosol than coacervates formed from HBpep(VPL)-SP.
  • the kinetics of disassembly of such coacervates is thus more efficient when in the cell cytosol.
  • the efficiency (of release) can be measured by a variety or means, such as measurement of fluorescence intensity of cargos over short time period, or viabilities of cells treated with toxic protein loaded coacervates (as shown in Figure 12 and Figure 15).
  • control coacervate composition e.g. HBpep(VPL)-SP.
  • the ability to measure the relative effect serum has on cellular entry/delivery can be determined as described in the Examples herein (see Example 2.2), e g. by the measuring effect 10% fetal calf serum (FCS) has on cellular entry of the coacervate formed from peptides of the first aspect of the invention compared to a suitable control (e g. HBpep(VPL)-SP).
  • FCS fetal calf serum
  • the isolated peptide of the first or eighth aspects of the invention displays faster cargo release than coacervates formed from HBpep(VPL)-SP.
  • increased cargo release occurs within 4 hours, such as within 2 hours of administration.
  • Increased cargo release can be an increase of 10%, 20%, 30%, 40%, 50%, 70%, 100%, 150%, 200%, 300%, 400%, 500% or more, relative to HBpep(VPL)SP.
  • the isolated peptide of the first or eighth aspects of the invention displays enhanced transfection efficiency than coacervates formed from HBpep(VPL)-SP.
  • Enhanced transfection can be measured by an increase in the amount of payload delivered into the cell (e.g., a nucleic acid, such as a gene, siRNA or ASO; a ribonucleoprotein complex, like CRISPR/CAS9; a polypeptide or protein, like an antibody, and the like).
  • Enhanced transfection efficiency can be an increase of 10%, 20%, 30%, 50%, 70%, 100%, 150%, 200%, 300%, 400%, 500% or more, relative to HBpep(VPL)-SP.
  • Aryl refers to cyclic aromatic groups with 6 to 14 carbon atoms, such as phenyl. If substituted, the substituents are defined as for alkyl above.
  • the self-immolative (SR) moiety comprises or includes a disulfide moiety/bond (-S-S-), i.e. disulfide bridge with a covalent bond between the two sulfur (S) atoms.
  • Said disulfide bond may provide a biologically relevant precursor to engineer specific intracellular release of the cargo upon exposure to specific conditions.
  • the disulfide bond may be reduced in a reducing environment, such that the disulfide bond is reduced to two thiols (-SH), i.e. dithiols, and trigger the autocatalytic cleavage of the self-immolative (SR) moiety.
  • the self-immolative (SR) moiety thus comprises a disulfide group that separates upon reduction into two thiols, with one being still attached to the lysine side chain and the other being released.
  • the remaining one thiol group on the lysine side chain then autocatalytically cleaves itself off such that the amino group of the lysine residue (K) is reformed, and the resulting restoration of the charged lysine residue (K) destabilizes the peptide coacervate leading to the subsequent dissolution of the coacervate phase and, if present, release of the recruited active agent/payload.
  • the minimum sequence length of a peptide of the invention is 16 amino acids or 17 when Z is present.
  • a preferred peptide has 5 pentapeptide motifs and so this would have 26 amino acids or 27 when Z is present.
  • the isolated peptide of the invention can comprise up to 4 consecutively linked peptide units and so the length of the isolated peptide can be as much as 108 amino acids long.
  • the isolated peptide of the first or eighth aspects of the invention is 15 - 45 or 16-45 amino acids long.
  • the isolated peptide is 25 or 26 or 27 amino acids long.
  • Z is tryptophan (W) or is absent
  • O is GHGX 1 Y (SEQ ID NO: 1)
  • G is glycine (G)
  • H histidine (H)
  • Y is tyrosine (Y)
  • each X 1 is independently selected from arginine (R), tyrosine (Y), lysine (K) and glutamine (E).
  • n is 0 - 5 m is 0 - 5 n+m is 3, 4, 5, 6, 7 or 8, optionally wherein at least two of the X 1 residues are different to each other.
  • the lysine residue (K) at position 16 is modified at an epsilon (s)-amino group with a self-immolative moiety, such as (SP) group.
  • the minimum sequence length of a peptide of the invention is 15 amino acids or 16 when X 2 or Z is present or 17 when X 2 and Z are present.
  • a preferred peptide has 5 pentapeptide motifs and so this would have 25 amino acids or 26 when X 2 or Z is present or 27 when X 2 and Z are present.
  • the peptides of the present invention form coacervates readily, in particular under neutral to alkaline conditions, i.e. pH values of 7 and higher. Stable solutions of these peptides without any distinct phase separation can be formed at low pH, for example less than 4.
  • the peptides may be prepared as stock solutions in slightly acidic solutions, such as 1-100 mM, for example about 10 mM acetic acid or other suitable weak acids.
  • peptide coacervate is formed by self-coacervation whereby a single type of peptide is formed in the coacervate composition (excluding any payload or ancillary peptide).
  • two of the types of peptide used to form the coacervates posses opposite net charges.
  • the peptides can assemble into coacervates driven by weak molecular interactions.
  • the inventors have found that strong interactions between the cationic and aromatic peptides can be formed which facilitate complex coacervation and inhibit or even reverse aggregation. These results suggest a potential strategy to mitigate or reverse aggregation by harnessing cation-ir interactions in aggregation-prone peptides.
  • At least one of the types of coacervate peptide used to form the coacervate possesses an aromatic amino acid (e.g., Tyr) at the X 1 position and another type of coacervate peptide possesses a cationic amino acid (e.g., Lys or Arg) at the X 1 position.
  • an aromatic amino acid e.g., Tyr
  • another type of coacervate peptide possesses a cationic amino acid (e.g., Lys or Arg) at the X 1 position.
  • a cationic amino acid e.g., Lys or Arg
  • the peptide with an aromatic amino acid at the X 1 position and the peptide with an cationic amino acid at the X 1 position are mixed in a ratio of between 1 :20 and 20:1 , such as between 1 :15 and 15:1 , 1 :10 and 10:1 , or 1 :8 and 8:1 (e.g. between 1 :1 and 1 :10).
  • Coacervates formed from mixtures comprising two type of peptide wherein one of the peptides possesses Tyr at the X 1 position and another possesses Arg at the X 1 position are particularly preferred.
  • the coacervate is formed from RP-K SF 7YP-K SP peptides to form a RP- K SP /YP-K SP complex (coacervate).
  • the RP-K sp and YP-K SP peptides are mixed in a ratio of between 1 :20 and 20:1 , such as between 1 :15 and 15:1 , 1 :10 and 10:1 , or 1 :8 and 8:1.
  • the active agent is provided in form of an aqueous solution, too, said solution may have a pH >7 and thus effect coacervate formation.
  • the solution of the active agent may be buffered with suitable buffering agents, such that the combined aqueous solutions of the active agent and the coacervate-forming peptides retain a pH >7.
  • the coacervate may be an aqueous liquid two phase formulation, as described above, i.e. a composition comprising (1) a coacervate colloidal phase comprising one or more peptides of the first aspect of the invention and the active agent; and (2) a dilute aqueous phase.
  • the coacervates formed in the above-described processes may have the form of droplets, for example microdroplets, having a substantially spherical shape with a diameter ranging from about 0.2 to about 5 pm, or may take the form of a condensed hydrogel.
  • compositions for delivery of an active agent comprising a peptide coacervate, wherein the peptide coacervate comprises:
  • the one or more isolated peptides in step (i) comprise a self-immolative moiety which autocatalytically cleaves itself upon exposure to specific conditions selected from the group consisting of: pH changes, redox changes, exposure to release agents, and combinations thereof.
  • the active agent is selected from the group comprising: proteins, (poly)peptides, carbohydrates, nucleic acids, lipids, (small) chemical compounds, nanoparticles, and combinations thereof.
  • the peptide coacervate is formed from the same type of peptide (i.e. one having the same amino acid sequence)
  • the peptide coacervate is formed from a combination of distinct type of peptide (i.e. two or more peptides having distinct amino acid sequences).
  • the peptide coacervate is formed from a combination of two distinct types of peptide.
  • the two types of peptide have opposite net charges.
  • the peptide coacervate is formed from a combination of a peptide having an aromatic amino acid at an X 1 position and a distinct peptide having a cationic amino acid at an X 1 position in the same relative position (i.e. relative to the position in the other type of peptide).
  • the aromatic amino acid is tyrosine (Y) and the cationic amino acid is arginine (R).ln particular embodiments, two distinct types of coacervateforming peptide are used in a ratio between 20:1 and 1 :20, such as between 10:1 and 1 :10.
  • a diagnostic active agent for inclusion in the coacervates of the invention could be a diagnostic enzyme that has bioactivity in certain diseased cells, or a peptide with NanoclickTM or FREP functions that can respond to molecules/enzyme on certain cells to give luminescence or fluorescence signals.
  • the coacervates of the invention are particularly suitable for intracellular delivery of a diagnostic moiety that cannot be uptaken by cells on its own.
  • the coacervates formed of the isolated peptides of the invention and an active agent may be formulated as compositions.
  • the composition may be a pharmaceutical or diagnostic formulation for administration to a subject. In various embodiments it can thus comprise one or more pharmaceutically or diagnostically acceptable excipients (such as diluents, auxiliaries, carriers and the like). Such formulations may additionally comprise further active agents that are not encapsulated in the coacervate phase.
  • such compositions are liquid compositions, including gels and pastes. “Liquid”, as used herein, particularly refers to compositions that are liquid under standard conditions (20° C. and 1013 mbar). In various embodiments, such liquid compositions are pourable.
  • the compositions may be in single dose or multi dose form. Suitable forms and packaging options are well known to those skilled in the art.
  • the pharmaceutical or diagnostic formulation can be adapted for and so may be suitable for human or veterinary use.
  • the coacervates are more efficient at coacervate disassembly when in the cell cytosol than coacervates formed from HBpep(VPL)-SP.
  • the coacervates are capable of enhanced cellular uptake compared to coacervates formed from HBpep(VPL)-SP.
  • a method for the recruitment of an active agent in a peptide coacervate comprising:
  • the aqueous solution of the active agent is buffered such that the combination of the aqueous solution of the active agent with the aqueous solution of the coacervate-forming peptides has a pH of > about 5.0 and ⁇ about 9.0, such as > about 6.5 and ⁇ about 9.0, or >about 7.0 and ⁇ about 8.0.
  • the peptide coacervate is formed from a combination of distinct type of coacervate-forming peptide (i.e. two or more peptides having distinct amino acid sequences).
  • the peptide coacervate is formed from a combination of two distinct types of coacervate-forming peptide.
  • the two types of peptide have opposite net charges.
  • the peptide coacervate is formed from a combination of a peptide having an aromatic amino acid at an X 1 position and a distinct peptide having a cationic amino acid at an X 1 position in the same relative position (i.e. relative to the position in the other type of peptide).
  • the aromatic amino acid is tyrosine (Y) and the cationic amino acid is arginine (R).
  • the volume ratio of the aqueous solution of the coacervate-forming peptides to the aqueous solution of the active agent is between 1 : 5 and 1 : 20.
  • the active agents in the combined aqueous solution are also in the form of an aqueous solution.
  • Said aqueous solution may have a pH >7.0, such as >8.0 and, in some embodiments, is buffered such that the combination of the aqueous solution of the active agent with the aqueous solution of the coacervate-forming peptides obtained in the combined aqueous solution has a pH >7.0, such as >8.0, as disclosed elsewhere herein.
  • the concentration of the coacervate-forming peptides in the provided aqueous solution is greater than about 0.3 mg/mL and may, for example, range from about 0.3 to about 100 mg/mL.
  • the combined solution after coacervate formation may be an aqueous liquid two phase formulation, comprising (1) a coacervate colloidal phase comprising the peptides derived from histidine-rich proteins and the active agent; and (2) a dilute aqueous phase.
  • the dilute aqueous phase may include a water-based liquid, as described above. It is peptide- depleted in that the majority of the peptides are located in the colloidal phase, e.g. 80 wt.-% or more of the peptides are present in the colloidal phase, for example at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99 wt.-%.
  • the dilute phase may thus contain residual amounts of peptides in the coacervate composition. As the coacervate formation is an equilibrium reaction, the exchange of peptides from the colloidal phase to the dilute phase and vice versa may be dynamic. However, in various embodiments, the above distribution applies.
  • a method for the delivery of an active agent comprising:
  • compositions including a peptide coacervate that comprises: a. one or more isolated peptides of the first and/or eighth aspects of the invention, optionally wherein the lysine residue (K), when present, is modified at an epsilon (E)- amino group with a self-immolative moiety; and b. an active agent, wherein the active agent is recruited in the peptide coacervate; and
  • lysine residue (K) when present, refers to the lysine that, when present, is the one located outside of the pentameric motif [i.e. not within the “O” motif, GHGX 1 Y (SEQ ID NO: 1) motif or not at the X 2 position].
  • the one or more isolated peptides of the first aspect of the invention in (a.) have the lysine residue (K) modified at an epsilon (E)- amino group with a self-immolative moiety.
  • the one or more isolated peptides of the eighth aspect of the invention in (a.) have the lysine residue (K) modified at an epsilon (E)- amino group with a self-immolative moiety.
  • the one or more isolated peptides of the eighth aspect of the invention in (a.) lack the lysine at X 2 .
  • the conditions that trigger the release of the active agent may be selected from those disclosed above for the composition for the delivery of the active agent.
  • compositions comprising a peptide coacervate may be exposed to or subjected to conditions that facilitate the release of the active agent from the coacervate phase. Said release may be facilitated by dissolution of the peptides of the coacervate phase, for example reversing the formation process by decreasing the pH, or degradation or disruption of the coacervate phase by suitable means.
  • a method for treating or diagnosing a condition or disease in a subject in need thereof comprising:
  • composition comprising a peptide coacervate to a subject, wherein the peptide coacervate comprises: a. one or more isolated peptides according to the first and/or eighth aspects of the invention, in particular wherein the lysine residue (K), when present, is modified at an epsilon (£)- amino group with a self-immolative moiety; and b. a pharmaceutical or diagnostic agent, wherein the pharmaceutical or diagnostic agent is recruited in the peptide coacervate; and
  • the conditions that trigger the release of the pharmaceutical or diagnostic agent may be selected from those disclosed above for the delivery methods.
  • the composition is administered to a subject in a therapeutically effective amount.
  • compositions described herein and comprising a peptide coacervate and a pharmaceutical or diagnostic agent, wherein the pharmaceutical or diagnostic agent is encapsulated in the coacervate are administered to said subject.
  • the administration may make use of any suitable administration route including oral administration or parenteral administration, for example intravenous, intramuscular, subcutaneous, epidural, intracerebral, intracerebroventricular, nasal, intraarterial, intraarticular, intracardiac, intradermal, intralesional, intraocular, intraosseous, intravitreal, intraperitoneal, intrathecal, intravaginal, transdermal, transmucosal, sublingual, buccal, and perivascular.
  • the administration may be systemic or localized, e.g. topically.
  • the conditions that trigger the release of the pharmaceutical or diagnostic agent may generally be selected from those disclosed above for the delivery methods.
  • the subject may be a mammal, for example a human.
  • an isolated peptide of the first and/or eighth aspect of the invention for preparing a coacervate composition, optionally wherein the coacervate composition comprises an active agent.
  • an isolated peptide of the first and/or eighth aspects of the invention in conjunction with an active agent for preparing a coacervate composition comprising the active agent.
  • a seventh aspect of the invention there is provided the isolated peptide of the first and/or eighth aspects of the invention or the composition of the second aspect of the invention for use in therapy or for use in a method of diagnosis practised on a mammal.
  • a peptide coacervate composition comprised of two types of coacervate-forming peptides wherein the two types of coacervateforming peptides are capable of interacting with each other via at least one ir-cationic interaction.
  • the ir-cationic interaction is formed between an aromatic amino acid present in one coacervate-forming peptide type and a cationic amino acid present in another coacervate-forming peptide type.
  • the aromatic amino acid is selected from tyrosine (Y), histidine (H), phenylalanine (F) or tryptophan (W).
  • the at least one n-cationic interaction is formed between arginine (R) and tyrosine (Y) amino acids.
  • the aromatic amino acid present in one coacervate-forming peptide type and a cationic amino acid present in another coacervate-forming peptide type are in the same relative location on the respective peptides.
  • the aromatic amino acid in one coacervate-forming peptide type and the cationic amino acid in the other coacervate-forming peptide are at the same X1 position of an isolated coacervate-forming peptide having the Formula (I), (II), (III), (IV) or (V).
  • the first coacervate-forming peptide has a different amino acid sequence to the second coacervate-forming peptide.
  • compositions and methods herein disclosed are further illustrated in the following examples and the accompanying Figures, which are provided by way of illustration and are not intended to be limiting the scope of the present disclosure.
  • Z is tryptophan (W) or is absent
  • G is glycine (G)
  • H is histidine (H)
  • Y is tyrosine (Y)
  • each X 1 is independently selected from alanine (A), glycine (G), serine (S), asparagine (N), histidine (H), arginine (R), aspartic acid (D), tyrosine (Y), and proline (P)
  • n is 0 - 5 m is 0 - 5 n+m is 3, 4, 5, 6, 7 or 8, optionally wherein at least two of the X 1 residues are different to each other; optionally wherein not all of the X 1 residues are proline (P).
  • B is GHGPY (SEQ ID NO: 2)
  • G is glycine (G)
  • H is histidine (H)
  • Y is tyrosine (Y)
  • P is proline (P);
  • each X 1 is independently selected from alanine (A), glycine (G), serine (S), asparagine (N), histidine (H), arginine (R), aspartic acid (D) and tyrosine (Y);
  • K is lysine (K) optionally modified with a self-immolative moiety
  • lysine residue (K) is modified at an epsilon (s)- amino group with a self- immolative moiety.
  • Z is tryptophan (W) or is absent
  • Z is tryptophan (W) or is absent
  • O is GHGX 1 Y (SEQ ID NO: 1)
  • G is glycine (G)
  • H histidine (H)
  • Y is tyrosine (Y)
  • each X 1 is independently selected from arginine (R), tyrosine (Y), lysine (K) and glutamine (E)
  • n is 0 - 5 m is 0 - 5 n+m is 3, 4, 5, 6, 7 or 8, optionally wherein at least two of the X 1 residues are different to each other.
  • composition for delivery of an active agent comprising a peptide coacervate, wherein the peptide coacervate comprises:
  • composition of embodiment 41 or 42, wherein the peptide coacervate is formed of a single type of isolated peptide.
  • composition of embodiment 44, wherein the peptide coacervate is formed of two types of isolated peptide that posses opposite net charges.
  • composition of embodiment 44, wherein the peptide coacervate is formed of two types of isolated peptide that posses opposite net charges.
  • composition of embodiment 44, wherein the peptide coacervate is formed of two types of isolated peptide wherein one peptide wherein one of the types of coacervate peptide possesses an aromatic amino acid (e.g., Tyr) at the X 1 position and other type of coacervate peptide possesses a cationic amino acid (e g., Lys or Arg) at the X 1 position.
  • aromatic amino acid e.g., Tyr
  • a cationic amino acid e g., Lys or Arg
  • composition of embodiment 47 wherein the peptide with an aromatic amino acid at the X 1 position and the peptide with an cationic amino acid at the X 1 position are mixed in a ratio of between 1 :20 and 20:1 .
  • composition of embodiment 51 wherein the protein or (poly)peptide is an antibody, antibody variant, antibody fragment or peptide.
  • composition of any one of embodiments 41 to 53, wherein the pH of the composition is > 5.0 and ⁇ 9.5, such as > about 6.5 and ⁇ about 9.0, or >about 7.0 and ⁇ about 8.0.
  • composition of any one of embodiments 41 to 55, wherein the coacervates formed are capable of enhanced cellular uptake compared to coacervates formed from HBpep(VPL)-SP.
  • a method for the delivery of an active agent comprising:
  • composition comprising a peptide coacervate to a subject, wherein the peptide coacervate comprises: a. one or more isolated peptides selected from the peptides of any one of embodiments 1 to 40 wherein, when present, the lysine residue (K) is modified at an epsilon (E)- amino group with a self-immolative moiety; and b. a pharmaceutical or diagnostic agent, wherein the pharmaceutical or diagnostic agent is recruited in the peptide coacervate; and
  • the pellets were dried under vacuum and re-dissolved by using 90% of 10 mM acetic acid and 10% acetonitrile for HPLC purification.
  • the purified peptides were isolated by lyophilization from certain HPLC elutes.
  • Molecular dynamics (MD) simulations were carried out for two peptides GP and RPY to understand how intermolecular interactions modulate the properties of coacervates.
  • systems containing 4, 10, and 30 peptide molecules were simulated, respectively.
  • Each system was subject to 3 replicates of simulations, with each replicate running for 1 s, resulting in a total simulation time of 18 s.
  • the required number of peptide molecules was randomly placed in a cubic box and solvated with water molecules.
  • 0.16 M NaCI was added to each system.
  • Each system was initially subject to 500 steps using steep descent energy minimization.
  • the giant unilamellar vesicles were prepared from 99.5% of 1-palmitoyl-2-oleoyl-sn- glycero-3-phosphocholine (POPC) and 0.5% of 1 ,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt) (Rhod-PE) using the gel-assisted method described in previous studies (Weinberger, A. et al. Biophys. J. 105, 154- 164 (2013).
  • POPC 1-palmitoyl-2-oleoyl-sn- glycero-3-phosphocholine
  • Rhod-PE 1 ,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt)
  • EGFP-loaded coacervates 0.3 mM HBpep-SP variants, 0.1 mg/mL EGFP
  • 100 pL of EGFP-loaded coacervates 0.3 mM HBpep-SP variants, 0.1 mg/mL EGFP
  • the cells were washed twice with a pH 5.0 phosphate buffer followed by PBS twice.
  • the treated cells were dissociated by trypsin for FACS.
  • the cells were pre-incubated for 1 hour and kept at low temperature during the 2 hours of uptake process.
  • the mean fluorescence intensity of treated HeLa cells measured by FACS was normalized to the control group that was treated only with EGFP-loaded Coacervates in the absence of inhibitors.
  • the recruitment of biomacromolecule cargos by peptide coacervates was performed simultaneously with inducing the LLPS.
  • Cargos were first dissolved or diluted in the optimal buffers to achieve the required concentration. Then, the peptide stock solutions were mixed with the cargo-contained buffer with the volume ratio of 1/9 to induce coacervation and cargo recruitment.
  • DMEM Dulbecco's modified Eagle medium
  • fetal bovine serum 100 units/mL of penicillin and 100 pg/mL of streptomycin.
  • the medium was replaced with 900 pL of fresh Opti-MEM.
  • 100 pL of freshly prepared protein- or peptide-loaded HBpep-SP coacervate suspensions (0.1 mg/mL of protein or 0.05 mg/mL of peptide, 1 mg/mL of HBpep-SP) were added into the Opti-MEM.
  • EGFP enhanced green fluorescent protein
  • luciferase luciferase
  • transfection was continued for another 20 hours before being imaged under a fluorescence microscope (for EGFP), or tested the luminescence by using Nano- Glo® Dual-Luciferase® kit and a microplate reader. Additionally, the transfection efficiency for EGFP was quantified by fluorescence-activated cell sorting (FACS).
  • FACS fluorescence-activated cell sorting
  • Two siRNAs including FAM labeled anti-PCSK9 and unlabeled anti-EGFP siRNA were used to evaluate the delivery efficiency of HBpep-SP peptides.
  • HeLa or HeLa-EGFP cells were cultured in 35 cm 2 dishes with full medium before the transfection. Then, the medium was replaced with 900 pL of Opti-MEM and 100 pL of freshly prepared siRNA-loaded coacervates (0.3 mM HBpep- SP variants, 200 nM siRNA). After 4 h of uptake, the cells treated with FAM-siRNA were imaged by fluorescence microscopy and the delivery efficiency was quantified by FACS.
  • the knock-down efficiency after 4 h of uptake, the HBpep-SP peptides containing medium was removed. The cells were washed with PBS twice and then cultured in 1.5 mL of full medium for another 20 h.
  • the PCSK9 mRNA knock-down efficiency was measured by reverse transcription-quantitative polymerase chain reaction analysis (RT-qPCR) and normalized by comparing it to glyceraldehyde 3-phosphate dehydrogenase (GADPH) mRNA.
  • RT-qPCR reverse transcription-quantitative polymerase chain reaction analysis
  • GADPH glyceraldehyde 3-phosphate dehydrogenase
  • HeLa cells were cultured in 35 cm 2 dishes until reaching 40% confluence. Then the medium was replaced with 900 pL of Opti-MEM and 100 pL of cargo-loaded HBpep-SP peptides. The final cargo concentration is 2 pg/mL for the all-in-one pDNA, 2 and 1 pg/mL forthe mRNA and sgRNA mixture, and 2 and 1 pg/mL for the Cas9 nuclease and sgRNA complex. After 4 h of uptake, the medium was discarded. The cells were washed with PBS twice and cultured in full media for another 44 h.
  • the efficiency of 48 h of transfection can be evaluated by using the T7 Endonuclease 1 (T7EI) assay.
  • T7EI T7 Endonuclease 1
  • the target genomic locus was amplified by PCR using Q5 Hot Start high-fidelity 2X master mix (NEB) and primers listed in Figure 26 and purified by PureLink PCR purification kit (Thermo Fisher Scientific).
  • 200 ng of PCR products were digested by T7EI and analyzed by 2% agarose gels before imaging with the gel documentation system.
  • the gray level of digested bands and undigested bands was measured by Imaged.
  • the indel percentage could be calculated by the following formula (Guschin, D.Y.
  • fraction cleaved the sum of each digested band intensity/(the sum of each digested band intensity + undigested band intensity).
  • the knock-out efficiency was also quantified by delivering Cas9 mRNA and EGFP-targeting siRNA into HeLa-EGFP cells following prior protocols. After 48 h of transfection, treated HeLa- EGFP cells were imaged under a fluorescence microscope, and their EGFP intensity decrease was measured by FACS. All fluorescence micrographs were imaged on live cells.
  • cytotoxicity of HBpep-SP peptides was determined by using the MTT assay [6], In detail, 10 4 HeLa cells in 100 pL of the full medium were cultured in 96-wells plates and incubated overnight. Then the media were replaced with 90 pL of Opti-MEM and 10 pL of coacervate suspensions (1 mg/mL of HBpep-SP). After 4 hours of cellular uptake, the media were removed and the cells were washed with PBS twice before adding 100 pL of the full medium. The cells were incubated for another 20 hours before 10 pL of 5mg/mL MTT dissolved in PBS was added.
  • HBpep-SP variants and saporin-loaded HBpep-SP variants were evaluated using the Cell Counting Kit-8 (CCK-8). As described previously (Cai, L. et al. ACS Omega 4, 12036-12042 (2019)), cells were cultured in 96-well plates with 100 pL of full media and incubated for 24 h.
  • the medium was then replaced with 100 pL of Opti-MEM containing saporin-loaded coacervates (various concentrations of saporin, 0.3 mM HBpep-SP variants) or various concentrations of HBpep-SP variants.
  • the medium was removed, and the cells were washed with PBS twice and cultured in 100 pL of fresh full medium.
  • the cells were incubated for another 20 h before changing the medium to the full medium containing 10% CCK- 8 solution. After 4 h of incubation, the cells were measured for absorbance at 460 nm using a microplate reader (Infinite M200 Pro, Tecan).
  • the relative cell viability for both the MTT assay and the CCK-8 were calculated as below.
  • Ab and Ac represent the absorbance of tested cells, no cells and control cells, respectively.
  • HFF Human foreskin fibroblast
  • RAW 264.7 cells were cultured in DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 pg/mL streptomycin under typical conditions (37 °C and 5% CO2).
  • Jurkat cells were cultured in RPMI-1640 Medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 pg/mL streptomycin.
  • the subculture started by detaching the cells with trypsin treatment, followed by centrifugation (1000 rpm, 5 min) to collect the cells. Then the pellets were resuspended with fresh media for subculture or experiments.
  • the Jurkat cells are suspension cells, the subculture was conducted by dilution of cell culture in fresh media to achieve the required cell density.
  • RAW 264.7 cells the cells were detached from the culture flask using a cell scraper (Corning) and collected by centrifugation (1000 rpm, 5 min). Then the pellets were resuspended with fresh media for subculture or experiments.
  • the AP variant forms microdroplets in a pH range from 7.0 to 8.0 ( Figure 2). Furthermore, changing V and L to G expands the coacervation pH to 9.0 without the formation of aggregates ( Figure 2).
  • GP coacervates exhibited the slowest recovery rate among the three tested coacervates, indicating a reduced molecular mobility, which is consistent with SFA measurements that denoted gel-like properties. Conversely, RPY coacervates displayed higher fluidity with the fastest recovery rate.
  • the inventors used various endocytic inhibitors and did not observe significant changes in the internalizations pathway as a result of residue mutations.
  • Coacervate disassembly (and concomitant release of cargos) is activated by GSH-induced disulfide bond reduction, which leads to cleavage of the sidechain grafted to the Lys residue of HBpeps (Sun, Y. et al. ACS Nano 17, 16597-16606 (2023)).
  • the disassembly kinetics can be monitored by measuring the concentration decay of non-reduced HBpep-SPs upon incubation with GSH by high-performance liquid chromatography (HPLC). This decay could be adequately fitted by a first-order kinetic model and occurred faster in RPY coacervates compared to GP coacervates, with a 1 8-fold higher rate constant.
  • Enhanced green fluorescent protein was first employed as cargo to study the cellular uptake and protein delivery efficiency of HBpep-SP variants.
  • EGFP Enhanced green fluorescent protein
  • AP and GP variants showed a better cellular uptake after 10 minutes, as more EGFP-loaded coacervates were observed inside the cells.
  • HBpep-SP variants exhibit slower release rates.
  • the GP variant showed more fluorescence puncta (attributed to intact coacervates) compared to the well-distributed signal inside the cytoplasm.
  • the variants with charged or polar sidechain behaved in a more complex fashion.
  • the HP variant showed both enhanced cellular uptake and cargo release properties.
  • the cells treated with EGFP-loaded HBpep(HP)-SP showed substantial uptake (Figure 7), which is significantly higher than the original VPL peptide ( Figure 5).
  • the HP coacervates released most of the EGFP cargo within 4 hours, also significantly faster than VPL.
  • R has a higher pKa, resulting in HBpep(RP)-SP to be more charged and hydrophilic at neutral pH.
  • the HBpep(RP)-SP coacervates tend to pre-maturely release the cargo before cellular internalization.
  • the HeLa cells treated with EGFP-loaded HBpep(RP)-SP coacervates showed homogenous but weak fluorescence signals.
  • R-PE red fluorescence protein also known as R-phycoerythrin
  • IgG IgG
  • RPY coacervates showed higher anticancer effects at the same cargo concentration compared to VPL or HP ( Figure 15). In all cases, the transfection efficiency followed the same performance trend of RPY > HP > VPL > GP > NP.
  • HBpep(VPL)-SP coacervates Another drawback of HBpep(VPL)-SP coacervates is its sensitivity to serum during cellular uptake. In the presence of 10% FBS, VPL coacervates lost the great ability to deliver EGFP into the cell ( Figure 19 and 31). However, variants with positively charged residues in their X- position, such as HP and RPY, showed improved tolerance to serum. This tackles one of the major issues that may obstruct the in vivo application of coacervates-based delivery system.
  • the HBpep-SP variant coacervates could also deliver other nucleic acid therapeutics like mRNA or siRNA.
  • the GP, HP and RPY coacervates all have better mRNA transfection efficiency than VPL and commercially available reagent Lipofectamine 3000 and transfected 95% (with a 1 .4-fold higher MFI compared to VPL), 99.5%, and 98.7% of cells, respectively.
  • the increased efficiency for GP coacervates compared to VPL in delivering nucleic acids can be attributed to the higher peptide concentration required to achieve full cargo recruitment for GP, which indicates weaker peptide-nucleic acid interactions, in turn leading to enhanced release.
  • GP may offer distinct advantages as a delivery vehicle for applications requiring slow release, particularly for nucleic acid therapeutics, which function at lower concentrations than protein therapeutics (Gupta, A., et al., Adv. Drug Delivery Rev. 178, 113834 (2021)). Additionally, in Jurkat T-cells and RAW264.7 macrophage cell lines, HP and RPY coacervates showed much better EGFP positivity for mRNA than Lipo3000 and VPL coacervates.
  • HeLa cells treated with FAM-siRNA loaded coacervates displayed increasing rates of positive cells and MFI in the order RPY > HP >VPL > GP >NP, where HP and RPY coacervates showed higher cellular uptake and fluorescence intensities of compared to those treated with FAM-siRNA loaded VPL coacervates and VPL, HP, and RPY all outperforming the highly specialized lipofectamine RNAiMAX (LipoRMAX) ( Figure 22).
  • the coacervates could deliver all three types of CRISPR/Cas9 genome editing modalities including all-in-one pDNA encoding both Cas9 nuclease and sgRNA sequences, the complex of sgRNA and mRNA encoding Cas9 nuclease, and the Cas9 ribonucleoprotein (RNP) complex ( Figure 23).
  • Variants like GP, HP and RPY showed better delivery efficiency and caused higher insertion-deletion (indel) frequencies compared to three commercially available reagents specialized for different modalities in delivering the all-in-one pDNA and mRNA/sgRNA gene editing machineries ( Figure 23).
  • the X position in the GHGXY (SEQ ID NO: 1) repeats of HBpep-SP backbone could be adjusted to all types of amino acid residues and their combinations (Table 3.1). Tuning the properties of the X residues not only expand the pH window for the peptide to phase separate, but also improved its performance as a delivery platform, including improved cellular uptake, cargo release and gene transfection efficiencies (Table 3.2). All variants showed improvements in at least one of these listed properties except YP. But the Y residue could be helpful when combined with other hydrophilic residues such as R and D to create phase separating peptides with even better performances, such as RPY shown above. Therefore, current data suggest multiple possibilities to customize the coacervates for any specific cargos with controllable release profiles by simply changing a few residues in the primary sequence of the phase separating peptides.
  • HBpep(VPL)-SP GHGVY-GHGVY-GHGPY-K(SP)-GHGPY-GHGLY-W (SEQ ID NO: 14).
  • HBpep(AP)-SP GHGAY-GHGAY-GHGPY-K(SP)-GHGPY-GHGAY- Hydrophobic W (SEQ ID NO: 15).
  • HBpep(SP)-SP GHGSY-GHGSY-GHGPY-K(SP)-GHGPY-GHGSY-W (SEQ ID NO: 26).
  • HBpep(NP)-SP GHGNY-GHGNY-GHGPY-K(SP)-GHGPY-GHGNY- Hydrophilic W (SEQ ID NO: 20).
  • HBpep(HP)-SP GHGHY-GHGHY-GHGPY-K(SP)-GHGPY-GHGHY- W SEQ ID NO: 17).
  • Positively-charged HBpep(RP)-SP GHGRY-GHGRY-GHGPY-K(SP)-GHGPY-GHGRY- W (SEQ ID NO: 18).
  • RP-K SP and YP-K SP can also interact with their less hydrophobic parent peptides, RP and YP, and influence the threshold concentration required to induce complex coacervation (Figure 34e).
  • the phase behavior of the complexes could be fine-tuned by adjusting the cationic-to-aromatic peptide ratio, resulting in various phase behaviors ranging from no phase separation to complex coacervation and, ultimately, aggregation (Figure 34f). This ability to modulate the phase behavior highlights the versatility of this peptide family to form complex coacervates under varying conditions.
  • FIG. 34h shows time-lapse microscopy images of YP-K sp aggregates changing in size and texture, which evolved over time into coacervate microdroplets, whereas the aggregates remained intact in the absence of RP-K sp .
  • Structural analyses using attenuated total reflection-Fourier transformed infrared spectroscopy (ATR-FTIR) revealed that YP-K SP formed p-turn structures, characterized by the amide I peak centered at 1676 coacervate -1 .
  • the peptide density profile showed a single peak for both YP- K sp and RP-K sp /YP-K sp mixture, while multiple small peaks appeared for RP-K sp , corresponding to several small clusters ( Figures 35d-e).
  • water density decreased but did not drop to zero, indicating the presence of significant numbers of "internal" water molecules.
  • HeLa cells treated with EGFP-loaded RP-K sp /YP or RP-K sp /YP-K sp coacervates exhibited over 90% uptake, along with higher MFI compared to the best-performing RP/YP coacervates.
  • This excellent improvement surpassed our previous efforts using self-coacervating peptides with the same self-immolative modification, which only achieved substantial cargo release after 24 hours. 31
  • the accelerated release observed here may be attributed to both the chemical cleavage of the self-immolative modification and the responsiveness of cation-n interactions in the crowded cellular environment.
  • the complex coacervate system can also deliver mRNA, another macromolecular therapeutic that has gained significant attention, particularly for its role in the development of COVID-19 vaccines.
  • mRNA another macromolecular therapeutic that has gained significant attention, particularly for its role in the development of COVID-19 vaccines.
  • the RP-K sp /YP coacervates with varying mix ratios effectively transfected HeLa cells with a reporter mRNA encoding EGFP, demonstrating a similar pattern to protein delivery: transfection efficiency increased as the YP content decreased.
  • the coacervates achieved the highest transfection efficiency at an 8:1 ratio, successfully transfecting 95% of treated HeLa cells.
  • Macrophage cells are increasingly studied for their pivotal roles in disease-related bioactivities, such as immune response, cancer development, and inflammation. 59 ' 61
  • their intrinsic resistance to foreign material transfections poses a significant challenge in developing targeted therapeutics. 62
  • the RP-K SF 7YP coacervates demonstrated excellent mRNA transfection capabilities in RAW264.7 cells, particularly at an 8:1 mix ratio, achieving 83.4% efficiency, significantly higher than the 22.8% efficiency obtained using the highly optimized Lipofectamine MessengerMax reagent ( Figure 38d).
  • CRISPR clustered regularly interspaced short palindromic repeats
  • Cas9 clustered regularly interspaced short palindromic repeats
  • Cas9/Cas9 tools The delivery of CRISPR/Cas9 tools is considered challenging as it involves delivering two components: the Cas9 nuclease and single guided RNA (sgRNA). 64 The most common way to tackle this is to deliver an all-in-one plasmid that encodes both the sequence of Cas9 and sgRNA. 65
  • plasmid based editing tools are associated with risks of genome integration and off-target effects.
  • SIRPa signal-regulatory protein alpha
  • T7EI T7 Endonuclease I
  • the inventors developed a novel strategy for designing a complex coacervate-based intracellular delivery system using the HBpep family of peptides.
  • the inventors systematically investigated how these substitutions affect both self- and complex coacervation behaviors.
  • the findings indicate that a low density of oppositely charged residues is unable to establish stable electrostatic interactions, resulting in homogeneous solutions of KP and EP and failure to form complex coacervates.
  • the strong cation-rr interactions between RP and YP prevent RP dissolution and YP aggregation, instead promoting and stabilizing the formation of complex coacervates.
  • the cation-TT interactions are disrupted by the protein-rich cellular environment, serving as a trigger for coacervate disassembly and cargo release in cells.
  • the inventors engineered complex coacervates capable of responding to intracellular redox conditions, thereby enhancing the controlled release of cargos.
  • Adjusting the ratio of cationic to aromatic peptides allowed precise control over the phase behavior and release kinetics of cargos from the coacervates, facilitating the efficient delivery of a diverse range of macromolecular therapeutics, including proteins, mRNA, and CRISPR/Cas9 gene editing tools.
  • this approach proved effective at genetically editing macrophages, a cell type notoriously resistant to transfection, highlighting the versatility and adaptability of our peptide-based complex coacervate system for immune cell therapies.

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Abstract

La présente invention concerne un peptide isolé comprenant la séquence d'acides aminés de formule (I) : (O)n-K-(O)m-Z ; dans laquelle K est une lysine éventuellement modifiée par une fraction auto-immolable Z est le tryptophane ou est absent ; O est GHGX1Y (SEQ ID NO : 1) ; G est la glycine, H est l'histidine, Y est la tyrosine ; chaque X1 est indépendamment choisi parmi l'alanine, la glycine, la sérine, l'asparagine, l'histidine (H), l'arginine (R), l'acide aspartique, la tyrosine et la proline ; n est compris entre 0 et 5 ; m est compris entre 0 et 5 ; n + m est égal à 3, 4, 5, 6, 7 ou 8, et éventuellement, tous les résidus X1 ne sont pas la proline (P). Un tel peptide est un peptide formant un coacervat. La présente invention concerne également des procédés de préparation de compositions de coacervat comprenant le peptide isolé, comprenant éventuellement également une ou plusieurs biomacromolécules (charges utiles). La présente invention concerne en outre les compositions de coacervat en tant que telles, et leurs utilisations, par exemple dans des méthodes de traitement ou de diagnostic.
PCT/SG2025/050203 2024-03-24 2025-03-20 Compositions de coacervat Pending WO2025207027A1 (fr)

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