Multilayer nanoparticle

Abstract

The present invention provides a multilayer nanoparticle comprising: a core, a cationic polymer, and a anionic polymer which are layered one on top of the other, and a nucleic acid-cationic peptide complex. The invention further provides a pharmaceutical composition, including a vaccine composition, comprising the multilayer nanoparticles, uses thereof for administration to a subject and methods for preparing the multilayer nanoparticles.

Description

[0001] MULTILAYER NANOPARTICLE

[0002] FIELD

[0003] The present application broadly relates to the field of nucleic acid delivery technology and vaccine technology, and in particular relates to nanoparticle delivery systems and vaccines. More particularly, the present application provides multilayer nanoparticles, a pharmaceutical composition, including a vaccine composition, comprising the multilayer nanoparticles, uses thereof for administration to a subject in need thereof and methods for preparing the same.

[0004] BACKGROUND

[0005] Layer-by-layer (LbL) nanoparticles refer to nanoparticles that are constructed using the layer-by- layer assembly technique. LbL assembly involves the sequential deposition of alternating layers of different materials onto a template or a core, resulting in a multilayered structure. The particle is first coated with a layer of material, typically through electrostatic interactions. This layer can be positively or negatively charged. Subsequent layers are then added by alternately exposing the particle to solutions containing materials with opposite charges. For example, if the initial layer around the core is positively charged, the next layer would be negatively charged, followed by a layer with a positive charge, and so on. This layer-by-layer deposition process can be repeated multiple times, allowing for the creation of a multilayered structure. While LbL nanoparticles containing siRNA or mRNA within the core of the nanoparticle have shown promising transfection efficiency for siRNA and mRNA, certain challenges persist in their development. These challenges encompass inefficient release of the LbL nanoparticle from endosomes, inadequate release of siRNA and mRNA, and inefficient translation of mRNA. As a result, there is a continued demand in the field for innovative solutions that address the efficient release of LbL nanoparticles from the endosomes and their encapsulated siRNA and mRNA, along with efficient translation of the mRNA.

[0006] SUMMARY

[0007] The present invention is based on an innovative multilayer nanoparticle comprising a core, at least one cationic polymer, and at least one anionic polymer, sequentially layered upon each other. Additionally, the nanoparticle includes a nucleic acid-cationic peptide complex, all integrated into a single nanoparticle. In particular, as evidenced in the experimental section of this specification which documents certain illustrative embodiments of the present invention, multilayer nanoparticle provides a superior uptake and translation efficiency of the delivered nucleic acid, such as mRNA. Moreover, the multilayer nanoparticle exhibits the ability to elicit enhanced immune cell responses, especiallyCD8+T cell responses. The cationic peptides of the nucleic acid-cationic peptide complex possess adjuvant properties, further boosting the immunogenicity of the multilayer nanoparticle. The versatile properties of these nanoparticles allow for the incorporation of additional cargo, such as adjuvants or small molecules, between the adjacent layers or in the core of the multilayer structure. Furthermore, as evidenced in the experimental section, the multilayer nanoparticle demonstrates improved cellular uptake, endosomal escape, translation efficiency, and reduced toxicity. Consequently, pharmaceutical compositions, particularly vaccines, that incorporate these novel multilayer nanoparticles have promising potential in preventing disease and reducing disease severity, thereby enhancing the overall global health impact of vaccination strategies.

[0008] Multilayer nanoparticles may find applications in various fields, including drug delivery, biosensing, catalysis, and imaging. The multilayered structure can also serve as a platform for controlled drug release, allowing for precise control over the release rate and duration. By incorporating specific functional molecules within the layers, the multilayer nanoparticles can be engineered for targeted drug delivery or diagnostic purposes. The layering process also provides opportunities for fine-tuning the surface properties, stability, and biocompatibility of these multilayer nanoparticles.

[0009] In view of these advantages, an aspect of the invention provides a multilayer nanoparticle comprising:

[0010] - a core;

[0011] - at least one cationic polymer and at least one anionic polymer which are layered one on top of the other; and

[0012] - a nucleic acid-cationic peptide complex.

[0013] A related aspect provides a pharmaceutical composition comprising the multilayer nanoparticle as defined herein and a pharmaceutically acceptable carrier, diluent, or excipient, optionally wherein the pharmaceutical composition is a vaccine.

[0014] A further aspect provides multilayer nanoparticle as defined herein or the pharmaceutical composition as defined herein for use as a medicament, or for use in therapy.

[0015] A further aspect provides multilayer nanoparticle as defined herein or the pharmaceutical composition as defined herein, for use in a method for delivering the nucleic acid to a subject; or where the nucleic acid encodes a protein, for use in a method for delivering the protein to a subject; or where the nucleic acid encodes an antigen, for use in a method for inducing an immune response to the antigen in a subject.

[0016] A related aspect provides a multilayer nanoparticle or the pharmaceutical composition for use as defined herein, wherein the antigen is causative of or associated with a disease in the subject, and inducing the immune response to the antigen in the subject is beneficial for prevention or treatment of the disease.

[0017] Also provided is an in vitro method for introducing a nucleic acid into a cell, comprising exposing the cell to the multilayer nanoparticle or the pharmaceutical composition.

[0018] An additional aspect provides a method for preparing a multilayer nanoparticle said method comprising: a) coating a core with at least one cationic polymer and at least one anionic polymer, whereby the at least one cationic polymer and at least one anionic polymer are layered one on top of the other; and b) adding at least one nucleic acid-cationic peptide complex to the coated core.

[0019] These and further aspects and preferred embodiments of the invention are described in the following sections and in the appended claims. The subject-matter of the appended claims is hereby specifically incorporated in this specification.

[0020] BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1. Schematic representation of the modified layer-by-layer polyelectrolyte nanoparticle (nanoparticle -LI). mRNA was complexed to LAH4-L1 (LI) at indicated LI to mRNA ratio (15: 1 - pg: pg) and subsequently applied as an outer layer (a). Characterization of the modified layer- by-layer polyelectrolyte nanoparticle (Sub-Ll). Successful layer application was confirmed by size (b) and charge (c) measurements via Zetaview®-NTA (n = 2). SEM images demonstrate the Nanoparticle (d) and Nanoparticle-Ll (e) particles at 14920x and 25550x magnification, respectively.

[0022] Figure 2. The washing step did not affect the characteristics of nanoparticle -LI particles. To ensure that no residual unbound LLmRNA complexes are present, a comparison of respectively unwashed (NW), washed (W) and supernatant (SN) of the washed Sub-Ll was conducted and characterized. 5 pg of mRNA-fLuc was formulated in each condition. The size (a) and charge (b) was determined via Zetaview®-NTA. (n = 2). Mock = no mRNA

[0023] Figure 3. nanoparticle - LI showed an enhanced transfection efficiency compared to unmodified nanoparticle in DC2.4 cells (a). DC2.4 cells were transfected with 250 ng mRNA-eGFP per 35,000 at indicated Ll:mRNA (15: 1 - pg:pg) and Nanoparticle-Ll to cell (25: 1) ratio. LI was included as a reference transfection agent. Unwashed (NW), washed (W) and supernatant (SN) of washed Nanoparticle-Ll were also used as transfection agents to ensure that the enhanced eGFP expression was induced by the modification of the nanoparticle and not by unbound LI (b). eGFP expression was followed up for 72h using IncuCyte® at 4x magnification and defined as green object count (GOC) per image.

[0024] Figure 4. Concentration-dependent effect of chloroquine (CQ) and ammonium chloride (NH4CI) stimulation in DC2.4 cells upon transfection with nanoparticle -LI (NW). DC2.4 were treated for Ih with indicated concentrations of CQ (20 - 0.16 pM) and NH4CI (625 - 39 pM). Subsequently, 250ng of mRNA-GFP were transfected per 75,000 DC2.4 cells at 25: 1 nanoparticle -LI to cell ratio and 15 : 1 L 1 :mRNA (pg : pg) complexes . The next day, cells were harvested, and transfection kinetics were studied during a course of 72h by IncuCyte at 4x magnification. The quantification of eGFP expression was defined by the green object count (GOC) per image. The mean GOC are illustrated in the above graph.

[0025] Figure 5. Concentration-dependent effect of ammonium chloride (NH4CI) stimulation in DC2.4 cells. DC2.4 were treated for Ih with indicated concentrations of NH4CI (375 - 2.25 pM). Subsequently, 250 ng of mRNA-GFP were transfected per 75,000 DC2.4 cells at 25: 1 nanoparticle -LI particle-to-cell ratio and 15: 1 LLmRNA (pg:pg) complexes. The next day, cells were harvested, and transfection kinetics were studied during a course of 72h by IncuCyte at 4x magnification. The quantification of eGFP expression was defined by the green object count (GOC) per image. The mean GOC are illustrated in the above graph, (n = 2)

[0026] Figure 6. Endosomal stimulation with 5 pM NH4CI did not significantly enhance transfection in DC2.4 cells. Briefly, prior to transfection, DC2.4 cells were treated with 5 pM NH4CI for Ih. Subsequently, DC2.4 cells were transfected with 250 ng mRNA-eGFP per 200,000 cells at indicated LLmRNA (15: 1 - pg:pg) and Nanoparticle-Ll to cells (25: 1) ratio. The next day, cells were harvested, and read-out was performed by flow cytometry for percentage (a) and MicroFlow Imaging (MFI) (b) of eGFP. mRNA complexed to LI and mock condition (no mRNA) were respectively used as positive and negative controls, (n = 3, mean ± SD). Two-way ANOVA, Sidak’s multiple comparison test: ** p < 0.01, *** p < 0.001, ns — not significant.

[0027] Figure 7. Uptake and translation ofNanoparticle-Ll. Confocal images of DC2.4 cells transfected with 250 ng Cy5-labelled mRNA-eGFP at indicated LLmRNA (15: 1 - pg:pg) and Nanoparticle- Ll to cells (25: 1) ratio. The commercially available transfection agent, jetMESSENGER® was used as a positive control. Images (a) were acquired at 63X magnification following DAPI staining. Uptake and translation kinetics of LI and Nanoparticle-Ll (NW - W and SN) were studied during a course of 72h via Incucyte® at 4x magnification (b). Cy5+and eGFP+were expressed as ROC (c) and GOC (d) per image. Cy5+(uptake) and eGFP signals were measured via flow cytometry at 24h - 48h and 72h post-transfection. Percentages of Cy5+eGFP+population and MFI of eGFP signals (f).

[0028] Figure 8. Maturation of DC2.4 cells after transfection with LI- or Nanoparticle-Ll -formulated tOVA mRNA at indicated LLmRNA (15: 1 - pg:pg) and Nanoparticle-Ll to cell (25: 1) ratio. DC2.4 cells were left untreated or transfected with mRNA encoding tOVA (modified RNA), or without mRNA (MOCK). The next day, cells were harvested and stained for DC markers and the maturation markers CD86 and CD40. Percentage CD86 and CD40 expression within the CDllc+DCs (a); cells were treated with R848 as a positive control; positive control for transfection was performed with jetMESSENGER®; positive control for antigen presentation where DCs were pulsed with SIINFEKL. Overlay graphs for CD86 and CD40 expression (b); n=l

[0029] Figure 9. Antigen presentation and maturation of GM -DCs after transfection with LI or Nanoparticle-Ll -formulated tOVA mRNA at indicated LLmRNA (15: 1 - pg:pg) and Nanoparticle-Ll to cell (25: 1) ratio. On day 5, GM-DCs were transfected with 500 ng of modified mRNA encoding for tOVA or without mRNA (MOCK). The next day, cells were harvested and stained for SIINFEKL presentation in MHC-I (MHC-I S) and the maturation markers CD86 and CD40 within the CDllc+DCs. Percentage (a) and MFI (b) for SIINFEKL presented in MHC-I. (c); cells were treated with R848 as a positive control; positive control for transfection was performed with jetMESSENGER®; positive control for antigen presentation where DCs were pulsed with SIINFEKL. Overlay graphs for CD86 and CD40 expression (d) n =1

[0030] Figure 10. Immunization with the layer-by-layer polyelectrolyte submicroparticles combined with the histidine-rich peptide-mRNA formulation (Nanoparticle - LI) results in the proliferation of CD8+OT-I T cells. CD45.1 mice were injected with CTV-labelled CD45.2+CD8+ OT-I splenocytes, and 2 days later they were immunized with 10 pg of modified mRNA encoding tOVA formulated to either LI or Nanoparticle-Ll. After 3 days, the spleen and lymph nodes were harvested, and single-cell suspensions were analyzed by flow cytometry. Gating was performed on viable cells; subsequently, we looked for CTV dilution in the CD45.2+CD3+CD8+T-cell population (a). Percentage of CD3+CD8+T-cell proliferation (b).

[0031] Figure 11. Particle characterizations measured viaZetaView®-NTA. Size (a) and zetapotential ( - potential) (b) were determined for Nanoparticle-Ll in every storage condition (x-axis). For Nanoparticle-Ll conditions, the LI peptide was complexed with 1.3 pg fluc-mRNA. Negative controls (Nanoparticle) do not contain fLuc-mRNA. Each sample contained approximately 20 million particles and was diluted in 1 mL of RNAse-free water. All individual data points represent their corresponding weighted average (n=2, mean). Translation efficiency of dendritic cells transfected with Nanoparticle stored in different conditions. DC2.4 cells were transfected with 250 ng eGFP-mRNA in Nanoparticle-Ll . eGFP+ (c) percentage and MFI (d) was determined via flow cytometry 24-48h post-transfection (n=2, mean). RT, room temperature. FZ, Mr. Frosty™ Freezing Container

[0032] Figure 12. Characterization of the modified layer-by-layer polyelectrolyte nanoparticle (Sub-Ll, Nanoparticle-LIR and Nanoparticle-RALA). Successful layer application was confirmed by size (a) and charge (b) measurements via Zetaview®-NTA (n = 2).

[0033] Figure 13. Transfection of 250 ng mRNA encoding eGFP in DC2.4 cells formulated with nanoparticle-Ll, nanoparticle -L1R and nanoparticle-RALA. 75,000 DC2.4 cells were transfected with 250 ng of mRNA encoding eGFP formulated with LI or the modified nanoparticle variants at indicated LI (R) or RALA (15: 1 or 14.5: 1 - pg: pg) and nanoparticle to cell (25: 1) ratio. The next day, cells were harvested, and read-out was performed by flow cytometry for percentage (a) and MFI (b) of eGFP. mRNA eGFP complexed to LAH4-L1 (R) and RALA and mock condition (no mRNA) were respectively used as positive and negative controls, (n = 2).

[0034] Figure 14. Characterization and transfection of mRNA and self-amplifying (sa) RNA encoding eGFP in DC2.4 cells formulated with Nanoparticle-Ll. Successful complexation with LI was confirmed by size (a) and charge (b) measurements via Zetaview®-NTA (n = 2). 125 ng of (sa)- mRNA encoding eGFP were transfected in 200,000 DC2.4 cells at indicated LLmRNA (15: 1 - pg:pg) and Nanoparticle-Ll to cell (25: 1) ratio. The next day, cells were harvested, and read-out was performed by flow cytometry for viability (a) percentage (b) and MFI (c) of GFP Sa-RNA eGFP complexed to LAH4-L1 and mock condition (no (sa)-mRNA) were respectively used as positive and negative controls, (n = 1).

[0035] Figure 15. Transfection with Gag-mRNA in DC2.4 cells. DC2.4 cells were transfected with 500 ng of modified mRNA Gag formulated in LAH4-L1 and Nanoparticle-Ll at indicated LLmRNA (15: 1 - pg:pg) and Nanoparticle-Ll to cell (25: 1) ratio. The next day, cells were harvested and stained for gag presentation. Percentage (a) and MFI (b) for Gag. Mock controls were performed using conditions without mRNA. (n = 2)

[0036] Figure 16. Transfection of 500 ng mRNA and saRNA encoding li80.tOVAin 75,000 DC2.4 cells. saRNA and mRNA were formulated with nanoparticle-Ll and LI. DC2.4 cells were transfected with each formulation at indicated LI : (sa)mRNA (15: 1 pg:pg) and nanoparticle to cell (25: 1) ratio. The next day, cells were harvested, and read-out was performed by flow cytometry for percentage of SIINFEKL presentation in MHC-I (MHC-I S) (a) and MFI (b).

[0037] Condition without mRNA (mock) was used as a negative control (n = 4).

[0038] Figure 17. Transfection and maturation of human monocyte-derived dendritic cells was tested with LI- orNanoparticle-Ll (SubP-Ll )-formulated eGFP mRNA at indicated LI :mRNA (15: 1 - pg:pg) and nanoparticle -LI to cells (25: 1) ratio. Mo-DCs were left untreated or transfected with mRNA encoding eGFP, or without mRNA (MOCK) in complete or serum-reduced medium. The next day, cells were harvested and stained for viability, DC markers and the maturation markers CD80, CD86, CD40 and CD83 within the CD1 lc+CDlc+DC population. Cells were treated with R848 as a positive control for maturation. Percentage (a) and MFI (b) of eGFP+within the CDl lc+CDlc+DC population. Overlay graphs for CD86, CD40 and CD83 expression (c) (« = 3)

[0039] Figure 18. Comparison of conventional nanoparticle- LI ( PARG SubP-Ll, comprising PARG as cationic polymer layer)) with a multilayered nanoparticle (PLL SubP-Ll) formulated with poly- L-lysine (PLL) as an alternative cationic polymer layer. Size and charge measurements were conducted via Zetaview®- NTA. Size (A) and charge (B) measurements of the different LbL SubP variants.

[0040] (n = 7 for SubP PARG and n = 3 for SubP PLL).

[0041] Figure 19. Comparison of the transfection efficiency and antigen presentation of conventional nanoparticle -LI ( PARG SubP-Ll, comprising PARG as cationic polymer layer)) with PLL SubP- Ll formulated with poly-L-lysine (PLL) as a positive layer. DC2.4 cells were transfected with 250 ng mRNA encoding for eGFP (a) or li80.tOVA (b) formulated in PARG SubP-Ll or PLL SubP-Ll. The next day, cells were harvested and read-out was performed by flow cytometry for percentage and MFI of eGFP (a) and SIINFEKL presentation in MHC-I (b) (n = 3).

[0042] Figure 20. Comparison of the transfection efficiency of conventional nanoparticle -LI ( PARG SubP-Ll (PARG as cationic polymer layer)) with PLL SubP-Ll formulated with poly-L-lysine (PLL) as a positive layer. Human monocyte-derived dendritic cells (mo-DCs) were transfected with 250 ng mRNA encoding for eGFP formulated in PARG SubP-Ll or PLL SubP-Ll PLL in complete or serum -reduced medium (Opti-MEM™). The next day, cells were harvested, and readout was performed by flow cytometry for percentage (a) and MFI (b) of eGFP within the CDl lc+cDCl+population, (n = 3 for PARG SubP-Ll and n = 1 for PLL SubP-Ll)

[0043] Figure 21. Comparison of the characterization and transfection efficiency of mRNA formulated with LAH4-L1 (LI), poly-L-lysine (PLL), conventional nanoparticle-L 1 (SubP-Ll) and PLGA nanoparticles (PLGA NPs) (San Juan et al.). DC2.4 cells were transfected with 250 ng mRNA encoding eGFP (mRNA-eGFP) formulated with LI or SubP-Ll particles and 0.5, 1 and 2 pg of mRNA-eGFP formulated with PLL and PLGA NPs (a). The next day, cells were harvested and read-out was performed by flow cytometry for percentage and MFI eGFP (c). (n = 1)

[0044] Figure 22. Comparison of the transfection efficiency of pDNA formulated with LAH4-L1 (LI), nanoparticle -LI (SubP-Ll) and PLGA NPs. HEK 293T cells were transfected with 500 ng of pDNA encoding for eGFP formulated in LI, SubP-Ll or PLGANPs. Different ratios of pDNAto LI (pg: g) were used for LI and SubP-Ll. The next day, cells were harvested and read-out was performed by flow cytometry. Unformulated pDNA and polyethyleneimine (PEI) formulated pDNA were included as a negative and positive control for transfection respectively. Representative contour plots of HEK 293T cells transfected with unformulated pDNA and pDNA formulated with SubP-Ll (1: 15 ratio) and PEI (a). Percentage and MFI of eGFP acquired via flow cytometry (b) at 3 days post-transfection, (n = 3)

[0045] Figure 23. Comparison of the characterisation and transfection efficiency of conventional nanoparticle -LI (CaCOs SubP-Ll, comprising CaCO, cores) with nanoparticle-L 1 comprising PLGA-based cores (PLGA SubP-Ll). Schematic representation of the incorporation of PLGA as cores of the PLGA SubP-Ll (a). Size and charge measurements after each layer application, acquired with the Zetaview®-NTA. (b) DC2.4 cells were transfected with 500 ng mRNA encoding eGFP formulated with CaCO, SubP-Ll and its variant PLGA SubP-Ll containing PLGA cores. The next day, cells were harvested, and read-out was performed by flow cytometry for percentage and MFI eGFP. (c)

[0046] (« = 3)

[0047] Figure 24. Comparison of the transfection efficiency of LEmRNA and conventional nanoparticle-L 1 (SubP-Ll) and SubP-Ll containing PLL+as the outermost layer (SubP PLL+). SubP PLL+NPs were formulated as: CaCO, cores / DEXS / PARG / DEXS / PLL:mRNA, wherein PLL+was complexed to mRNA prior to addition to the DEXS layer. Poly-L-lysine mRNA complexes (PLL+) and LAH4-Ll:mRNA complexes (LLmRNA) were also tested without formulation into a nanoparticle. Schematic representation of the incorporation PLL+as the outermost layer of the LbL SubP formulation (a). DC2.4 cells were transfected with 0.5, 1 or 2 pg mRNA encoding eGFP formulated with classic SubP-Ll and its variant containing PLGA cores. The next day, cells were harvested, and read-out was performed by flow cytometry for percentage and MFI of eGFP (b) (n = 3).

[0048] Figure 25. Inflammasome activation in GM-DCs treated with LPS, LAH4-L1 (A) and SubP-Ll (B). GM-DCs were generated by culture with 20 ng / mL rm GM-CSF for 6 days. On day 6, GM- DCs were primed with 0.5 pg / mL LPS for 3h, followed by 1 h incubation with 10-25 pM necrosulfonamide (NSA), 0.01-1 pM Belnecasan (VX-769) or 0.01-1 pM MCC.950 before treatment with 15 pg / mLLAH4-Ll or nanoparticle-Ll (SubP-Ll) containing 15 pg / mLof LAH4- L1. The supernatant was harvested after 1 h and stored at -20 °C and analysed with ELISA for IL- ip. ( / ? = !)

[0049] Figure 26. Comparison of the transfection efficiency of nanoparticle-Ll (SubP-Ll) containing 2 or 3 polyelectrolyte layers. DC2.4 cells were transfected with 500 ng mRNA encoding eGFP formulated with nanoparticle-Ll (SubP-Ll). The next day, cells were harvested, and read-out was performed by flow cytometry for percentage (a) and MFI of eGFP (b) (n=2).

[0050] Figure 27. PrimeFlow® RNA assay of DC2.4 after transfection with Rhodamine B labelled SubP- Ll (RhB SubP-Ll). DC2.4 cells were transfected with 250 ng of mRNA encoding eGFP formulated in Rhodamine B-SubP-Ll. After 0,5, 1, 2, 4, 6, and 24 h, DC2.4 cells were harvested and stained with the fixable viability dye eFluor 450. Subsequently, cells were fixated and permeabilised. The next day, in situ hybridization was conducted using an eGFP-specific probe to target untranslated mRNA-eGFP present in the cytoplasm. Uptake (RhB+), and translation (eGFP+) efficiency and indication of the endosomal release of the particles (mRNA-eGFP) were acquired via flow cytometry. Representative contour plots of DC2.4 cells transfected with RhB SubP-Ll at 4h timepoint (a). Percentages and MFI of the uptake (RhB+), untranslated mRNA- eGFP and eGFP protein (eGFP+) (b).

[0051] DESCRIPTION OF EMBODIMENTS

[0052] As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise.

[0053] The terms “comprising”, “comprises” and “comprised of’ as used herein are synonymous with “having”, “have”, “including”, “includes”, “containing”, or “contains”, and are inclusive or open- ended and do not exclude additional, non-recited members, elements or method steps. The terms also encompass “constituted of’, “consists in”, “consisting of’, and “consists of’, and also the terms “consisting essentially of’, “consisting essentially in” and “consists essentially of’, which enjoy well-established meanings in patent terminology.

[0054] The recitation of numerical ranges by endpoints includes all integer numbers and, where appropriate, fractions subsumed within the respective ranges, as well as the recited endpoints. This applies to numerical ranges irrespective of whether they are introduced by the expression “from. . . to. . . ” or the expression “between. . . and. . . ” or another expression. Any numerical range recited herein is intended to include all sub-ranges subsumed therein.

[0055] The terms “about” or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of + / - 10 % or less, preferably + / - 5 % or less, more preferably + / - 1 % or less, and still more preferably + / - 0.1 % or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” or “approximately” refers is itself also specifically, and preferably, disclosed.

[0056] Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order, unless specified. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

[0057] Whereas the terms “one or more” or “at least one”, such as one or more members or at least one member of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any >3, >4, >5, >6 or >7 etc. of said members, and up to all said members. In another example, “one or more” or “at least one” may refer to 1, 2, 3, 4, 5, 6, 7 or more.

[0058] As used herein, the term “and / or” when used in a list of two or more items, means that any one of the listed items can be employed by itself or any combination of two or more of the listed items can be employed. For example, if a list is described as comprising group A, B, and / or C, the list can comprise A alone, B alone, C alone, A and B in combination, A and C in combination, B and C in combination, or A, B, and C in combination.

[0059] The discussion of the background to the invention herein is included to explain the context of the invention. This is not to be taken as an admission that any of the material referred to was published, known, or part of the common general knowledge in any country as of the priority date of any of the claims.

[0060] Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. All documents cited in the present specification are hereby incorporated by reference in their entirety. In particular, the teachings or sections of such documents herein specifically referred to are incorporated by reference. Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the invention. When specific terms are defined in connection with a particular aspect of the invention or a particular embodiment of the invention, such connotation or meaning is meant to apply throughout this specification, i.e., also in the context of other aspects or embodiments of the invention, unless otherwise defined.

[0061] In the following passages, different aspects or embodiments of the invention are defined in more detail. Each aspect or embodiment so defined may be combined with any other aspect(s) or embodiment(s) unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0062] Reference throughout this specification to “one embodiment”, “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the appended claims, any of the claimed embodiments can be used in any combination.

[0063] As corroborated by the experimental section, which illustrates certain representative embodiments of the present invention, the inventors provide advantageous applications of certain multilayer nanoparticles, pharmaceutical compositions, particularly vaccines, methods to manufacture these and their use in medicine. The present multilayer nanoparticle displays one or more advantages, the multilayer nanoparticle demonstrates improved cellular uptake, endosomal escape, translation efficiency, reduced toxicity, and exhibits the ability to elicit enhanced immune cell responses, especially CD8+T cell responses. The cationic peptides of the nucleic acid-cationic peptide complex possess adjuvant properties, further boosting the immunogenicity of the multilayer nanoparticle. The versatile properties of these nanoparticles allow for the incorporation of additional cargo, such as adjuvants or small molecules, between the adjacent layers or in the core of the multilayer structure.

[0064] An aspect of the invention thus provides a multilayer nanoparticle comprising:

[0065] - a core; and

[0066] - at least one cationic polymer and at least one anionic polymer which are layered one on top of the other; and

[0067] - a nucleic acid-cationic peptide complex.

[0068] As used herein, the term “multilayered nanoparticle” refers to a nanosized particle composed of different layers of cationic and anionic polymers as described herein. It is understood that the multilayered nanoparticle formulation may be influenced by, but not limited to, the selection of the different polymer components, the degree of charges of the polymers and peptides, the nature of the PEGylation, and the like.

[0069] As used herein, the term “cationic polymer” refers to a polymer comprising or consisting of a positively charged polyelectrolyte. Positively charged polyelectrolytes can comprise cationic groups including primary or secondary amines. They may be derived from natural or unnatural poly-amino acids wherein one or more monomer possesses a cationic moiety such as amino or guanidinium groups. Exemplary monomers of natural polyaminoacids include lysine, ornithine, guanidine or any combination thereof. Examples of positively charged polyelectrolytes include, but are not limited to, poly(cystamine bisacrylamide-co-4-amino-l -butanol (pABOL), trimethylammoniumbenzylchitosan (TMAB-CS) aliphatic polyamine polymers, such as polyethyleneimine and poly (allylamine), and cationic peptides such as, poly(lysine) (PLL) and poly(arginine) or a salt thereof. Preferably, the positively charged polyelectrolyte comprises poly- L-arginine.

[0070] As used herein, the term “anionic polymer” refers to a polymer comprising or consisting of a negative charged polyelectrolyte. Negative charged polyelectrolytes can comprise carboxyl groups such as carboxylic acid groups and carboxylate salt groups. For example, carboxylated polyelectrolytes can be a carboxyalkyl cellulose, such as a carboxymethyl cellulose or carboxy ethyl cellulose. Other carboxylated polyelectrolytes include anionic polysaccharides that are natural, synthetic, or semi-synthetic in origin. Exemplary negative charged polysaccharides include, but are not limited to, dextran sulphate (DEXS), hyaluronic acids, carboxymethyldextran, carboxyalkyl starches, alginic acids, carboxymethyl or butyl glucans, chitosans or a salt thereof. In certain embodiments, carboxylated polyelectrolytes can comprise polyacrylic acid, polymaleic acid, polyaspartic acid, any combination or salt thereof. Preferably, the negative charged polysaccharides comprises dextran sulphate (DEXS). In embodiments, the multilayer nanoparticle comprises:

[0071] - a core;

[0072] - at least one cationic polymer layer and at least one anionic polymer layer which are layered one on top of the other, wherein the outermost layer of the nanoparticle is an anionic polymer layer; and

[0073] - a nucleic acid-cationic peptide complex associated with the outermost anionic polymer layer, wherein the cationic peptide of the nucleic acid-cationic peptide complex is an amphipathic peptide.

[0074] In particular, the multilayer nanoparticle may be capable of facilitating the introduction of the nucleic acid comprised by the nucleic acid-cationic peptide complex into a cell. Preferably, the multilayer nanoparticle may be capable of facilitating the introduction of the nucleic acid comprised by the nucleic acid-cationic peptide complex into a cell, wherein the cationic peptide of the nucleic acid-cationic peptide complex is an amphipathic peptide.

[0075] Hence, the multilayer nanoparticle may enhance the delivery or uptake of nucleic acid into a cell, such that when a quantity of the nucleic acid, comprised by the multilayer nanoparticle is contacted with the cell, the uptake of the nucleic acid by the cell will be enhanced, increased, or more efficient (i.e., a greater proportion of the nucleic acid will be taken up by the cell) compared to when the same quantity of the same nucleic acid, not comprised by the multilayer nanoparticle (in particular free nucleic acid in solution), is contacted with a cell of the same cell type under otherwise the same conditions. In particular embodiments, the multilayer nanoparticle may enhance the delivery or uptake of nucleic acid into a cell, such that when a quantity of the nucleic acid, comprised by the multilayer nanoparticle is contacted with the cell, the uptake of the nucleic acid by the cell will be enhanced, increased, or more efficient (i.e., a greater proportion of the nucleic acid will be taken up by the cell) compared to when the same quantity of the same nucleic acid comprised by the nucleic acid-cationic peptide complex, is contacted with a cell of the same cell type under otherwise the same conditions. In a certain embodiment, at least one nucleic acidcationic peptide complex is associated with the anionic polymer, such as associated by means of an electrostatic interaction between the cationic peptide and the anionic polymer.

[0076] As used herein, the term “electrostatic interaction” involves the interaction between opposite electric charges, such as cationic (positive) and anionic (negative) charges.

[0077] In embodiments, the first layer surrounding the core of the nanoparticle can be a cationic polymer or an anionic polymer. For example, the first layer surrounding the core of the nanoparticle can either be a cationic polymer or an anionic polymer. For instance, if the initial layer is a cationic polymer, the subsequent layer will have the opposite charge, resulting in an anionic polymer layer being applied onto the cationic polymer layer. Subsequently, this anionic layer can be further covered with yet another layer having the opposite charge to the cationic polymer layer, and so forth until the multilayer nanoparticle contains a desired number of layers. In a preferred embodiment, the outermost layer of the nanoparticle comprises the anionic polymer.

[0078] In embodiments, the nanoparticle comprises two layers of polymers surrounding the core, wherein the multilayer nanoparticle comprises one layer of a cationic polymer and one layer of anionic polymer. For example, the initial layer surrounding the core is a cationic polymer, the subsequent layer applied onto the initial cationic polymer layer is an anionic polymer layer. For example, starting from the core towards the periphery of the nanoparticle, the order of the layers may be C- A, where “C” stands for cationic polymer and “A” stands for anionic polymer.

[0079] In embodiments, the nanoparticle comprises three layers of polymers surrounding the core, wherein the multilayer nanoparticle comprises one layer of a cationic polymer and two layers of anionic polymer. For example, the initial layer surrounding the core is an anionic polymer, the subsequent layer applied onto the initial anionic polymer layer is a cationic polymer layer, and a further anionic polymer layer is applied onto the cationic polymer layer. For example, starting from the core towards the periphery of the nanoparticle, the order of the layers may be A-C-A, where “C” stands for cationic polymer and “A” stands for anionic polymer.

[0080] In embodiments, the nanoparticle comprises four layers of polymers surrounding the core, wherein the multilayer nanoparticle comprises two layers of a cationic polymer and two layers of anionic polymer. For example, starting from the core towards the periphery of the particle, the order of the layers may be C-A-C-A or A-C-A-C, where “C” stands for cationic polymer and “A” stands for anionic polymer.

[0081] In embodiments, the nanoparticle comprises five layers of polymers surrounding the core, wherein the multilayer nanoparticle comprises two layers of a cationic polymer and three layers of anionic polymer or two layers of a anionic polymer and three layers of cationic polymer. For example, starting from the core towards the periphery of the particle, the order of the layers may be C-A-C-A-C or A-C-A-C-A, where “C” stands for cationic polymer and “A” stands for anionic polymer.

[0082] As used herein, the term "outermost layer" refers to the surface layer of a nanoparticle that can comprise a cationic or an anionic polymer. The outermost layer is typically positioned on the exterior of the multilayer nanoparticle. The outermost layer of the multilayer nanoparticle can be directly exposed to the external surroundings. In a certain embodiment, at least one nucleic acid-cationic peptide complex is associated with the nanoparticle, particularly with at least one anionic polymer layer of the nanoparticle, such as with one or two anionic polymer layers of the nanoparticle. In a preferred embodiment, the outermost layer comprises the anionic polymer and the nucleic acid-cationic peptide complex is associated with the anionic polymer of the outermost layer, preferably at least one nucleic acid-cationic peptide complex is disposed on the outer surface of the outermost anionic polymer layer.

[0083] In embodiments, several types of cores can be utilized in various applications. A variety of cores with different shapes, sizes and / or materials can be coated with one or more layers of polymers as disclosed herein. In a certain embodiment, the core can be solid or hollow or porous. These cores may exhibit diverse shapes, such as, but not limited to, spheres, spheroids, cylinders, ellipsoids, cubes, cuboids, or cones. In certain embodiments, the core can adopt various geometries or symmetries, including planar, circular, rounded, tubular, hexagonal, octagonal, and other regular or irregular geometries. In some embodiments, a collection of cores have an average greatest dimension (e.g. diameter) of less than 900 nanometers (nm). In some embodiments, a collection of particle cores have a greatest dimension (e.g. diameter) of about at least 800 nm, about at least 700 nm, about at least 600 nm, about at least 500 nm, about at least 400 nm, about at least 300 nm, about at least 250 nm, about at least 200 nm, about at least 150 nm, about at least 100 nm, about at least 90 nm, about at least 80 nm, about at least 70 nm, about at least 60 nm, about at least 50 nm, about at least 40 nm, about at least 30 nm, about at least 20 nm, about at least 10 nm, or about at least 5 nm. In some embodiments, a collection of particle cores have a greatest dimension of less than or more than about 400 nm. In some embodiments, a collection of particle cores have a greatest dimension in between of any two values above. In some embodiments, a greatest dimension is a hydrodynamic diameter. For the purpose of achieving desirable characteristics and consistent performance, it is advantageous to employ a collection of cores that demonstrate uniformity in terms of size, shape, and composition. In such instances, each core will exhibit similar properties. In certain embodiments, a collection of cores with homogeneity in diameters (for example hydrodynamic diameters) is employed. The term "homogeneity in diameters" denotes a core collection wherein at least about 80 %, at least about 90 %, or at least about 95 % (by number) of the cores possess a diameter (or maximum dimension) falling within 5 %, 10 %, or 20 % of the average diameter (or average maximum dimension). The average diameter of the core collection with homogeneity in diameters may fall within the aforementioned ranges. To further enhance uniformity, the cores may have a polydispersity index of less than 0.3, less than 0.25, less than 0.2, less than 0.15, less than 0.1, less than 0.05, less than 0.01, or less than 0.005. For instance, the polydispersity index of the cores used in this invention may range from about 0.005 to 0.3, about 0.005 to 0.2, about 0.01 to 0.15, or about 0.05 to 0.1. In some embodiments, the polydispersity index of the cores can range between of any two values above. It is important to note that particle cores exhibiting homogeneity, particularly concerning size, are expected to offer increased repeatability and higher accuracy for various applications. In some instances, a collection of cores may be deliberately designed to be heterogeneous with regard to size, shape, and composition.

[0084] As used herein, the term “polydispersity index” (“PDI)” generally refers to a numerical measure used to quantify the distribution of core sizes within a collection of cores. The PDI indicates the uniformity or heterogeneity of core sizes within a given collection of cores. The PDI can be calculated with the following formula: PDI= Dw / Dn, wherein Dwrepresents the weight-average core diameter and Dnrepresents the number-average core diameter. For example, a lower PDI indicates a more narrow and consistent distribution of cores sizes, suggesting that the cores in the collection are relatively similar in size. On the other hand, a higher PDI indicates a broader range of cores sizes, implying a greater degree of heterogeneity within the collection.

[0085] In a certain embodiment, the core can be an organic or inorganic core. Suitable cores can be biocompatible and / or biodegradable. In certain embodiments, inorganic cores include, but are not limited to, calcium salts, carbonate salts, plastics, ceramics, silicon, metals {e.g., gold, silver, iron, cobalt, zinc, cadmium, nickel, gadolinium, chromium, copper, manganese, palladium, tin, and alloys thereof), metal-coated materials, metal oxides, and combinations thereof. In a certain embodiment, the core contains CaCO, preferably the core consists of CaCOs. In certain embodiments, the organic cores can be composed of or contain polymers, such as polypeptides, and / or polysaccharides. Additionally, synthetic derivatives of natural polymers may also serve as constituents of polymeric cores. For instance, the polymeric cores may comprise a variety of polymers, including but not limited to poly(arylates), poly(anhydrides), poly(hydroxy acids), polyesters, poly(ortho esters), poly(alkylene oxides), polycarbonates, polypropylene fumarates), poly(caprolactones), polyamides, polyamino acids, polyacetals, polylactides, polyglycolides, poly(dioxanones), polyhydroxybutyrate, polyhydroxyvalerate, poly(vinyl pyrrolidone), polycyanoacrylates, polyurethanes, and polysaccharides.

[0086] In a certain embodiment, nucleic acid of the nucleic acid-cationic peptide complex is a deoxyribonucleic acid (DNA) molecule or an ribonucleic acid (RNA) molecule or a hybrid RNA / DNA molecule, such as an RNA molecule selected from the group consisting of messenger RNA (mRNA), self-amplifying RNA (saRNA), short interfering RNA (siRNA), genomic RNA (gRNA), non-coding RNA, small non-coding RNA, micro RNA (miRNA), circular RNA, transamplifying RNA (taRNA), a synthetic analogue of RNA, and combinations thereof. As used herein, the term “self-amplifying RNA” or “self-replicating” RNA generally refers to a synthetic RNA molecule engineered with the dual purpose of encoding a target antigen — typically a protein of interest, and carrying the machinery for autonomous replication, preferably derived from an Alphavirus including Venezuelan equine encephalitis virus (VEEV), Semliki Forest virus (SFV), and Sindbis virus, within cells in a subject. SaRNA is able to generate multiple copies of itself and the target antigen. By doing so, saRNA can trigger an immune response in the body of a subject. The saRNA structure typically includes a region for encoding the target antigen, replicase machinery (such as RNA polymerase and helicase), and regulatory elements that collectively enable efficient replication and protein expression. The helicase is an enzyme responsible for unwinding the saRNA's double -stranded regions during replication. By breaking the hydrogen bonds between the base pairs, helicase creates a single -stranded template that can be used for RNA synthesis. Examples of helicases include, but are not limited to, HIV-1 Tat Helicase, E. coli Replicative Helicase (DnaB), Human DNA Helicase (RecQ), Hepatitis C Virus NS3 Helicase, Flavivirus NS3 Helicase, SARS-CoV-2 Helicase (nspl3), the RNA polymerase is an enzyme enables synthesis of a complementary RNA strand using the single -stranded saRNA as a template. The polymerase enables production of a new RNA molecule that can then serve as a template for further replication. Regulatory elements of saRNA can include untranslated regions (UTRs), internal ribosome entry sites (IRES), Poly(A) tail, internal regulatory sequences, splice sites, kozak sequence and / or promoter sequences.

[0087] The term “nucleic acid” as used throughout this specification typically refers to a polymer (preferably a linear polymer) of any length composed essentially of nucleoside units. A nucleoside unit commonly includes a heterocyclic base and a sugar group. Heterocyclic bases may include inter alia purine and pyrimidine bases such as adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U) which are widespread in naturally-occurring nucleic acids, other naturally- occurring bases (e.g., xanthine, inosine, hypoxanthine) as well as chemically or biochemically modified (e.g., methylated), non-natural or derivatized bases. Exemplary modified nucleobases include without limitation 5 -substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5- propynyluracil and 5-propynylcytosine. Nucleoside units may be linked to one another by any one of numerous known inter-nucleoside linkages, including inter alia phosphodiester linkages common in naturally-occurring nucleic acids, and further modified phosphate- or phosphonate-based linkages such as phosphorothioate, alkyl phosphorothioate such as methyl phosphorothioate, phosphorodithioate, alkylphosphonate such as methylphosphonate, alkylphosphonothioate, phosphotriester such as alkylphosphotriester, phosphoramidate, phosphoropiperazidate, phosphoromorpholidate, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphorothioate; and further siloxane, carbonate, sulfamate, carboalkoxy, acetamidate, carbamate such as 3’-N-carbamate, morpholino, borano, thioether, 3 ’-thioacetal, and sulfone intemucleoside linkages. Preferably, inter-nucleoside linkages may be phosphate-based linkages including modified phosphate-based linkages, such as more preferably phosphodiester, phosphorothioate or phosphorodithioate linkages or combinations thereof. The term “nucleic acid” also encompasses any other nucleobase containing polymers such as nucleic acid mimetics, including, without limitation, peptide nucleic acids (PNA), peptide nucleic acids with phosphate groups (PHONA), locked nucleic acids (LNA), morpholino phosphorodiamidate -backbone nucleic acids (PMO), cyclohexene nucleic acids (CeNA), tricyclo-DNA (tcDNA), and nucleic acids having backbone sections with alkyl linkers or amino linkers (see, e.g., Kurreck 2003 (Eur J Biochem 270: 1628-1644)). “Alkyl” as used herein particularly encompasses lower hydrocarbon moieties, e.g., C1-C4 linear or branched, saturated or unsaturated hydrocarbon, such as methyl, ethyl, ethenyl, propyl, 1 -propenyl, 2- propenyl, and isopropyl. Nucleic acids as intended herein may include naturally occurring nucleosides, modified nucleosides or mixtures thereof. A modified nucleoside may include a modified heterocyclic base, a modified sugar moiety, a modified inter-nucleoside linkage or a combination thereof. A nucleic acid can be naturally occurring, e.g., present in or isolated from nature, can be recombinant, i.e., produced by recombinant DNA technology, and / or can be, partly or entirely, chemically or biochemically synthesised. A “nucleic acid” can be double-stranded, partly double stranded, or single -stranded. Where single-stranded, the nucleic acid can be the sense strand or the antisense strand. In addition, nucleic acid can be circular or linear.

[0088] As used herein, the term “nucleic acid-cationic peptide complex” refers to nucleic acid having been complexed to cationic peptide by an electrostatic interaction between the cationic peptide and the negatively charged nucleic acid(s). In certain embodiments, the cationic peptide comprises or consists of one or more amino acids which display a positive charge at physiological pH (such as pH about 7.4), such as histidine, lysine and / or arginine, and optionally one or more amino acids which are not charged at physiological pH, such as preferably one or more apolar amino acids, such as valine, leucine, isoleucine, cysteine, and / or methionine, preferably leucine and / or alanine. In certain embodiments, the cationic peptide comprises no amino acids which are negatively charged at physiological pH, such as aspartic acid or glutamic acid, or comprises a limited number, such as only 1, 2 or 3, negatively charged amino acids, such that the overall charge of the peptide is positive. In certain embodiments, the cationic peptide comprises or consists of one or more histidine, lysine and / or arginine and one or more apolar amino residues such as leucine and / or alanine. The cationic peptide can comprise amphipathic peptides including, but are not limited to histidine-rich amphipathic peptides such as LAH4-L1 SEQ ID NO: 1 (KKALLAHALHLLALLALHLAHALKKA) or a known functional derivative thereof or arginine-rich amphipathic peptides such as LAH4-L1R SEQ ID N0:2 (RRALLAHALHLLALLALHLAHALRRA) or a known functional derivative thereof or RALA SEQ ID NO: 3 (WEARLARALARALARHLARALARALRACEA) or a known functional derivative thereof. RALA is also known to be a derivative of GALA SEQ ID NO: 4 (WEAALAEALAEALAEHLAEALAEALEALAA) In a preferred embodiment, the cationic peptide of the nucleic acid-cationic peptide complex is an arginine-rich amphipathic peptide such as LAH4-L1R .

[0089] Derivates of LAH4-L1 and LAH-L1R include, but are not limited to,:

[0090] LAH4: KKALLALALHHLAHLALHLALALKKA (SEQ ID NO: 5)

[0091] LAH4-L2: KKALLALALHHLALLALHLAHALKKA (SEQ ID NO:6)

[0092] LAH4-L3: KKALLALALHHLALLAHHLALALKKA (SEQ ID NO:7)

[0093] LAH4-L4iso: KKALLHLALLHAALLAHHLALALKKA (SEQ ID NO: 8)

[0094] LAH4-L5: KKALLHLALLHAALLAHLAALHLKKA (SEQ ID NO:9)

[0095] L AH4-L6iso : KKALLHLALLLAALHAHLAALHLKKA (SEQ ID NO : 10)

[0096] LAH4-A1 : KKALLAHALHLLAALALHLAHLLKKA (SEQ ID NO: 11)

[0097] LAH4-A2: KKALLLAALHHLAALALHLAHLLKKA (SEQ ID NO: 12)

[0098] LAH4-A3: KKALLLAALHHLLALAHHLAALLKKA (SEQ ID NO: 13)

[0099] LAH4-A4: KKALLHAALAHLLALAHHLLALLKKA (SEQ ID NO: 14)

[0100] LAH4-A5 : KKALLHALLAHLAALLHALLAHLKKA (SEQ ID NO : 15)

[0101] LAH4-A6iso: KKALLHALLAALLAHLHALLAHLKKA (SEQ ID NO: 16)

[0102] LAH4-A4-K1N : KALLHAALAHLLALAHHLLALLKKA (SEQ ID NO: 17)

[0103] LAH4-A4-K3N : KKKLLHAALAHLLALAHHLLALLKKA (SEQ ID NO: 18)

[0104] LAH4-A4-dKC : KKALLHAALAHLLALAHHLLALLA (SEQ ID NO: 19)

[0105] LAH4-A4-dlaa : KKALLHAALAHLLALAHHLLALLK (SEQ ID NO: 20)

[0106] LAH4-A4-d2aa : KKLLHAALAHLLALAHHLLALLK (SEQ ID NO:21)

[0107] LAH4-A4-d2Caa : KKALLHAALAHLLALAHHLLALK (SEQ ID NO:22)

[0108] LAH4-A4-d3aa : KKLHAALAHLLALAHHLLALLK (SEQ ID NO:23)

[0109] LAH4-A4-d5aa : KKLHAALAHLLALAHHLLAK (SEQ ID NO:24) LAH2-A6 : KKALLHAALAHLLALAAALLALLKKA (SEQ ID NO: 25)

[0110] K2-L10A12-K2 : KKALLAAALAALLALAAALLALLKKA (SEQ ID NO: 26)

[0111] LAH4-A4-Leu: KKLLLHALLAHLLALLHHLLALLKKL (SEQ ID NO:27).

[0112] LAH4-Ll-dKC: KKALLAHALHLLALLALHLAHALA (SEQ ID NO:28)

[0113] LAH4-L0: KKALLAHALAHLALLALHLALHLKKA (SEQ ID NO:29)

[0114] As used herein, the term “amphipathic peptides” refer to peptides comprising hydrophilic residues and hydrophobic residues within the same molecule. Hydrophobic residues can comprise, but are not limited to, leucine and alanine residues. Hydrophilic residues can comprise, but are not limited, histidine and arginine. In certain embodiments, the amphipathic peptides, in particular cationic amphipathic peptides, may comprise positively charged amino acids such as histidine, arginine, and / or lysine, and apolar amino acids such as leucine and / or alanine at a ratio between 7: 1 and 1:7 (by number), such as about 6: 1, about 5: 1, about 4: 1, about 3: 1, about 2: 1, about 1: 1, about 1:2, about 1:3, about 1:4, about 1:5 or about 1:6 positively charged : apolar, such as preferably about 1 : 2 positively charged : apolar. In certain embodiments, the amphipathic peptides may comprise histidine, arginine, and / or lysine, and further comprise leucine, alanine, cysteine, and / or tryptophan. Preferably, alanine may be more abundant than histidine, e.g., a ratio between 12: 1 or 3:2 or 2: 1 alanine : histidine (by number), preferably at a ratio of 2: 1 alanine : histidine (by number). In certain embodiments, the amphipathic peptides may comprise histidine, leucine, alanine, and lysine residues of which histidine and lysine residues are present at a ratio between 7: 1 and 1:7 (by number), such as about 6: 1, about 5: 1, about 4: 1, about 3: 1, about 2: 1, about 1: 1, about 1:2, about 1:3, about 1:4, about 1:5 or about 1:6 histidine: lysine. Preferably, at a ratio of 1: 1 histidine: lysine (by number). In certain embodiments, the amphipathic peptides may comprise histidine, leucine, alanine, and lysine residues of which alanine and lysine residues are present at a ratio between 7: 1 and 1:7 (by number), such as about 6: 1, about 5: 1, about 4: 1, about 3: 1, about 2: 1, about 1: 1, about 1:2, about 1:3, about 1:4, about 1:5 or about 1:6 alanine: lysine. Preferably, at a ratio of 2: 1 alanine: lysine (by number). In certain embodiments, the amphipathic peptides may comprise histidine, leucine, alanine, and lysine residues of which alanine and leucine residues are present at a ratio between 7:6 and 6:7 (by number), such as about 6:5, about 5:4, about 4:3, about 3:2, about 2: 1, about 1: 1, about 1:2, about 2:3, about 3:4, about 4:5 or about 5:6 alanine: leucine. Preferably, at a ratio of 4:5 alanine: leucine (by number). In certain embodiments, the amphipathic peptides may comprise histidine, leucine, alanine, and lysine residues of which histidine and leucine residues are present at a ratio between 7:6 and 6:7 (by number), such as about 6:5, about 5:4, about 4:3, about 3:2, about 2: 1, about 1: 1, about 1:2, about 2:3, about 3:4, about 4:5 or about 5:6 histidine: leucine. Preferably, at a ratio of 4:5 histidine: leucine (by number).

[0115] In certain embodiments, the amphipathic peptides may comprise histidine, leucine, alanine, and arginine residues of which histidine and arginine residues are present at a ratio between 7: 1 and 1:7 (by number), such as about 6: 1, about 5: 1, about 4: 1, about 3: 1, about 2: 1, about 1: 1, about 1:2, about 1:3, about 1:4, about 1:5 or about 1:6 histidine: arginine. Preferably, at a ratio of 1: 1 histidine: arginine (by number). In certain embodiments, the amphipathic peptides may comprise histidine, leucine, alanine, and arginine residues of which alanine and arginine residues are present at a ratio between 7: 1 and 1:7 (by number), such as about 6: 1, about 5: 1, about 4: 1, about 3: 1, about 2: 1, about 1: 1, about 1:2, about 1:3, about 1:4, about 1:5 or about 1:6 alanine: arginine. Preferably, at a ratio of 2: 1 alanine: arginine (by number). In certain embodiments, the amphipathic peptides may comprise histidine, leucine, alanine, and arginine residues of which alanine and leucine residues are present at a ratio between 7:6 and 6:7 (by number), such as about 6:5, about 5:4, about 4:3, about 3:2, about 2: 1, about 1: 1, about 1:2, about 2:3, about 3:4, about 4:5 or about 5:6 alanine: leucine. Preferably, at a ratio of 4:5 alanine: leucine (by number). In certain embodiments, the amphipathic peptides may comprise histidine, leucine, alanine, and lysine residues of which histidine and leucine residues are present at a ratio between 7:6 and 6:7 (by number), such as about 6:5, about 5:4, about 4:3, about 3:2, about 2: 1, about 1: 1, about 1:2, about 2:3, about 3:4, about 4:5 or about 5:6 histidine: leucine. Preferably, at a ratio of 4:5 histidine: leucine (by number).

[0116] In certain embodiments, the amphipathic peptides may comprise or consist of histidine, leucine, alanine, and lysine residues. In such embodiments, histidine and leucine residues may be present at a number ratio of 4:5 histidine: leucine and / or at a number ratio of 1 : 1 histidine: alanine and / or at a number ratio of 4:5 histidine: leucine and / or at a number ratio of 1 : 1 histidine: lysine. In such embodiments, the amphipathic peptides may comprise or consist of about 5% to 20% (by number or count), about 6% to 19%, about 7% to 18%, about 8% to 18%, about 9% to 18%, about 10% to 18%, about ll%to 18%, about 12% to 18%, about 13%to 17%, about 14% to 16% of histidine of the overall length of the amphipathic peptides and / or about 25% to 35%% (by number or count), about 26% to 34%, about 27% to 33%, about 28% to 32%, about 29% to 31% of alanine of the overall length of the amphipathic peptides, and / or about 27% to 42%% (by number or count), about 28% to 41%, about 29% to 40%, about 30% to 39% of leucine of the overall length of the amphipathic peptides, and / or about 10% to 20%% (by number or count), about 11% to 19%, about 12% to 18%, about 13% to 17%, about 14% to 16% of lysine of the overall length of the amphipathic peptides. In certain particularly preferred embodiments, the amphipathic peptides may comprise, consist essentially of or consists of an amino acid sequence having a sequence identity of at least 90% to LAH4-L1 SEQ ID NO: 1 (KKALLAHALHLLALLALHLAHALKKA) or a known functional derivative thereof and / or to LAH4-L1R SEQ ID NO:2 (RRALLAHALHLLALLALHLAHALRRA) or a known functional derivative thereof.

[0117] In certain embodiments, the amphipathic peptides may comprise, consist essentially of or consists of an amino acid sequence having a sequence identity of preferably at least 91%, at least 92%, at least 93% or at least 94% to SEQ ID NO: 1, more preferably a sequence identity of at least 95%, such as at least 96%, at least 97%, at least 98% or at least 99% to SEQ ID NO: 1. In certain embodiments, the amphipathic peptides may comprise, consist essentially of or consists of an amino acid sequence having a sequence identity of preferably at least 91%, at least 92%, at least 93% or at least 94% to SEQ ID NO: 2, more preferably a sequence identity of at least 95%, such as at least 96%, at least 97%, at least 98% or at least 99% to SEQ ID NO: 2.

[0118] Where an amino acid sequence differs, varies or diverges from a certain amino acid sequence - for example, where the former amino acid sequence is said to display a certain degree or percentage of sequence identity to the latter amino acid sequence, or where the former amino acid sequence is said to differ by a certain number of amino acids, such as at most 5, at most 4, at most 3, at most 2, or at most 1 single amino acid, from the latter amino acid sequence - such sequence variation may be constituted by one or more amino acid additions (e.g., a single amino acid or a stretch of two or more contiguous amino acids may be added at one position of an amino acid sequence or each independently at two or more positions of an amino acid sequence), deletions (e.g., a single amino acid or a stretch of two or more contiguous amino acids may be deleted at one position of an amino acid sequence or each independently at two or more positions of an amino acid sequence), and / or or substitutions (e.g., a single amino acid or a stretch of two or more contiguous amino acids may substitute a single one or a stretch of two or more contiguous amino acids at one position of an amino acid sequence or each independently at two or more positions of an amino acid sequence). Preferably, the one or more amino acid substitutions and / or additions, in particular one or more single amino acid substitutions and / or additions, may be hydrophilic and / or positively charged amino acid substitutions and / or additions.

[0119] In certain embodiments, C-terminal residue of the amphipathic peptides is a alanine residue; or the N-terminal and C-terminal residues of the amphipathic peptides are both alanine residues. The N-terminal residue of the amphipathic peptides is a lysine residue or a arginine residue. In certain, embodiments, the overall charge of the cationic peptides at physiological pH may be between +1 and +20 mV, such as between +2 and +18 mV, between +3 and +15 mV, between +4 and +12 mV, between +5 and +10 mV, such as, for example +6, +7, +8, or +9 mV.

[0120] In certain embodiments, the overall length of the cationic peptides, such as the amphipathic peptides, may be between 5 and 100 amino acids, such as preferably between 10 and 50 amino acids or between 10 and 40 amino acids, more preferably between 15 and 35 amino acids, such as between 20 and 30 amino acids, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids, preferably the overall length of the amphipathic peptides is 26 amino acids.

[0121] In certain embodiments, the nucleic acid-cationic peptide complex comprises a cationic peptide and a nucleic acid, preferably mRNA, of which the cationic peptide and the nucleic acid are present at a ratio between 1: 1 and 20: 1 (by micrograms), such as about 20: 1, about 15: 1, about 10: 1, about 9: 1, about 8: 1, about 7: 1, about 6: 1 about 5: 1, about 4: 1, about 3: 1, about 2: 1, about 1 : 1 cationic peptide : nucleic acid. Preferably, at a ratio of 10: 1 cationic peptide : nucleic acid, (by number).

[0122] In a certain embodiment, the nucleic acid-cationic peptide complex is conjugated to or associated with one or more layers of the multilayer nanoparticle, such as to the outermost layer of the multilayer particle, via electrostatic interaction or via covalent bond, optionally by means of a linker such as a cleavable linker.

[0123] In a certain embodiment, a hydrophilic, non-immunogenic polymer moiety is conjugated to the nucleic acid-cationic peptide complex and / or to one or more layers of the multilayer nanoparticle, such as to the outermost layer of the multilayer particle.

[0124] Non-limiting examples of suitable hydrophilic, non-immunogenic polymers include, for example, homopolymers selected from the group consisting of poly(alkyl-oxazoline), polysaccharide, poly(N-vinylpyrrolidone), poly(sarcosine), polyvinyl alcohol (PVA), poly(acrylic acid), poly (methacrylic acid), poly (acrylamide) and polyethylene glycol (PEG). Alternatively, or in addition, the hydrophilic, non-immunogenic polymer can be selected from heteropolymers comprising at least one monomeric subunit from two or more of the polymers selected from the group consisting of poly(alkyl-oxazoline), polysaccharide, poly(N -vinylpyrrolidone), poly(sarcosine), polyvinyl alcohol (PVA), poly(acrylic acid), poly(methacrylic acid), poly(acrylamide) and polyethylene glycol (PEG). The polymer moiety preferably is a polyethylene glycol (PEG) modified polymer.

[0125] A PEG modified polymer generally refers to polymers having one or more PEG chains covalently attached. PEGs are polymers that are non-ionic, non-toxic, biocompatible and highly hydrophilic. Non-limiting examples of suitable PEGylated polymers for conjugation to the nucleic acidcationic peptide complex, include, without limitation, PEGylated dialkylamines, PEGylated phosphatidylethanolamines, PEGylated phosphatidic acids, PEGylated ceramides, PEGylated diacylglycerols, PEGylated dialkylglycerols, and mixtures thereof. Preferably, the cationic peptide of the nucleic acid-cationic peptide complex is PEGylated.

[0126] In a certain embodiment, the multilayer nanoparticle comprises an adjuvant, such as an adjuvant selected from a group consisting of atoll-like receptor (TLR)- agonist, RIG-I like receptor (RLR)- agonist, Nod-like receptor protein (NLRP)- agonist, iNKT-agonist or derivates thereof, bacterial lipopolysaccharide or derivates thereof, synthetic polynucleotides, and combinations thereof.

[0127] As used herein, the term “adjuvant” refers to any agent with immunostimulatory properties that can be used to stimulate the immune system to increase the response to a concomitantly administered antigen / immunogen. There are many suitable adjuvants, for example but not limited to, stimulators of pattern recognition receptors, such as Toll-like receptors, RIG-1 and Nod-like receptor protein (NLRP), iNKT agonist or derivates thereof, stimulator of interferon genes (STING), mineral salts, such as alum, monophosphoryl lipid (MPL) A of Enterobacteria, such as Escherichia coli, Salmonella minnesota, Salmonella iyphimurium. or Shigella flexneri, Matrix M, alpha galactosylceramide, polyphosphazene, MF59, Poly(lactic-co-glycolic acid) (PLGA), PLA, amino alkyl glucosaminidine phosphates, Glucopyranosyl lipid A (GLA), Poly (EC), CpG, IC31, liposomes and liposomal formulations such as AS01, ASO4 / A1(OH)3, cyclic dinucleotide, amidobenzimidazoles, synthesized or specifically prepared microparticles and microcarriers such as bacteria-derived outer membrane vesicles (OMV) of N. gonorrheae, Chlamydia trachomatis and others, or chitosan particles, depot-forming agents, such as Pluronic® block co-polymers, synthetic polynucleotides, such as muramyl dipeptide, aminoalkyl glucosaminide 4-phosphates, such as RC529, or proteins, such as bacterial lipopolysaccharide or derivates thereof, including toxoids or toxin fragments.

[0128] In preferred embodiments, the adjuvant comprises one or more toll-like receptor (TLR) agonists. In particular, the adjuvant may comprise one or more agonists for TLRs 1,2, 3, 4, 5, 7, 8, 9 or any combination thereof, preferably a TLR 7 and 8 (TLR7 / 8) agonist or a TLR 9 agonist. Non -limiting examples of TLR agonists include, without limitation, imidazoquinolines, adenine derivatives and immunostimulatory DNA or RNA. In particular embodiments, the adjuvant comprises an imidazoquinoline (IMDQ) compound such as, e.g., imiquimod, resiquimod, imidazoquinoline amines, imidazopyridine amines, MGN1703, SD-101, 6,7-fused cycloalkylimidazopyridine amines, and 1,2-bridged imidazoquinoline amines. In certain embodiments, the adjuvant is conjugated to a polymer on the surface of the multilayer nanoparticle or partially or fully encapsulated in between layers of the multilayer nanoparticle or the core of multilayer nanoparticle. The polymer may be conjugated to the adjuvant via a covalent conjugation, for example through a linker such as a disulfide bridge, or via non-covalent interactions such as hydrogen bonds.

[0129] The term “encapsulate” as used herein means to enclose within the multilayer nanoparticle, preferably enclose completely within the multilayer nanoparticle.

[0130] In a certain embodiment, the multilayer nanoparticle contains an additional cargo, preferably a small molecule, and is preferably located in the core. Small molecules can include cyclic dinucleotides.

[0131] The small molecule is typically an active agent, preferably a therapeutic or diagnostic agent. The small molecule can also be an imaging agent, such as a fluorescent agent. In certain embodiments, said small molecule selected from the group consisting of a chemotherapeutic agent, an antibiotic, an antifungal agent, and an antiviral agent. Preferably said chemotherapeutic agent is a small molecule is selected from the group consisting of amsacrine, bifonazole, bicalutamide, buparlisib, camptothecin, celecoxib, daunorubicin, dasatinib, danazol, doxorubicin, fenofibrate, glibenclamide, imatinib, lapatinib, and paclitaxel. Preferably said an antiviral agent include, but are not limited to, antiretroviral agents such as nucleoside analogues with reverse transcriptase activity (such as tenofovir, emtricitabine, lamivudine, abacavir, stavudine, didanosine, zidovudine), the nonnucleoside reverse transcriptase inhibitors (such as delavirdine, efavirenz, etravirine, nevirapine and rilpivirine), protease inhibitors (atazanavir, darunavir, indinavir, ritonavir, tipranavir and many others), and miscellaneous agents such as maraviroc, enfuvirtide (fusion inhibitor), and integrase inhibitors (raltegravir, elvitegravir and dolutegravir), and viral polymerase and protease inhibitors remdesivir, galidesivir, favipiravir, ribavirin, sofosbuvir, tenofovir / emtricitabine, baloxavir marboxil, EIDD-2801, lopinavir / ritonavir; virus- / host-directed viral entry and fusion inhibitors arbidol chloroquine / hydroxychloroquine, chlorpromazine, camostat mesylate, nafamostat mesylate, bromhexine and agents with diverse / unclear mechanism of actions as oseltamivir, triazavirin, ivermectin, nitazoxanide, and niclosamide. The size of a small molecule is subject to variation. In embodiments the small molecule has the size of 1000 Da or below, preferably 800 Da or lower, more preferably 600 Da or lower, again more preferably 500 Da or lower, again more preferably 400 Da or lower, again more preferably 300 Da or lower, again more preferably 200 Da or lower, again more preferably 100 Da or lower. The small molecule can be hydrophobic or hydrophilic small molecule, preferably a polar or non-polar small molecule. Additionally, the small molecule can have a low solubility in aqueous media. In embodiments, the multilayer nanoparticle comprises:

[0132] - a core; and

[0133] - at least one layer of cationic polymer and at least one layer of negatively charged polysaccharides which are layered one on top of the other; and

[0134] - a nucleic acid-cationic peptide complex.

[0135] In embodiments, the multilayer nanoparticle comprises:

[0136] - a core; and

[0137] - at least one layer of cationic polymer and at least one layer of DEXS which are layered one on top of the other; and

[0138] - a nucleic acid-cationic peptide complex.

[0139] In embodiments, the multilayer nanoparticle comprises:

[0140] - a core; and

[0141] - at least one layer of cationic peptides and at least one layer of anionic polymers which are layered one on top of the other; and

[0142] - a nucleic acid-cationic peptide complex.

[0143] In embodiments, the multilayer nanoparticle comprises:

[0144] - a core; and

[0145] - at least one cationic polymer layer, such as a cationic peptide, and at least one anionic polymer layer which are layered one on top of the other, and

[0146] - a nucleic acid-cationic peptide complex, wherein the cationic peptide of the nucleic acid-cationic peptide complex is an amphipathic peptide.

[0147] In embodiments, the multilayer nanoparticle comprises:

[0148] - a core; and

[0149] - at least one layer of poly-L-arginine and at least one layer of anionic polymers which are layered one on top of the other; and

[0150] - a nucleic acid-cationic peptide complex.

[0151] As taught above, the peptides of the nucleic acid-cationic peptide complex may be amphipathic peptides.

[0152] In embodiments, the multilayer nanoparticle comprises:

[0153] - a core; and

[0154] - at least one layer of poly-L-arginine and at least one layer of anionic polymers which are layered one on top of the other; and - a nucleic acid-LAH4-Ll complex.

[0155] In embodiments, the multilayer nanoparticle comprises:

[0156] - a core; and

[0157] - at least one layer of poly-L-arginine and at least one layer of anionic polymers which are layered one on top of the other; and

[0158] - a nucleic acid-LAH4-LlR complex.

[0159] In embodiments, the multilayer nanoparticle comprises:

[0160] - a core; and

[0161] - at least one layer of cationic peptides and at least one layer of anionic polymers which are layered one on top of the other; and

[0162] - a nucleic acid-LAH4-LlR complex.

[0163] In a preferred embodiment, the multilayer nanoparticle comprises:

[0164] - a core; and

[0165] - at least one layer of poly-L-arginine and at least one layer of DEXS which are layered one on top of the other; and

[0166] - a nucleic acid-LAH4-LlR complex.

[0167] In another preferred embodiment, the multilayer nanoparticle comprises:

[0168] - a core; and

[0169] - at least one layer of poly-L-arginine and at least one layer of DEXS which are layered one on top of the other; and

[0170] - a nucleic acid-LAH4-Ll complex.

[0171] In a certain embodiment, the multilayer nanoparticle has a maximum diameter (the multilayer nanoparticles as taught herein may be typically substantially spherical) or a maximum dimension (also, greatest dimension) of less than 900 nm, preferably from about 250 to 800 nm, more preferably from about 300 to 700 nm, even more preferably from about 400 to 650 nm. In some embodiments, the maximum diameter of the multilayer nanoparticle can range between any two values above. The maximum diameter or maximum dimension of the multilayer nanoparticle can be measured from cross-section of the multilayer nanoparticle, preferably a mid-section. In this context, the diameter of a nanoparticle refers to the length measured from the outermost layer on one side of the cross-section of the nanoparticle to the outermost layer on the opposite side of the cross-section of the nanoparticle; a dimension of a nanoparticle refers to the length measured from the outermost layer on one side of the cross-section of the nanoparticle to the outermost layer on the opposite side of the cross-section of the nanoparticle, with maximum dimension being the largest one of said dimensions (i.e., length). In general, multilayer nanoparticle size may be subject to variation. In certain embodiments, a collection of multilayer nanoparticles can have an average greatest dimension (e.g. diameter) of less than 1000 nanometres (nm). In some embodiments, a collection of multilayer nanoparticles have an average greatest dimension (e.g. diameter) of less than 900 nm, about 100 to 850 nm, about 250 to 800 nm, about 300 to 700 nm, about 350 to 700 nm, about 400 to 650 nm, about 450 to 600 nm, or about 500 to 550 nm. In some embodiments, a collection of multilayer nanoparticles have an average greatest dimension (e.g. diameter) in between of any two values above. In some embodiments, a collection of multilayer nanoparticles have an average greatest dimension (e.g. diameter) of less than 900 nm, of less than 800 nm, less than 750 nm, less than 700 nm, less than 650 nm, less than 600 nm, 550 nm, less than 500 nm, or less than 450 nm. In some embodiments, the average greatest dimension diameter of the multilayer nanoparticle can range between of any two values above. In some embodiments, a greatest dimension is a hydrodynamic diameter. Techniques for measuring the size of nanoparticles are known to the skilled person and include, for example, dynamic light scattering (DLS)) . In embodiments, the mean diameter of the multilayer nanoparticle is determined by dynamic light scattering. Parameters characterising a collection of dispersed nanoparticles which can be measured by these methodologies include in particular a Z-average size and polydispersity index (PDI) of the particles. Hence, the above values referring to the average diameter or average maximum dimension of the nanoparticles may also apply to the Z-average size of the nanoparticles as measured particularly by DLS. In a preferred embodiment, Zetaview® (Particle Metrix) is used to determine the size of the multilayer nanoparticle. The size of the multilayer nanoparticle is determined according to the Brownian motion. The mean square displacement per time interval is quantified and determines the diffusion coefficient per time interval for each individual multilayer nanoparticle. The diffusion coefficient is taken into the Stokes-Einstein equation to calculate the size multilayer nanoparticle.

[0172] A further aspect of the invention provides a pharmaceutical composition, comprising one or more multilayer nanoparticles as described herein and a pharmaceutically acceptable carrier, diluent or excipient. Such pharmaceutical compositions are particularly suitable as vaccines. In particular embodiments, the pharmaceutical composition is a vaccine composition or a vaccine.

[0173] The term “pharmaceutically-acceptable carrier” means one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration to a subject contemplated by the present application. The term “carrier” denotes an organic or inorganic ingredient, natural or synthetic, with which the lipid nanoparticles and any other optional agent(s) are combined to facilitate administration. The pharmaceutical composition may further comprise one or more pharmaceutically acceptable ingredients, as known to the skilled person, including, but not limited to polymeric excipients, for instance chitosan derivatives or salts thereof; lipid excipients, such as phospholipids; surfactants; solvents; buffering agents, and the like. The components of the pharmaceutical compositions are commingled in a manner that precludes interaction that would substantially impair their desired pharmaceutical efficiency.

[0174] The type of carrier and other ingredients will vary depending on the mode of administration. When it is desirable to deliver the pharmaceutical compositions as envisaged herein systemically, it may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form. Pharmaceutical parenteral formulations may include aqueous solutions of the ingredients. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Alternatively, injection suspensions may be prepared as oil-based suspensions such as are known in the art or that will be readily apparent to those of ordinary skill in the art based on this disclosure.

[0175] In the context of the present invention, the term “vaccine” refers to any formulation intended to provide adaptive immunity (antibody and / or T cell response) against an antigen, e.g. to prevent or treat a disease, disorder, or addiction. Vaccines can be prophylactic (e.g., to prevent or ameliorate the effects of future abuse or overdose) or therapeutic (e.g, to actively treat or reduce the symptoms of an ongoing disease or disorder).

[0176] Another aspect of the invention provides the multilayer nanoparticle or the pharmaceutical composition, for (therapeutic) use in a method for delivering the nucleic acid to a subject; or where the nucleic acid encodes a protein, for use in a method for delivering the protein to a subject; or where the nucleic acid encodes an antigen, for use in a method for inducing an immune response to the antigen in a subject.

[0177] The term “protein” generally encompasses macromolecules comprising one or more polypeptide chains. The term “polypeptide” generally encompasses linear polymeric chains of amino acid residues linked by peptide bonds. A “peptide bond”, “peptide link” or “amide bond” is a covalent bond formed between two amino acids when the carboxyl group of one amino acid reacts with the amino group of the other amino acid, thereby releasing a molecule of water. Especially when a protein is only composed of a single polypeptide chain, the terms “protein” and “polypeptide” may be used interchangeably to denote such a protein. The terms are not limited to any minimum length of the polypeptide chain. Polypeptide chains consisting essentially of or consisting of 50 or less (< 50) amino acids, such as < 45, < 40, < 35, < 30, < 25, < 20, < 15, < 10 or < 5 amino acids may be commonly denoted as a “peptide”. In the context of proteins, polypeptides or peptides, a “sequence” is the order of amino acids in the chain in an amino to carboxyl terminal direction in which residues that neighbour each other in the sequence are contiguous in the primary structure of the protein, polypeptide or peptide. The terms may encompass naturally, recombinantly, semi-synthetically or synthetically produced proteins, polypeptides or peptides. Hence, for example, a protein, polypeptide or peptide can be present in or isolated from nature, e.g., produced or expressed natively or endogenously by a cell or tissue and optionally isolated therefrom; or a protein, polypeptide or peptide can be recombinant, i.e., produced by recombinant DNA technology, and / or can be, partly or entirely, chemically or biochemically synthesised. Without limitation, a protein, polypeptide or peptide can be produced recombinantly by a suitable host or host cell expression system and optionally isolated therefrom (e.g., a suitable bacterial, yeast, fungal, plant or animal host or host cell expression system), or produced recombinantly by cell-free translation or cell-free transcription and translation, or non-biological peptide, polypeptide or protein synthesis. The terms also encompasses proteins, polypeptides or peptides that carry one or more co- or post-expression-type modifications of the polypeptide chain(s), such as, without limitation, glycosylation, lipidation, acetylation, amidation, phosphorylation, sulphonation, methylation, PEGylation (covalent attachment of polyethylene glycol typically to the N-terminus or to the side-chain of one or more Lys residues), ubiquitination, sumoylation, cysteinylation, glutathionylation, oxidation of methionine to methionine sulphoxide or methionine sulphone, signal peptide removal, N-terminal Met removal, conversion of proenzymes or pre-hormones into active forms, etc. Such co- or post-expression-type modifications may be introduced in vivo by a host cell expressing the proteins, polypeptides or peptides (co- or post-translational protein modification machinery may be native to the host cell and / or the host cell may be genetically engineered to comprise one or more (additional) co- or post-translational protein modification functionalities), or may be introduced in vitro by chemical (e.g., PEGylation) and / or biochemical (e.g., enzymatic) modification of the isolated proteins, polypeptides or peptides.

[0178] In certain embodiments, the antigen is causative of or associated with a disease in the subject, and inducing the immune response to the antigen in the subject is beneficial for prevention or treatment of the disease.

[0179] In certain embodiments, the disease can be an infectious disease, for example human immunodeficiency virus (HIV). Gag mRNA can be used as the nucleic acid for the nucleic acidcationic peptide complex in the multilayer nanoparticle. Gag mRNA can be complexed to an amphipathic peptide, preferably to LAH4-L1. As used herein, the terms “subject” or “patient” are generally used interchangeably and refer to animals, preferably warm-blooded animals, more preferably vertebrates, even more preferably mammals, still more preferably primates, and specifically includes human patients and nonhuman mammals and primates. Preferred subjects or patients are human subjects.

[0180] The terms “treat” or “treatment” encompass both the therapeutic treatment of an already developed disease or condition, such as the therapy of an already developed disease, disorder, addiction or overdose, as well as prophylactic or preventive measures, wherein the aim is to prevent or lessen the chances of incidence of an undesired affliction, such as to prevent occurrence, development and progression of disease, disorder, addiction or overdose.

[0181] A 'therapeutic amount' or ‘therapeutically effective amount’ as used herein refers to the amount of multilayer nanoparticle effective to treat a disease or disorder in a subject, i.e., to obtain a desired local or systemic effect. The term thus refers to the quantity of multilayer nanoparticle that elicits the biological or medicinal response in a tissue, system, animal, or human that is being sought by a researcher, veterinarian, medical doctor or other clinician. Such amount will typically depend on the multilayer nanoparticle and the severity of the disease or disorder, but can be decided by the skilled person, possibly through routine experimentation.

[0182] In embodiments, the number of the multilayer nanoparticles ranges from 1 to 1012multilayer nanoparticles per dose, preferably from about 1 to about 109multilayer nanoparticles per dose, such as from about 10 to about 109, from about 102to about 109or from about 103to about 109, from about 103to about 108, from about 103to about 107, from about 103to about 106multilayer nanoparticles per dose. In some embodiments, the number of the multilayer nanoparticles can range between of any two values above.

[0183] Administration of the pharmaceutical composition described herein may be once, twice, three times, four times, or more times at appropriate time intervals to evoke and maintain the desired immune response. The time intervals between different doses (e.g., between the primary dose and second dose, or between the second dose and a third dose) may not be the same, and the time interval between each two doses may be determined independently. The dose of the vaccine composition, the number of doses administered to the subject, and the time intervals between two doses of the vaccine composition may be determined by a person skilled in the medical art. An appropriate dose and a suitable duration and frequency of administration will be determined by factors such as the condition of the patient, age of the patient, the type and severity of the patient’s disease or disorder to be treated or prevented, the particular composition of the multilayer nanoparticle, and the method of administration. Optimal doses may generally be determined using experimental in vitro assays, in vivo animal models, and / or human clinical trials. When administered in a liquid form, suitable dose sizes may vary with the size (i.e., weight or body mass) of the patient, but will typically range from about 100 pl to about 1 ml, such as about 100 pl, about 200 pl, about 300 pl, about 400 pl, about 500 pl, about 600 pl, about 700 pl, about 800 pl, about 900 pl, or about 1 ml (comprising an appropriate dose) for a 10-100 kg subject. The use of the minimum dosage that is sufficient to provide effective therapy and / or prophylaxis is usually preferred. Patients may generally be monitored for therapeutic or prophylactic effectiveness using assays suitable for the condition being treated or prevented, which assays will be familiar to those having ordinary skill in the art and are described herein.

[0184] Any route of administration can be used to deliver the composition to the subject. Indeed, although more than one route can be used to administer the composition, a particular route can provide a more immediate and more effective reaction than another route. For example, the vaccine compositions described herein may be administered via a route including parenteral, oral, enteral, transdermal / transmucosal, and inhalation. The term enteral, as used herein, is a route of administration in which the agent is absorbed through the gastrointestinal tract or oral mucosa, including oral, rectal, and sublingual. The term parenteral, as used herein, describes administration routes that bypass the gastrointestinal tract, and are typically administered by injection or infusion, including intraarterial, intradermal, subdermal, intramuscular, intranasal, intraocular, intraperitoneal, intravenous, subcutaneous, submucosal, intravaginal, intrastemal, intracavemous, intrathecal, intrameatal, and intraurethral injection. The term transdermal / transmucosal, as used herein, is a route of administration in which the agent is administered through or by way of the skin, including topical. The term inhalation encompasses techniques of administration in which an agent is introduced into the pulmonary tree, including intrapulmonary or transpulmonary and includes intranasal administration. Preferably, the composition is administered via injection.

[0185] Another aspect of the invention provides an in vitro or ex vivo method for introducing a nucleic acid into a cell, comprising exposing the cell to the multilayer nanoparticle or the pharmaceutical composition.

[0186] Yet another aspect of the invention provides a method for preparing a multilayer nanoparticle according to the invention comprising : a) coating a core with at least one cationic polymer and at least one anionic polymer, whereby the at least one cationic polymer and at least one anionic polymer are layered one on top of the other; and b) adding at least one nucleic acid-cationic peptide complex to the coated core. In embodiments, the core is first coated with a layer of cationic polymer, followed by coating the core containing a layer of cationic polymer with a layer of anionic polymer.

[0187] In embodiments, coating the core with layers of anionic and cationic polymers involves sequentially applying these layers with opposite charges on top of each other until the desired number of layers is achieved. Applying layers of anionic and cationic polymers preferably involves sequentially immersing the core or the coated core in a solution containing anionic or cationic polymers.

[0188] In embodiments, after the step b), the multilayer nanoparticles are immersed in EDTA to dissolve the core.

[0189] It should be appreciated that in the description of illustrative embodiments of the invention, various features of the invention are sometimes grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure and aiding in the understanding of one or more of the various inventive aspects.

[0190] The present application also provides aspects and embodiments as set forth in the following Statements. In these statements, the wording “The multilayer nanoparticle according to Statement 1, wherein... ” or “The multilayer nanoparticle according to any one of Statements 2 to 6, wherein... ” also discloses and may be replaced by the simple wording “In certain embodiments...”.

[0191] Statement 1. A multilayer nanoparticle comprising:

[0192] - a core;

[0193] - at least one cationic polymer and at least one anionic polymer which are layered one on top of the other; and

[0194] - a nucleic acid-cationic peptide complex.

[0195] Statement 2. The multilayer nanoparticle according to statement 1, wherein at least one nucleic acid-cationic peptide complex is associated with the anionic polymer, such as associated by means of an electrostatic interaction between the cationic peptide and the anionic polymer.

[0196] Statement 3. The multilayer nanoparticle according to statement 1 or 2, wherein the outermost layer of the nanoparticle comprises the anionic polymer.

[0197] Statement 4. The multilayer nanoparticle according to any one of the previous statements, wherein the outermost layer comprises the anionic polymer and the at least one nucleic acid-cationic peptide complex is associated with the anionic polymer of the outermost layer. Statement 5. The multilayer nanoparticle according to statement 4, wherein at least one nucleic acid-cationic peptide complex is disposed on the outer surface of the outermost anionic polymer layer.

[0198] Statement 6. The multilayer nanoparticle according to any one of the previous statements, wherein the anionic polymer is an anionic polysaccharide, such as, dextran sulfate, polyacrylic acid or a salt thereof.

[0199] Statement 7. The multilayer nanoparticle according to any one of the previous statements, wherein the cationic polymer comprises poly-L-arginine, poly(cystamine bisacrylamide-co-4-amino-l- butanol) (pABOL), trimethylammoniumbenzylchitosan (TMAB-CS) or a salt thereof.

[0200] Statement 8. The multilayer nanoparticle according to any one of the previous statements, wherein the cationic peptide of the nucleic acid-cationic peptide complex is an amphiphatic peptide, preferably LAH-L1 having the sequence set forth in SEQ ID NO: 1 (KKALLAHALHLLALLALHLAHALKKA) or LAH-L1R having the sequence set forth in SEQ ID NO:2 (RRALLAHALHLLALLALHLAHALRRA).

[0201] Statement 9. The multilayer nanoparticle according to any one of the previous statements, wherein the core contains CaCOs.

[0202] Statement 10. The multilayer nanoparticle according to any one of the previous statements, wherein the nucleic acid of the nucleic acid-cationic peptide complex is a DNA molecule or an RNA molecule or a hybrid RNA / DNA molecule, such as an RNA molecule selected from the group consisting of mRNA, saRNA, siRNA, gRNA, non-coding RNA, small non-coding RNA, long non-coding RNA, miRNA, circular RNA, taRNA, a synthetic analogue of RNA, and combinations thereof.

[0203] Statement 11. The multilayer nanoparticle according to any one of the previous statements, wherein the multilayer nanoparticle comprises an adjuvant, such as an adjuvant selected from a group consisting of a toll-like receptor (TLR)- agonist, RIG-I like receptor (RLR)- agonist, Nodlike receptor protein (NLRP)- agonist, iNKT agonist or derivates thereof, bacterial lipopolysaccharide (LPS) or derivates thereof, synthetic polynucleotides, and combinations thereof.

[0204] Statement 12. The multilayer nanoparticle according to any one of the previous statements, wherein the nucleic acid-cationic peptide complex is conjugated to one or more layers of the multilayer nanoparticle, such as to the outermost layer of the multilayer particle. Statement 13. The multilayer nanoparticle according to any one of the previous statements, wherein the nanoparticle contains an additional cargo, preferably a small molecule, and preferably located in the core.

[0205] Statement 14. The multilayer nanoparticle according to any one of the previous statements, wherein multilayer nanoparticle has a diameter of less than 900 nm, preferably from about 250 to 800 nm, more preferably from about 300 to 700 nm, even more preferably from about 400 to 650 nm.

[0206] Statement 15. A pharmaceutical composition comprising the multilayer nanoparticle as defined in any one of statements 1 to 14 and a pharmaceutically acceptable carrier, diluent, or excipient, optionally wherein the pharmaceutical composition is a vaccine.

[0207] Statement 16. The multilayer nanoparticle according to any one of statements 1 to 14, or the pharmaceutical composition according to statementl5, for use in medicine.

[0208] Statement 17. The multilayer nanoparticle as defined in any one of statements 1 to 14 or the pharmaceutical composition as defined in statementl5, for use in a method for delivering the nucleic acid to a subject; or where the nucleic acid encodes a protein, for use in a method for delivering the protein to a subject; or where the nucleic acid encodes an antigen, for use in a method for inducing an immune response to the antigen in a subject.

[0209] Statement 18. The multilayer nanoparticle or the pharmaceutical composition for use according to claim 17, wherein the antigen is causative of or associated with a disease in the subject, and inducing the immune response to the antigen in the subject is beneficial for prevention or treatment of the disease.

[0210] Statement 19. The multilayer nanoparticle or the pharmaceutical composition for use according to any one of statements 17 or 18, wherein the subject is human.

[0211] Statement 20. An in vitro or ex vivo method for introducing a nucleic acid into a cell, comprising exposing the cell to the multilayer nanoparticle as defined in any one of statements 1 to 14 or the pharmaceutical composition as defined in statement 15.

[0212] Statement 21. A method for preparing a multilayer nanoparticle according to any one of statements 1 to 14 said method comprising: a) coating a core with at least one cationic polymer and at least one anionic polymer, whereby at least one cationic polymer and at least one anionic polymer are layered one on top of the other; and b) adding at least one nucleic acid-cationic peptide complex to the coated core.

[0213] EXAMPLES

[0214] 1. Generation of the layer-by-layer polyelectrolyte nanoparticles

[0215] Biodegradable nanoparticle were generated according to the layer-by-layer (LbL) technique. An in-house protocol, adapted from Tarakanchikova et al. 2020, ( )

[0216] , was applied to synthesize the functionalized nanoparticle (Figure la).

[0217] The particle synthesis was conducted under strict RNAse-free conditions. All solutions were purchased in RNAse-free form or, when not commercially available, treated with 0.1 % diethylpyrocarbonate (DEPC, CeHioOs, Sigma-Aldrich, St-Louis, Missouri, USA). For one synthesis, 0.386 mL of 0.33 M sodium carbonate (Na2COs, Sigma Aldrich, St-Louis, Missouri, USA) in 83 % ethylene glycol (C2H8O2, Sigma Aldrich, St-Louis, Missouri, USA) was added dropwise in a small beaker to 2 mL of 0.33 M calcium chloride (CaCU Sigma Aldrich, USA) in 83 % ethylene glycol. The solutions were allowed to precipitate for 20 min at 1000 rpm under magnetic stirring. Subsequently, the obtained calcium carbonate (CaCOs) submicrocores were collected, spun down and washed once with ImL of 99 % ethanol (VWR® International, Radnor, Pennsylvania, USA) and twice with 1 mL of RNAse-free water (Versylene® Fresenius Kabi, Belgium). Each centrifugation step was conducted at 21,130 x g for 90 seconds.

[0218] Next, the positively charged (poly-L-arginine, PARG) and negatively charged (dextran sulphate, DEXS) polyelectrolytes were applied according to the LbL technique. Briefly, cores were dissolved in 0.400 mL PARG (1 mg / mL in 0.05 sodium chloride Sigma Aldrich, St-Louis, Missouri, USA ) or DEXS (Img / mL in 0.05 sodium chloride, Sigma Aldrich, St-Louis, Missouri, USA ) and incubated on ice for 7 minutes. Subsequently, the particles were, spun down at 21,130 x g for 90 sec and washed twice with 1 mL RNAse-free water. This process was repeated after each layer application and until the desired number of layers was reached. In this case, two layers of DEXS and one layer of PARG were applied (core-DEXS-PARG-DEXS).

[0219] To obtain the combination of the nanoparticle with LAH4-L1 SEQ ID NO: 1 (LI, KKALLAHALHLLALLALHLAHALKKA), LAH4-L1R SEQ ID NO:2 (L1R, RRALLAHALHLLALLALHLAHALRRA) (GenScript, Piscataway, NJ, USA) or RALA SEQ ID NO: 3 (WEARLARALARALARHLARALARALRACEA) mRNA encoding enhanced green fluorescent protein (eGFP-mRNA, L-7601) (TriLink Biotechnologies, San Diego, USA), Cy5- labeled mRNA encoding enhanced green fluorescent protein (Cy5-mRNA-eGFP, L-7701) (TriLink Biotechnologies, San Diego, USA), modified mRNA encoding Firefly luciferase (Fluc- mRNA, 5 moU, L-7202) (TriLink Biotechnologies, San Diego, USA) or modified mRNA encoding li80.tOVA (a truncated form of ovalbumine) (TriLink Biotechnologies, San Diego, USA) were complexed to L1(R) by adding mRNAto L1(R) in a 15: 1 ratio and to RALA in a 14, 5: 1 ratio (pg peptide to pg mRNA). MRNA-peptide complex formation was ensured after 15 min incubation at room temperature. Subsequently, Ll(R)-mRNA and RALA-mRNA complexes were applied as an outer layer to the nanoparticle and allowed to complex for 15 min at room temperature.

[0220] SaRNA encoding eGFP and li80.tOVA were generated in-house. T7-VEEV-eGFP (saRNA-eGFP plasmid was a gift from prof. Steven Dowdy (Addgene plasmid #58977; http: / / n2t.nct / addgene:58977; RRID:Addgene_58977)). T7-VEEV-li80.tOVA (saRNA-li80tOVA plasmid) was created by inserting the li80.tOVA construct in the T7-VEE-eGFP backbone by restriction cloning with Xbal and Clal. A deletion in the T7 promotor sequence was required for the capping system CleanCap® Reagent AU (TriLink BioTecnologies, USA). The deletion was performed with a site-directed-mutagenesis (SDM) with Q5® Site-Directed Mutagenesis Kit (New England Biolabs, USA) according to the manufacturer’s protocol obtaining the T7-VEE- GFP and T7-VEE-li80.tOVA o.H521_ldelG variant. Subsequently, plasmids were linearized with Mlul (Thermo Scientific™, Waltham, Massachusetts, USA) at 37 °C for 2 hours and purified using Monarch® PCR & DNA Cleanup kit (New England Biolabs, Ipswich, USA), with transcriptional capping using CleanCap® Reagent AU (Trilink Biotechnologies, San Diego, U SA) . The obtained IVT saRNA was treated with Turbo™ DNAse (Invitrogen™, U SA) at 2 U / pL for 15 minutes at 37 °C to remove DNA template and then purified with Monarch® RNA cleanup kit (New England Biolabs, Ipswich, USA). IVT product yield and integrity were confirmed by Nanodrop® ND- 1000 Spectrophotometer (Thermo Scientific™, Waltham, USA) and gel electrophoresis.

[0221] 2. Characterization of the modified nanoparticle

[0222] 2.1. Nanoparticle-Ll have the expected size and charge

[0223] As a first step, Nanoparticle-Ll (SubP-Ll) were characterised using the Zetaview-NTA®. In general, Nanoparticle were expected to have a size ranging between 400 and 700 nm. Compared to the unmodified Nanoparticle, Nanoparticle-Ll had an average size of 650 nm, 559 nm and 484 nm respectively (Figure lb). Furthermore, the zetapotential before and after the application of the cationic peptide layer was measured. The zetapotential increased from -30 mV to +18 mV (Nanoparticle-Ll) (Figure 1c). This value corresponded to the charge of the Ll-mRNA complexes, indicating that the complexation of each peptide to DEXS was successful. Next, SEM images were recorded to evaluate the morphology of the particles. The sponge-like surface corresponds to the nature of the CaCO , cores and the formation of vaterite particles (Figures Id and e).

[0224] 2.2. Washing step did not affect the characterization of nanoparticle-Ll

[0225] Although the size and the charge of the generated Nanoparticle-Ll corresponded to the expected values, it needed to be confirmed that no residual LI was still present in the samples. The generated Nanoparticle-Ll were therefore subjected to an additional washing step. Briefly, the generated Nanoparticle-Ll was spun down and the supernatant (SN) was collected. Subsequently, the pellet containing the washed Nanoparticle-Ll (W) were reconstituted in RNAse-free water (Figure 2a). Interestingly, the characteristics of the washed particles (520 nm and +28 mV) remained within the expected range and were comparable to the unwashed particles (650 nm, + 18mV). However, the collected supernatant contained residual unbound LI as the size and charge (250 nm, +19 mV) correspond to the LI complexes (200 nm, + 15mV) (Figure 2).

[0226] 2.3. LI enhances the translation efficiency of nanoparticle

[0227] Next, the translation efficiency of nanoparticle, nanoparticle-Ll (NW, W and SN) and LI was compared. Mock conditions were taken along as a negative control and eGFP expression was followed up for 72h via the Incucyte® (Sartorius, Gottingen, Germany). EGFP expression was expressed as green object count (GOC) per image. Interestingly, nanoparticle-Ll outperformed nanoparticles not containing LI (Figure 3a) confirming the beneficial effect of applying mRNA as complexes with LI in the outer layer.

[0228] To assess whether the washing step and the unbound LI in the supernatant would affect the translation efficiency, the eGFP expression of washed particles and the collected supernatant was compared with the unwashed particles. As expected, the washing step did influence the translation efficiency and induced lower GOC compared to the unwashed particles (Figure 3b). Furthermore, although the residual LI was able to induce eGFP expression, the GOC was lower compared to the unwashed nanoparticle-Ll conditions.

[0229] 2.4. Concentration-dependent effect of chloroquine diphosphate and ammonium chloride on the translation efficiency of nanoparticle-Ll

[0230] The use of endosomal escape stimulators was explored to investigate whether the translation efficiency of Nanoparticle-Ll can be further improved. Ammonium chloride (NH4CI) and chloroquine diphosphate (CQ) were used to enhance the endosomal escape of the polyelectrolyte particles. In the acidic milieu of the endosome, NH4CI and CQ become protonated and trapped. This way NH4CI and CQ can act as a proton sponge, inducing endosomal rupture.

[0231] Immortalized murine dendritic cells (DC2.4, kindly provided by Prof. P. Midoux from the Centre National de Recherche Scientifique (CNRS) and the Universite d’ Orleans) were preincubated for Ih before transfection with Nanoparticle-Ll and LI with CQ (0.16 pM - 20 pM) and NH4CI (39 pM - 625 pM). Although CQ did not improve the translation efficiency, the stimulation with 39 pM NH4CI showed comparable GOC (eGFP expression) with unstimulated Nanoparticle-Ll (Figure 4). Therefore, a lower concentration range, (37.5 to 2.25 pM NH4CI) was tested. Interestingly, for the unwashed Nanoparticle-Ll, a dose-dependent effect was observed. For instance, when comparing unstimulated conditions with 4.5 pM NH4CI stimulation, an increase from 1200 to 2000 GOC per image respectively was observed. A limited effect of NH4CI was however observed for washed particles and LI (Figure 5). Based on these data, prestimulation with 5p M NH4CI was conducted in the following experiments and eGFP expression was assessed via flow cytometry. Overall, the unwashed particles showed higher eGFP expression percentage (40 % eGFP, Figure 6a) and median fluorescence intensity (MFI) (Figure 6b) compared to the washed particles (25 % eGFP) and the supernatant (10% eGFP). Furthermore, 5 pM NH4CI did not significantly increase the translation efficiency of the washed and unwashed Nanoparticle-Ll .

[0232] 2.5. Nanoparticle-Ll particles are efficiently taken up and induce efficient translation efficiency in DC2.4 cells

[0233] To simultaneously assess the uptake and translation of mRNA encoding eGFP, DC2.4 cells were transfected with Cy5 labelled mRNA encoding eGFP. Cy5 and eGFP positivity were evaluated via confocal microscopy, Incucyte® and flow cytometry.

[0234] First, the uptake and translation efficiency of the particles were studied via confocal microscopy. For this, the commercially available transfection reagent jetMESSENGER® (Polyplustransfection®, Illkirch, Strasbourg, France) was used as a positive control. Both LI -mRNA and Nanoparticle-Ll (NW) particles were taken up by DC2.4 cells as red signals were observed inside the cytoplasm. Moreover, the green signal indicates successful transfection efficiency in both conditions (Figure 7a)

[0235] To evaluate the kinetics of the Nanoparticle, Cy5+and eGFP+were followed up for 72h via Incucyte®. Cy5+and eGFP+were respectively expressed as red object count (ROC) and green object count (GOC) per image. Already after 4h, Nanoparticle-Ll (NW and W) and LI are efficiently taken up by DC2.4 cells (Figure 7b). Although this signal decreased at 8h posttransfection, the signal remained detectable for 72h. Furthermore, after 8 h, eGFP expression (Figure 7c) started to increase in both Nanoparticle-Ll (NW and W) and LI, indicating the initiation of eGFP translation. The course of GOC remained stable for 48 h for LI and Nanoparticle-Ll (W and NW). Although the supernatant also induced Cy5+and eGFP+, the area of the ROC and GOC curve was smaller compared to LI and Nanoparticle -LI (NW and W) suggesting that the induced effect is limited.

[0236] Cy5+(uptake) and eGFP signal were measured via flow cytometry at 24 h - 48 h and 72 h posttransfection. Both the percentages of the Cy5+eGFP+population and the MFI of eGFP decreased over time but remained stable. Overall, submicroparticles induce both higher uptake and translation efficiency than LI alone.

[0237] 3. In vitro and in vivo immunogenicity of nanoparticle-Ll

[0238] 3.1. Nanoparticle-Ll induce antigen presentation and maturation of DC2.4 cells and GM-DCs

[0239] For further application, it was assessed whether transfection with modified mRNA encoding li80.tOVA (a truncated form of ovalbumin (tOVA, Trilink®, San Diego, California, USA) would induce antigen presentation in DC2.4 cells. DC2.4 cells were transfected with 500 ng of modified mRNA encoding li80.tOVA was formulated in respectively Nanoparticle-Ll, LI or jetMESSENGER® (a previously tested transfection agent showing very efficient translation efficiency in DC2.4 cells). Transfection with both Nanoparticle-Ll and LI resulted in a comparable percentage (35 % and 25 % respectively) of SIINFEKL presentation in MHC-I (Figures 8a and b). Interestingly, an up-regulation of the Cluster of Differentiation 40 (CD40) and CD86 (Figures 8c and d) was observed, upon transfection with nanoparticle-Ll, indicating that nanoparticle-Ll can induce dendritic cell (DC) maturation.

[0240] Next, a similar assay using murine bone marrow-derived dendritic cells (GM-DCs) was performed. DCs were subjected to the same treatment as described above. Although transfection of Nanoparticle-Ll, LI and jetMESSENGER® induced a low percentage of SIINFEKL presentation in MHC-I (10 %, 9 % and 8.5 % respectively) (Figures 9a and b), a shift in the MFI of the co -stimulatory molecules CD40 and CD86 in the nanoparticle-Ll condition was observed (Figure 9c). This indicates that the LbL formulation can induce DC maturation in primary DCs.

[0241] 3.2. Nanoparticle-Ll induce the proliferation of OT-I CD8+T cells in vivo

[0242] In the next step, in vivo immunogenicity of nanoparticle-Ll and LI was assessed. Briefly, CD45.2+CD8+OT-I T cells were isolated, labelled with cell trace violet (CTV) and injected in the tail vein of CD45.U mice. Two days after the adoptive transfer, mice were immunized intramuscularly with 10 pg of modified li80.tOVA mRNA formulated in Nanoparticle-Ll or LI. As a positive control, 2.5 pg of LNP -formulated modified li80.tOVA mRNA was used. Three days later, the proliferation of transferred OT-I splenocytes was assessed in the spleen and the mesenteric and inguinal lymph nodes. Nanoparticles showed 100 % proliferation in the spleen, inguinal and mesenteric lymph nodes indicating systemic immunization (not shown). When comparing LI with nanoparticle -LI it was observed that both nanoparticle -LI and LI can induce local immunization as the proliferation percentage was the highest in the ipsilateral lymph nodes (Figure 10b).

[0243] 4. Nanoparticle-Ll remain stable for up to 7 days

[0244] The stability of the nanoparticles at different storage temperatures was evaluated (4°C, room temperature (18 - 21 °C), 37°C, -20 °C and -80 °C). Size and charge measurements were conducted after 1, 3 and 7 days of storage. Particle characterization was conducted to evaluate whether the storage conditions would affect the complexation of LI.

[0245] Compared with freshly prepared particles, both the size and charge of the nanoparticle -LI remained within the expected ranges, except for 3 days old particles at 4 °C (Figure Ila). Interestingly, 7-day-old particles carried a remarkably increased positive charge compared to fresh nanoparticle -LI (+37 mV vs + 3.6mV) (Figure 11b). All other conditions followed a similar trend as their fresh variant.

[0246] Finally, the transfection efficiency of the stored nanoparticle was examined. Overall, results show a relatively stable eGFP+percentage for day 3 and 7 particles in all conditions. However, the MFI increased up to 3 days and returned to reference levels on day 7 (Figures 11c and d).

[0247] 5. Use of other cationic peptides

[0248] Nanoparticle-LIR and nanoparticle -RALA were characterised using the Zetaview-NTA®. Compared to the unmodified particles, nanoparticle -L1R and nanoparticle-RALA have an average size of 559 nm and 484 nm respectively (Figure 12a). Furthermore, measurement of the zetapotential before and after the application of the cationic peptide layer showed that the zetapotential increases from -30 mV to + 31mV (nanoparticle-LlR) and + 4.9mV (nanoparticle- RALA). These values correspond to the charges of LIR-mRNA and RALA-mRNA, indicating that the complexation of each peptide to DEXS was successful (Figure 12b).

[0249] Next, the transfection efficiency of both particles in DC2.4 cells was evaluated. Indeed, both alternatives were also able to induce eGFP expression. Although both nanoparticle -L1R and nanoparticle-RALA did not outperform nanoparticle -LI . Transfection with nanoparticle-LlR and nanoparticle-RALA however, did not enhance the translation efficiency of nanoparticle (Figures 13a and b). 6. Nanoparticle-Ll as an saRNA-based platform

[0250] It was investigated whether the formulation of self-amplifying mRNA encoding eGFP would induce efficient gag expression in DC2.4 cells. Characterization of complexed saRNA with LI at 15: 1 ratio (pg peptide: pg RNA), showed that saRNA was efficiently packed. Both size and charge lay in the expected range (Figures 14a and b). Interestingly, although nanoparticle-Ll saRNA induced lower eGFP expression compared to mRNA (25 % and 40 % respectively), saRNA-GFP induced a more efficient GFP expression, showcasing the self-amplifying nature of the RNA but also proving that nanoparticle-Ll can be applied as a formulation platform (Figures 14d and e).

[0251] 7. mRNA-based vaccines for infectious diseases

[0252] MRNA is a versatile tool that can be used in the vaccine development of any infectious disease. Since nearly any protein can be introduced via mRNA, its application potential is very wide. Nanoparticle-Ll was investigated as a potential HIV vaccine candidate. As a first step, it was evaluated whether the formulation of modified gag mRNA with nanoparticle-Ll would induce efficient gag expression in DC2.4 cells. Interestingly, although nanoparticle-Ll induced lower gag expression compared to LI (25 % and 33 % respectively), the gag expression per cell was more efficient compared to LI. Similar to the previous transfection experiment, 5 pM NFLC1 did not enhance the translation efficiency of nanoparticle-Ll (Figure 15).

[0253] 8. Efficient SIINFEKL presentation in DC2.4 cells upon transfection with nanoparticle-Ll-saRNA-OVA

[0254] Preliminary in vitro assay using saRNA encoding eGFP has shown that Nanoparticle-Ll is a suitable transfection agent. For further application, it was assessed whether transfection with saRNA encoding li80.tOVA (in-house generated) would induce antigen presentation in DC2.4 cells and outperform modified mRNA-OVA. For this, 500 ng saRNA or modified mRNA encoding li80.tOVA was complexed with LI at a 15: 1 ratio (pg peptide:pg saRNA) and applied as an outer layer to the multilayer nanoparticles. Transfection of saRNA and mRNA encoding li80.tOVA with LI resulted in a comparable percentage and MFI (6 % and 4.5 % and 25768 and 26453 respectively) of SIINFEKL presentation in MHC-I respectively (Figures 16b and c). Interestingly, saRNA formulated with Nanoparticle-Ll induces higher MFI (36.992 and 18055 respectively) compared with mRNA despite a lower percentage (6.5 % and 15 % respectively).

[0255] 9. Transfection and maturation of human monocyte-derived dendritic cells

[0256] Transfection and maturation of human monocyte -derived dendritic cells (Mo-DCs) were tested with LI- or SubP-Ll -formulated eGFP mRNA at indicated LLmRNA (15: 1 - pg:pg) and SubP- LI to cells (25: 1) ratio. Mo-DCs were left untreated or transfected with mRNA encoding eGFP, or without mRNA (MOCK) in complete or serum-reduced medium. The next day, cells were harvested and stained for viability (not shown), DC markers and the maturation markers CD80, CD86, CD40 and CD83 within the CDllc+CDlc+DC population. Cells were treated with R848 as a positive control for maturation. Percentage (Figure 17a) and MFI (Figure 17b) of eGFP+within the CDllc+CDlc+DC population. Overlay graphs for CD86, CD40 and CD83 expression (Figure 17c).

[0257] 10. Comparison of conventional Nanoparticle (PARG) with SubP formulated with poly- L-lysine (PLL) as an alternative positive layer

[0258] Comparison of conventional nanoparticle, comprising PARG as cationic polyelectrolyte layer (CaCOs cores / PARG / DEXS / PARG), with a nanoparticle formulated with poly-L-lysine (PLL, 1 mg / mL in 0.05 sodium chloride Sigma Aldrich, St-Louis, Missouri, USA )) as an alternative positive layer (CaCOs cores / PLL / DEXS / PLL). The size and charge of both nanoparticle were measured via Zetaview®- NTA. Size (A) and charge (B) measurements of the different nanoparticle -LI and variants (Figure 18 a, b, c)

[0259] 11. Comparison of the transfection efficiency and antigen presentation of conventional SubP-Ll (PARG) with SubP-Ll formulated with poly-L-lysine (PLL) as a positive layer

[0260] The transfection efficiency and antigen presentation of conventional nanoparticle (PARG SubP- Ll) and a nanoparticle formulated with poly-L-lysine (PLL SubP-Ll) as a positive layer were determined. DC2.4 cells were transfected with 250 ng mRNA encoding for eGFP or li80.tOVA formulated in PARG SubP-Ll or PLL SubP-Ll. The next day, cells were harvested and read-out was performed by flow cytometry for percentage and MFI of eGFP (a and b) and SIINFEKL presentation in MHC-I (c and d) (Figure 19).

[0261] 12. Comparison of the transfection efficiency of conventional SubP-Ll (PARG) with SubP-Ll formulated with poly-L-lysine (PLL) as a positive layer in Mo-DCs

[0262] The transfection efficiency of conventional nanoparticles (PARG SubP-Ll) and nanoparticles formulated with poly-L-lysine (PLL SubP-Ll) as a positive layer. Mo-DCs were transfected with 250 ng mRNA encoding for eGFP formulated in PARG SubP-Ll or PLL SubP-Ll in complete or serum -reduced medium (Opti-MEM™ Thermo Scientific™, Waltham, Massachusetts, USA). The next day, MO-DCs were harvested, and read-out was performed by flow cytometry for percentage (a) and MFI (b) of eGFP within the CDllc+cDCU population. As shown in Figure 20, PARG SubP-Ll has a better transfection efficiency than PLL SubP-Ll (Figure 21). 13. Characterization and transfection efficiency of mRNA formulated with LAH4-L1 (LI), poly-L-lysine (PLL), conventional LbL SubP-Ll (SubP-Ll) and PLGA nanoparticles (PLGA NPs)

[0263] MRNA formulated with LAH4-L1 (LI), poly-L-lysine (PLL), conventional nanoparticle -LI (SubP-Ll) and PLGA nanoparticles with PLL as the cationic polyelectrolyte layer (PLGA 10 mg / mL in RNAse free water Sigma Aldrich, St-Louis, Missouri, USA). PLGA nanoparticles were formulated according to San Juan et al.) were characterization and the transfection efficiency was determined. The nanoparticle-Ll (SubP-Ll) was formulated as: CaCO, cores / DEXS / PARG / DEXS / LAH4-Ll:mRNA and the PLL nanoparticles (NPs) were formulated (according to San Juan et al.) as: PLGA cores / PLL / PGA / PLL / mRNA or DNA / PLL. DC2.4 cells were transfected with 0.25 pg mRNA encoding eGFP (mRNA-eGFP) formulated with LI or nanoparticle-Ll (SubP-Ll) and 0.5, 1 and 2 pg of mRNA-eGFP formulated with PLL and PLL NPs. The next day, cells were harvested and read-out was performed by flow cytometry for percentage and MFI eGFP (c). As shown in Figure 21, nanoparticle-Ll (SubP-Ll) containing 0.25 pg of mRNA encoding eGFP has a better transfection efficiency than PLL NPs and PLL complexed to mRNA (PLL:mRNA) containing 0.5, 1 or 2 pg of mRNA-eGFP.

[0264] 14. Comparison of the transfection efficiency of pDNA formulated with LAH4-L1 (LI), SubP-Ll and PLGA NPs

[0265] Further, the transfection efficiency of pDNA formulated with LAH4-L 1 (L 1), SubP-L 1 and PLGA NPs (Sao Paolo et al.) was determined. HEK 293T cells were transfected with 500 ng of pDNA encoding for eGFP (kindly provided by prof. Karine Breckpot from the Laboratory for Molecular and Cellular Therapy at Vrije Universiteit Brussel ) formulated in LI, SubP-Ll or PLGA NPs. The SubP-L 1 nanoparticle was formulated as: CaCO, cores / DEXS / PARG / DEXS / LAH4- LEpDNA and the PLGA NPs were formulated as: PLGA cores / PLL / PGA / PLL / DNA / PLL. Different ratios of pDNA to LI (pg:pg) were used for LI and SubP-Ll. The next day, cells were harvested and read-out was performed by flow cytometry. Unformulated pDNA and polyethyleneimine (PEI, Sigma Aldrich, St-Louis, Missouri, USA) formulated pDNA were included as a negative and positive control for transfection respectively. Representative contour plots of HEK 293T cells transfected with unformulated pDNA and pDNA formulated with SubP- Ll (1: 15 ratio) and PEI. Percentage and MFI of eGFP acquired via flow cytometry (b) at 3 days post-transfection. As shown in Figure 22, SubP-L 1 also demonstrate high transfection efficiency when formulated with pDNA . 15. Comparison of the characterisation and transfection efficiency of conventional SubP-Ll with SubP-Ll containing PLGA-based cores.

[0266] Conventional SubP-Ll (CaCOs SubP-Ll) NPs and SubP-Ll containing PLGA-based cores (PLGA SubP-Ll) were characterisation and their transfection efficiency was determined. Schematic representation of the incorporation of PLGA as cores of the SubP-Ll formulation (PLGA SubP-Ll) is shown in Figure 23a. Size and chaige measurements after each layer application, acquired with the Zetaview®-NTA (Figure 23b). DC2.4 cells were transfected with 500 ng mRNA encoding eGFP formulated with SubP-Ll NPs and PLGA SubP-Ll NPs. The next day, cells were harvested, and read-out was performed by flow cytometry for percentage and MFI eGFP (Figure 23c). Both SubP-Ll NPs and PLGA SubP-Ll NPs demonstrate similar transfection efficiency as shown in figure 23.

[0267] 16. Comparison of the transfection efficiency of Ll:mRNA and PLL+: mRNA and different nanoparticles

[0268] The conventional nanoparticle-Ll (SubP-Ll) were formulated as: CaCO, cores / DEXS / PARG / DEXS / LAH4-Ll:mRNA, wherein LAH4-L1 was complexed to mRNA prior to addition to the DEXS layer. SubP PLL+NPs were formulated as: CaCO, cores / DEXS / PARG / DEXS / PLL:mRNA, wherein PLL+was complexed to mRNA prior to addition to the DEXS layer. Poly-L-lysine mRNA complexes (PLL+) and LAH4-Ll:mRNA complexes (LEmRNA) were also tested without formulation into a nanoparticle. DC2.4 cells were transfected with 0.5, 1 or 2 pg mRNA encoding eGFP formulated with SubP-Ll and SubP PLL+. The next day, cells were harvested, and read-out was performed by flow cytometry for percentage and MFI of eGFP (b). Figure 24 demonstrates that the amphipathic peptide LAH4-L1 enhances the transfection efficiency.

[0269] 17. Inflammasome activation

[0270] Inflammasome activation was tested in GM-DCs treated with LPS, LAH4-L1 (A) and SubP-Ll (B). GM-DCs were generated by culture with 20 ng / mL rm GM-CSF for 6 days. On day 6, GM- DCs were primed with 0.5 pg / mL LPS for 3h, followed by 1 h incubation with 10-25 pM necrosulfonamide (NSA, Selleck Chemicals, Houston, United States), 0.01-1 pM Belnecasan (VX-769, Bio-connect, Huissen, The Netherlands) or 0.01-1 pM MCC.950 (a selective inhibitor of the NLRP3 inflammasome, Bio-connect, Huissen, The Netherlands) before treatment with 15 pg / mL LAH4-L1 or SubP-Ll containing 15 pg / mL of LAH4-L1. The supernatant was harvested after 1 h and stored at -20 °C and analysed with ELISA for IL- 1 . (Figure 25) 18. Comparison of the transfection efficiency of SubP-Ll containing 2 or 3 polyelectrolyte layers.

[0271] The transfection efficiency of SubP-Ll NPs containing 2 or 3 polyelectrolyte layers was determined. SubP-Ll NPs with 2 layers of polyelectrolytes were formulated as: CaCO, cores / DEXS / PARG / DEXS. SubP-Ll NPs with 3 layers of polyelectrolytes were formulated as: CaCOs cores / PARG / DEXS. DC2.4 cells were transfected with 500 ng mRNA encoding eGFP formulated with SubP-Ll NPs with 2 or 3 layers. The next day, cells were harvested, and readout was performed by flow cytometry for percentage (a) and MFI of eGFP (b). Figure 26 demonstrated similar transfection efficiency for SubP-Ll NPs with 2 and 3 layers.

[0272] 19. PrimeFlow® RNA assay

[0273] PrimeFlow® RNA assay (Thermo Scientific™, Waltham, Massachusetts, USA) of DC2.4 after transfection with Rhodamine B labelled SubP-Ll (RhB SubP-Ll). Briefly, DC2.4 cells were transfected with 250 ng of mRNA encoding eGFP formulated in Rhodamine B-SubP-Ll . After 0,5, 1, 2, 4, 6, and 24 h, DC2.4 cells were harvested and stained with the fixable viability dye eFluor 450. Subsequently, cells were fixated and permeabilized using appropriate buffers provided in the kit. The next day, in situ hybridization was conducted using an eGFP-specific probe to target untranslated mRNA-eGFP present in the cytoplasm. Uptake (RhB+), and translation (eGFP+) efficiency and indication of the endosomal release of the particles (mRNA- eGFP) were acquired via flow cytometry. Representative contour plots of DC2.4 cells transfected with RhB SubP-Ll at 4h timepoint (Figure 27a). Percentages and MFI of the uptake (RhB+), untranslated mRNA-eGFP and eGFP protein (eGFP+) (Figure 27b).

Claims

CLAIMS1.A multilayer nanoparticle comprising:- a core;- at least one cationic polymer layer and at least one anionic polymer layer which are layered one on top of the other, wherein the outermost layer of the nanoparticle is an anionic polymer layer; and- a nucleic acid-cationic peptide complex associated with the outermost anionic polymer layer, wherein the cationic peptide of the nucleic acid-cationic peptide complex is an amphipathic peptide.

2. The multilayer nanoparticle according to claim 1, wherein at least one nucleic acid-cationic peptide complex is associated with the anionic polymer, such as associated by means of an electrostatic interaction between the cationic peptide and the anionic polymer, or wherein the nucleic acid-cationic peptide complex is conjugated to one or more layers of the multilayer nanoparticle, such as to the outermost layer of the multilayer particle.3.The multilayer nanoparticle according to claim 1 or 2, wherein the outermost layer of the nanoparticle comprises the anionic polymer, optionally wherein the outermost layer comprises the anionic polymer and the at least one nucleic acid-cationic peptide complex is associated with the anionic polymer of the outermost layer.4.The multilayer nanoparticle according to claim 3, wherein at least one nucleic acid-cationic peptide complex is disposed on the outer surface of the outermost anionic polymer layer.5.The multilayer nanoparticle according to any one of the previous claims, wherein the anionic polymer is an anionic polysaccharide, such as, dextran sulfate, polyacrylic acid or a salt thereof.

6. The multilayer nanoparticle according to any one of the previous claims, wherein the cationic polymer comprises poly-L-arginine, poly(cystamine bisacrylamide-co-4-amino-l -butanol) (pABOL), trimethylammoniumbenzylchitosan (TMAB-CS) or a salt thereof.

7. The multilayer nanoparticle according to any one of the previous claims, wherein the cationic peptide of the nucleic acid-cationic peptide complex is an amphiphatic peptide, preferably LAH4-LI having the sequence set forth in SEQ ID NO: 1 (KKALLAHALHLLALLALHLAHALKKA) or LAH4-L1R having the sequence set forth in SEQ ID NO:2 (RRALLAHALHLLALLALHLAHALRRA) .

8. The multilayer nanoparticle according to any one of the previous claims, wherein the nucleic acid of the nucleic acid-cationic peptide complex is a DNA molecule or an RNA molecule or a hybrid RNA / DNA molecule, such as an RNA molecule selected from the group consisting of mRNA, saRNA, siRNA, gRNA, non-coding RNA, small non-coding RNA, long non-coding RNA, miRNA, circular RNA, taRNA, a synthetic analogue of RNA, and combinations thereof.9.The multilayer nanoparticle according to any one of the previous claims, wherein: the multilayer nanoparticle comprises an adjuvant, such as an adjuvant selected from a group consisting of a toll-like receptor (TLR)- agonist, RIG-I like receptor (RLR)- agonist, Nodlike receptor protein (NLRP)- agonist, iNKT agonist or derivates thereof, bacterial lipopolysaccharide (LPS) or derivates thereof, synthetic polynucleotides, and combinations thereof; and / or the nanoparticle contains an additional cargo, preferably a small molecule, and preferably located in the core.

10. A pharmaceutical composition comprising the multilayer nanoparticle as defined in any one of claims 1 to 9 and a pharmaceutically acceptable carrier, diluent, or excipient, optionally wherein the pharmaceutical composition is a vaccine.

11. The multilayer nanoparticle according to any one of claims 1 to 9, or the pharmaceutical composition according to claim 10, for use in medicine.

12. The multilayer nanoparticle as defined in any one of claims 1 to 9 or the pharmaceutical composition as defined in claim 10, for use in a method for delivering the nucleic acid to a subject; or where the nucleic acid encodes a protein, for use in a method for delivering the protein to a subject; or where the nucleic acid encodes an antigen, for use in a method for inducing an immune response to the antigen in a subject, optionally wherein the subject is human.

13. The multilayer nanoparticle or the pharmaceutical composition for use according to claim 12, wherein the antigen is causative of or associated with a disease in the subject, and inducing the immune response to the antigen in the subject is beneficial for prevention or treatment of the disease.

14. An in vitro or ex vivo method for introducing a nucleic acid into a cell, comprising exposing the cell to the multilayer nanoparticle as defined in any one of claims 1 to 9 or the pharmaceutical composition as defined in claim 10.

15. A method for preparing a multilayer nanoparticle according any one of claims 1 to 9 said method comprising: a) coating a core with at least one cationic polymer and at least one anionic polymer, whereby the at least one cationic polymer and at least one anionic polymer are layered one on top of the other; and b) adding at least one nucleic acid-cationic peptide complex to the coated core.

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