Systems, compositions, and methods for delivering functional or therapeutic proteins to target cells

Abstract

A system for obtaining a virus-like particle (VLP) comprises a first nucleic acid comprising a first nucleotide sequence encoding a first fusion polypeptide that comprises a retroviral gag polypeptide of the VLP and an adaptor polypeptide of a first kind; and a second nucleic acid comprising a second nucleotide sequence encoding a second fusion polypeptide that comprises an adaptor polypeptide of the first kind or of a second kind, and a functional or therapeutic polypeptide; wherein the adaptor polypeptides of the first and / or the second kind are configured to interact with each other such that during formation of the VLP in a VLP packaging cell the second fusion polypeptide is packaged into the VLP.

Description

New PCT-ApplicationBaldauf, Hanna-MariSalinas Illarena, AlejandroVossius Ref.: AK2314 PCTSYSTEMS, COMPOSITIONS, AND METHODS FOR DELIVERING FUNCTIONAL OR THERAPEUTIC PROTEINS TO TARGET CELLSFIELD OF INVENTION5

[0001] The present disclosure relates to virus-based systems and methods for producing and delivering functional or therapeutic proteins packaged in virus-like particles to target cells.INTRODUCTION

[0002] Intracellular protein delivery is challenging, because cellular membranes 0 are intrinsically non-permissive for large biomolecules like proteins. It is shown herein, that by exploiting the ability of viruses and engineered virus-like particles (VLPs) to fuse with target cells and by engineering various components of such VLPs it is possible to deliver a broad range of functional or therapeutic cargo proteins of interest to target cells to exert a wide variety of effects in vitro and in vivo. The novel protein delivery 5 platform described herein allows specific cargo protein loading into VLPs, quantification, and maturation of the cargo proteins to an essentially un-modified state, thereby preserving original cargo protein function as good as possible while obstructing VLP assembly as less as possible by overcoming direct fusion of viral structural proteins to cargo proteins as used currently in other VLP-based protein delivery tools.0

[0003] Most other viral vectors or VLPs have traditionally been used for DNA or RNA delivery. Direct protein deliveiy poses several advantages over the traditional method, including precise dosing of cargo protein without reliance on transcription and / or translation rates of target cells, as well as precise quality control of final cargo protein product before deliveiy avoiding e.g. misfolding of proteins of interest upon 5 RNA translation in target cells.

[0004] Exemplary prior art that describes the technical background of the present disclosure is given by: Schneider, C., Oellerich, T., Baldauf, HM. et al. SAMHD1 is a biomarker for cytarabine response and a therapeutic target in acute myeloid leukemia. Nat Med 23, 250-255 (2017)SUMMARY

[0005] The inventors of the present disclosure have developed novel VLPs (as well as novel systems and methods for producing such VLPs) that are derived from natural occurring retroviruses such as HIV-i. Aspects disclosed herein enable packaging of certain cargo proteins of interest inside the VLPs with high efficiency, specificity, and without significantly obstructing VLP assembly in suitable packaging cells. The VLPs disclosed herein can then be harvested, concentrated, and used for transferring the packaged proteins to target cells for inducing a wide range of effects depending on the type of packaged proteins and target cell. These novel VLPs can potentially be used both in research to deliver proteins to cells in culture and in patients to deliver therapeutically relevant proteins to treat diseases. An exemplary overview of aspects disclosed herein is illustrated in Fig. 1.

[0006] In some implementations of the present disclosure, engineered versions of a retroviral structural protein Gag, e.g., an HIV-1 structural protein Gag, fused to an adaptor domain (also designated adaptor polypeptide herein) in combination with a cargo protein of interest (also designated functional or therapeutic polypeptide herein) fused to a corresponding adaptor domain allow to package the cargo protein of interest inside retroviral derived, e.g., an HIV-1 derived VLP with high efficiency, and specificity without obstructing VLP assembly in a packaging cell due to a non-covalent effectively attractive interaction between both adaptor domains of both fusion polypeptides acting during VLP assembly in or by a suitable packaging cell. Herein, the adaptor polypeptide of the first kind preferably comprises or consists of SEQ ID NO: 12 (“ENQQLEQKNSQLKQEISQLEQEISQLEY”) or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto, and / or the adaptor polypeptide of the second kind preferably comprises or consists of SEQ ID NO: 13 (“KNSQLKEENSQLEEKIQQLKEKIQQLKY”) or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto. HIV-i derived VLP is preferably an HIV-i-derived Gag, and most preferably an HIV-i-derived Gag comprising or consisting of SEQ ID NO: 16 (“MGARASVLSGGELDRWEKIRLRPGGKKKYKLKHIVWASRELERFAVNPGLLETSEG CRQILGQLQPSLQTGSEELRSLYNTVATLYCVHQRIEIKDTKEALDKIEEEQNKSKKKA QQAAADTGHSNQVSQNYPIVQNIQGQMVHQAISPRTLNAWVKWEEKAFSPEVIPMF SALSEGATPQDLNTMLNTVGGHQAAMQMLKETINEEAAEWDRVHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMTHNPPIPVGEIYKRWIILGLNKIVRMYSPTSILDIR QGPKEPFRDYVDRFYKTLRAEQASQEVKNWMTETLLVQNANPDCKTILKALGPGATL EEMMTACQGVGGPGHKARVLAEAMSQVTNPATIMIQKGNFRNQRKTVKCFNCGKEG HIAKNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSHKGRPGNFLQSRPEP TAPPEESFRFGEETTTPSQKQEPIDKELYPLASLRSLFGSDPSSQ”) or a sequence being at least 8o% identical, preferably at least 90% identical, and most preferred at least 95% thereto.While in the appended examples the HIV-i-derived Gag of SEQ ID NO: 16 is illustrated as VLP, a plethora of alternatives are known for the prior. Non-limiting but preferred examples are retroviral-derived Gags selected any one of SEQ ID NOs 23 to 36 or a sequence being at least 80% identical, preferably at least 90% identical, and most preferred at least 95% thereto.

[0007] Nucleotide and amino acid sequence analysis and alignment in connection with the present invention are preferably carried out using the NCBI BLAST algorithm (Stephen F. Altschul, Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), Nucleic Acids Res. 25:3389-3402). BLAST can be used for nucleotide sequences (nucleotide BLAST) and amino acid sequences (protein BLAST). The skilled person is aware of additional suitable programs to align nucleic acid sequences. As defined herein, sequence identities of at least 80% identical, preferably at least 90% identical, and most preferred at least 95% are envisaged by the invention. However, also envisaged by the invention are with increasing preference sequence identities of at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.8%, and 100%.

[0008] In some implementations, a detector polypeptide / protein tag can be included in the cargo protein polypeptide for precise quantification of packaged proteins as well as one or more protease cleavage sites for adaptor domain cleavage during VLP maturation. In contrast to other previously described retrovirus-derived protein delivery particles, the VLPs can be engineered to be devoid of other viral components such as a reverse transcriptase and / or integrase enzymes of HIV-1 or similar, which could have an adverse side effect. Furthermore, certain surface proteins, transmembrane proteins and / or glycoproteins can be included into or on the VLPs hull, e.g., to enable binding and fusion to target cells expressing specific surfacemarkers. Herein, the detector polypeptide preferably comprises of consist of SEQ ID NO: 20 (“VSGWRLFKKIS”) or a sequence being at least 8o%, and preferably at least 90% identical thereto. With respect to the sequences being at least 8o% identical to SEQ ID NO: 20 it to be understood that these sequences retain the capability of SEQ ID NO: 20 to be a detector polypeptide.

[0009] More specifically, in some aspects, the present disclosure provides a first system for obtaining a virus-like particle (VLP) comprising a first nucleic acid (e.g., in form of a plasmid or similar construct) comprising a first nucleotide sequence encoding a first fusion polypeptide that comprises a retroviral gag polypeptide of the VLP and an adaptor polypeptide of a first kind, and a second nucleic acid comprising a second nucleotide sequence encoding a second fusion polypeptide that comprises an adaptor polypeptide of the first kind or of a second kind, and a functional or therapeutic polypeptide. The adaptor polypeptides of the first and / or the second kind are configured to interact with each other such that (e.g., via a non-covalent effectively attractive interaction; see Fig. 2, panel C for an example) during formation / assembly of the VLP in or at a VLP packaging cell the second fusion polypeptide is packaged into the VLP. Examples of the first fusion polypeptide and the second fusion polypeptide are shown in Fig. 2 (polypeptides Gag-Adp and Adp-Det-CS-CP, respectively; see also SEQ ID NO: 2 and SEQ ID NO: 7, respectively). Corresponding nucleotide sequences are given in SEQ ID NO: 1 (first nucleotide sequence) and SEQ ID NO: 6 (second nucleotide sequence), respectively. Wherever herein reference is made to a “system” also and alternatively a “kit” is contemplated herein. A system generally designates a combination of interacting components, while a kit generally designates a combination of parts that are assembled by user. Both options are applicable herein, while the system is preferred.

[0010] In connection with the different fusion polypeptides as described herein it is to be understood that the heterologous fusion partners can either be directly or indirectly fused to each other. Indirectly is preferred. In case of an indirect fusion preferably peptide linkers are used for the fusion, such that a GS-linkers or the linkers being described below.

[0011] In some implementations, the second fusion polypeptide may further comprise a protease cleavage site arranged in between the adaptor polypeptide of thefirst kind or of the second kind and the functional or therapeutic polypeptide. This protease cleavage site is for maintaining original cargo protein function as good as possible by delivering the cargo protein in a nearly unmodified state after adaptor cleavage. Further, the VLP may be a HIV-i derived VLP and / or the protease cleavage site maybe selected from the group consisting of wild-type HIV-i protease cleavage sites and engineered derivatives thereof. Herein, the HIV-i protease cleavage sites and / or engineered derivatives preferably comprises or consists of any one of SEQ ID NOs 14 and 85-87 or a sequence being at least 80%, and preferably at least 90% identical thereto. With respect to the sequences being at least 80% identical to any one of SEQ ID NOs 14 and 85-87 it is to be understood that these sequences retain the capability of SEQ ID NOs 14 and 85-87 to be a HIV-i protease cleavage site. Herein, the HIV-i protease cleavage sites and / or engineered derivatives most preferably comprises or consists of SEQ ID NO: 14 (“SATIMMORGN) or a sequence being at least 80%, and preferably at least 90% identical thereto. With respect to the sequences being at least 80% identical to SEQ ID NO: 14 it is to be understood that these sequences retain the capability of SEQ ID NO: 14 to be a HIV-i protease cleavage site.

[0012] In some implementations, the adaptor polypeptide of the first kind may be at least in part complementary to the adaptor polypeptide of the second kind or at least in part self-complementary. This partial complementarity causes a sufficiently strong non-covalent interaction between the first and the second polypeptides during VLP assembly that is at the same time weak enough not to obstruct VLP maturation after VLP assembly. For instance, some parts of the corresponding adaptor domains may be oppositely polarized or charged (see Fig.2). For example, the adaptor polypeptide of the first kind and / or the adaptor polypeptide of the second kind may comprise at least partially complementary coiled-coil (CC) domains, preferably complementary coiled-coil (CC) domains. Coiled-coils are a-helical structural protein motifs that wrap each other into supercoiled helices. They play essential roles as subunits that oligomerize protein complexes involved in biological processes or that form structural elements of biological materials (see, for review, Park (2020), Int J Mol Sci., 21(IO):3584). The complementary coiled-coil (CC) domains are preferably dimeric coiled-coil (CC) domains. The structure and interface of a dimeric coiled-coil domainsare known. A helical wheel diagram that presents amino acid residues located at the□ Hydrophobic 0 Polar [] Charged characteristic positions labeled abcdefg isAmino acids a and d are hydrophobic amino acids, g and e are charged amino acids and amino acids b, c and f are polar amino acids. The hydrophobic amino acids form the inner part of the coiled-coil while the polar amino acids form the outer part. The charged amino acids g and e interact with each other; i.e. a is positively charged and d is negatively charged or vice versa. In accordance with a preferred implementation, the adaptor polypeptide of the first kind comprises or consists of SEQ ID NO: 12 (“ENQQLEQKNSQLKQEISQLEQEISQLEY”) or sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto, and the adaptor polypeptide of the second kind SEQ ID NO: 13 (“KNSQLKEENSQLEEKIQQLKEKIQQLKY”) or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto. With respect to the sequences being at least 80% identical to SEQ ID NO: 12 and 13 it is to be understood that these sequences are preferably dimeric coiled-coil (CC) domains, i.e. they retain the capability of SEQ ID NOs 12 and 13 to bind to each other to form a dimeric coiled-coil structure.

[0013] The inventors found that it is beneficial for some implementations that the adaptor polypeptide of the first kind and / or the adaptor polypeptide of the second kind may comprise at least 15, preferably at least 20, and more preferably at least 25 amino acids to ensure a strong enough interaction and / or that the adaptor polypeptide of the first kind and / or the adaptor polypeptide of the second kind may comprise at most 200, preferably at most 100, more preferably at most 50, and even more preferably at most 30 amino acids to prevent the interaction to become too strong and / or to minimize space required for the adaptor domains inside the VLP and / or to avoid making too large modifications to Gag preventing VLP assembly. Alsocontemplated herein are the ranges that can be formed by the afore-described minimum and maximum lengths of the adaptor polypeptide of the first kind and / or the adaptor polypeptide of the second kind. Examples of the ranges are with increasing preference 15 to 100 amino acids, 20 to 50 amino acids, and 25 to 30 amino acids. In this manner, cargo protein packaging can be optimized while simultaneously minimizing detrimental effects on VLP assembly and maturation.

[0014] In some implementations, the system described above may further comprise a third nucleic acid comprising a third nucleotide sequence encoding a third fusion polypeptide that comprises an adaptor polypeptide of the first kind or of the second kind, and a protease polypeptide that is adapted to cleave the protease cleavage site (see Fig. 2, polypeptide Adp-PR for an example as well as SEQ ID NO: 11). A corresponding nucleotide sequence is given in SEQ ID NO: 10. Optionally, the protease polypeptide may comprise a viral protease, preferably an HIV-i derived protease.In some implementations, the protease polypeptide of the third fusion polypeptide may comprise a natural protease cleavage site arranged at the N-terminus of the protease polypeptide. This mimics the natural protease environment, in which the protease fully liberates itself from the natural HIV-i Gag-Pol polypeptide during virion maturation. The protease cleavage site herein preferably comprises or consists of SEQ ID NO: 14 (“SATIMMQRGN) or a sequence being at least 80%, and preferably at least 90% identical thereto, or SEQ ID NO: 15 (“ERQANFLGKI”)=or a sequence being at least 80%, and preferably at least 90% identical thereto. While the protease cleavage sites of SEQ ID NOs 14 and 15 are illustrated by the appended examples, additional suitable protease cleavage sites from HIV-i are known. Non-limiting but preferred examples are the protease cleavage sites of SEQ ID NOs 37 to 46 or a sequence being at least 80%, and preferably at least 90% identical thereto. With respect to the sequences being at least 80% identical to SEQ ID NOs 14 and 15 or any one of SEQ ID NOs 37 to 45 it is to be understood that these sequences retain the capability of SEQ ID NOs 14 and 15 or any one of SEQ ID NOs 37 to 45 to be a HIV-i protease cleavage site.

[0015] The system described above may also comprise a fourth nucleic acid comprising a fourth nucleotide sequence encoding a first surface protein of the VLP adapted to enable fusion of the VLP with a target cell (see Fig. 2 for an example). In some implementations, the first surface protein may be further adapted to enable binding of the VLP to the target cell and / or the first surface protein may compriseVesicular Stomatitis Virus Glycoprotein (VSV-G) or derivatives thereof or other viral glycoproteins known to be pseudotyped with lentiviral particles.

[0016] In other implementations, the fourth nucleotide sequence or a fifth nucleotide sequence of a fifth nucleic acid may further encode a second surface protein of the VLP adapted to enable binding of the VLP to one or more selected target cell types, here, optionally, the first surface protein may be mutated such that it is no longer adapted to enable binding of the VLP to the target cell but is still capable of being presented at the surface of the VLP and fusing with the target cell once binding occurred. Further, the second surface protein of the VLP preferably comprises in addition one or more cell-type specific antibody fragments. In this case, the second surface protein is generally composed of an intracellular domain fused to a transmembrane domain which incorporates into the VLPs and an extracellular domain mediating binding to target cells like an antibody fragment (preferably scFv).Preferably, this fusion protein comprises or consists of the second surface protein of the VLP including one of the chains of the cell-type specific antibody fragments, such that one or more cell-type specific antibody fragments can be formed on the surface of the VLP that are bound to the VLP. The antibody fragments are preferably Fab or Fab’ fragments, Fd, F(ab')2, Fv or scFv fragments, single domain VH or V-like domains, such as VhH or V-NAR-domains. In the working examples scFv and the intracellular and transmembrane domains of CD8a and PDFGRb are illustrated. In more detail, the fusion proteins IgK-scFvGemtuzumab-HA-CD8a (nucleotide sequence SEQ ID NO: 70 and amino acid sequence SEQ ID NO: 71) and IgK-scFvGemtuzumab-HA-IgGq-PDFGRb (nucleotide sequence SEQ ID NO: 72 and amino acid sequence SEQ ID NO: 73) are exemplified. In addition, it is shown in Fig. 14 that the VLPs can be engineered to fuse with cells expressing specific surface markers only. Panel a depicts how the broadly tropic VSV-G leading to unspecific fusion of VLPs can be mutated and coexpressed with scFv antibody fragments on the VLP surface to modulate the fusion specificity, while Panel b illustrates that VLPs pseudotyped with VSV-G WT can transduce 5 different cell lines very efficiently, while VSV-G K47Q R354A only has limited fusogenicity or no fusogenicity in some cell lines.

[0017] In this manner, cell-type specific protein delivery can be achieved for a wide range of cargo proteins and target cell types enabling new therapy paradigms for awide range of prevalent diseases. In this connection it to be understood that a functional polypeptide is a polypeptide that can exert a biological function within a cell. A therapeutic polypeptide is a polypeptide when being administer to a subject having a disease exerts a curative effect on at least one sign or symptom of the disease. For example, in a first class of therapeutic applications, the functional or therapeutic polypeptide maybe selected from the group consisting of bacterial toxins such as diphteria toxin, or cytotoxic proteins and cell death-inducing proteins such as Granzyme B, Caspase-3, or the intracellular domain of CD95 or BAX adapted for killing of cell lines in vitro or killing of cancerous cells in vivo. In the latter in vivo case the subject has cancer and the therapeutic polypeptide exerts a curative effect on at least one sign or symptom of cancer by the killing of cancerous cells.

[0018] In a second class of applications, the functional or therapeutic polypeptide may be selected from the group consisting of: enzymes for intracellular catalysis of chemical reactions such as beta-lactamase, an immunomodulatory protein for the downregulation (e.g. FOXP3) or the upregulation of immune function (e.g. constitutively active STING or cGAS mutants), a protein that influences the degradation of specific proteins in the target cells to modulate protein levels such as Parkin with many cellular targets or Vpx inducing SAMHD1 degradation, designer nucleases or recombinases for genomic editing of cell lines in vitro or gene therapy purposes in vivo such as Cas9, Casi2a, or I-Crel, intrabodies for intracellular binding of target proteins, and fluorescent or light emitting proteins such as GFP or Renilla luciferase.

[0019] In a third class of applications the functional or therapeutic polypeptide may be selected from the group consisting of transcription factors to induce or repress gene expression such as the tunable dCas9-VP64 (induction), KRAB-dCas9 (repression) polypeptides, or the master regulator of autophagy TFEB, RNA-binding proteins complexed with RNA molecules to influence gene expression in target cells such as the M2 coat protein bound to custom RNA molecules, signaling cascade components to trigger cellular responses like the NF-KB inhibitor IKBCX, intracellular vaccination agents like the Papillomavirus E7 protein, tumor suppressor proteins such as p53 or PTEN, and anti-aging or lifespan-enhancing proteins such as the telomerase enzyme or SIRT1.IO

[0020] In some implementations, the second fusion polypeptide may further comprises a detector polypeptide that may be arranged between the adaptor polypeptide of the first kind or the second kind and the protease cleavage site or that may be arranged between the protease cleavage site and the functional or therapeutic polypeptide or that maybe arranged at an end of the adaptor polypeptide of the first kind or the second kind or at an end of the functional or therapeutic polypeptide. As discussed in more detail below, such a detector polypeptide may be used for quantification and characterization of obtained and functional VLPs. For example, the detector polypeptide may comprise an epitope tag capable of producing a bioluminescent signal and / or an (entire) luciferase producing a bioluminescent signal.

[0021] As mentioned above, the first, the second, the third, the fourth and / or the fifth nucleic acid may be comprised in a vector, for example, they may be part of a respective transfection plasmid capable of transfecting suitable VLP packaging cells (see Fig. 1). The vector is preferably an expression vector capable of expressing the first, the second, the third, the fourth and / or the fifth nucleic acid. The term “vector” in accordance with the invention means preferably a plasmid, cosmid, virus, bacteriophage or another vector used e.g. conventionally in genetic engineering which carries the nucleic acids of the invention. The nucleic acids of the invention may, for example, be inserted into several commercially available vectors. Non-limiting examples include prokaiyotic plasmid vectors, such as of the pUC-series, pBluescript (Stratagene), the pET-series of expression vectors (Novagen) or pCRTOPO (Invitrogen) and vectors compatible with an expression in mammalian cells like pREP (Invitrogen), pCAGGS (e.g. Addgene), pcDNAs (Invitrogen), pCEPq (Invitrogen), pMCineo (Stratagene), pXTi (Stratagene), pSG5 (Stratagene), EBO-pSV2neo, pBPV-1, pdBPVMMTneo, pRSVgpt, pRSVneo, pSV2-dhfr, PIZD35, pLXIN, pSIR (Clontech), pIRES-EGFP (Clontech), pEAK-10 (Edge Biosystems) pTriEx-Hygro (Novagen) and pCINeo (Pro mega). The nucleic acid molecules inserted into the vector can e.g. be synthesized by standard methods, or isolated from natural sources. Ligation of the coding sequences to transcriptional regulatory elements and / or to other amino acid encoding sequences can also be carried out using established methods. Transcriptional regulatory elements (parts of an expression cassette) ensuring expression in cells are well known to those skilled in the art. These elements comprise regulatory sequences ensuring the initiation of transcription (e. g., translation initiation codon, promoters,such as naturally-associated or heterologous promoters and / or insulators; see above), internal ribosomal entry sites (IRES) (Owens, Proc. Natl. Acad. Sci. USA 98 (2001), 1471-1476) and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptional as well as translational enhancers. Preferably, the polynucleotide encoding the polypeptide / protein or fusion protein of the invention is operatively linked to such expression control sequences allowing expression in cells. The vector may further comprise nucleic acid sequences encoding secretion signals as further regulatory elements. Such sequences are well known to the person skilled in the art. Furthermore, depending on the expression system used, leader sequences capable of directing the expressed polypeptide to a cellular compartment may be added to the coding sequence of the polynucleotide of the invention. Such leader sequences are well known in the art. Furthermore, it is preferred that the vector comprises a selectable marker. Examples of selectable markers include genes encoding resistance to neomycin, ampicillin, hygromycine, and kanamycin. Specifically-designed vectors allow the shuttling of DNA between different hosts, such as bacteria-fungal cells or bacteria-animal cells (e. g. the Gateway system available at Invitrogen). An expression vector according to this invention is capable of directing the replication and the expression of the polynucleotide and the encoded peptide or fusion protein of this invention. Apart from introduction via vectors such as phage vectors or viral vectors (e.g. adenoviral, retroviral), the nucleic acid molecules as described herein above maybe designed for direct introduction or for introduction via liposomes into a cell.

[0022] Further aspects of the present disclosure relate to a second system for obtaining a virus-like particle (VLP) comprising a first nucleic acid comprising a first nucleotide sequence comprising a translational frameshift element, wherein, without translational frameshifting, the first nucleotide sequence (e.g., SEQ ID NO: 3) encodes a first fusion polypeptide (e.g., SEQ ID NO: 4) comprising a retroviral gag polypeptide of the VLP and an adaptor polypeptide of a first kind, wherein, with translational frameshifting, the first nucleotide sequence encodes a second fusion polypeptide (e.g., SEQ ID NO: 5) comprising the retroviral gag polypeptide of the VLP, and a protease polypeptide that is adapted to cleave a protease cleavage site, and a second nucleic acid comprising a second nucleotide sequence encoding a third fusion polypeptide that comprises an adaptor polypeptide of the first kind or of a second kind, and a functional or therapeutic polypeptide. The adaptor polypeptides of the first and / or the secondkind are configured to interact with each other such that during formation of the VLP in a VLP packaging cell the third fusion polypeptide is packaged into the VLP - as described in detail above for similar systems. The above-described implementations and preferred embodiments as described in connection with the first system apply mutatis mutandis to the second system as far as being amenable for combination therewith. Moreover, a schematic representation of the switch from the Gag-FS-CCp to the Gag-FS-PR open reading frames at the ribosomal shift area is shown in Fig. 10.

[0023] In some implementations, the translational frameshifting element is preferably a ribosomal frameshift region, wherein the ribosomal frameshift region preferably comprises of consists of SEQ ID NO: 21 C’agagacaggctaattttttagggaagatctggccttcccacaagggaaggccaggga”) or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto, wherein the translational frameshifting element is within the first nucleic acid C-terminal of the nucleic acid sequence encoding the retroviral gag polypeptide of the VLP and N-terminal of the nucleic acid sequences encoding the adaptor polypeptide of a first kind and the protease polypeptide that is adapted to cleave a protease cleavage site. Ribosomal frameshifting refers to a translational recoding phenomenon where ribosomes shift into a new reading frame by +1 or -1 nucleotide, typically allowing synthesis of an extended fusion protein by bypassing an in-frame stop codon. It involves specific mRNA elements that are designated herein “translational frameshifting elements". In viruses, ribosomal frameshifting maybe programmed to occur at particular sites (i.e. the translational frameshifting elements). Ribosomal frameshifting allows to encode multiple types of proteins from the same mRNA.Translational frameshifting elements are known from various virus types, such as HIV-1 (human immunodeficiency virus), RSV (Rous sarcoma virus) [8] and the influenza virus. While the ribosomal frameshift region of SEQ ID NO: 21 is illustrated in the examples a number of alternative ribosomal frameshift regions are known, Nonlimiting but preferred examples are the ribosomal frameshift regions select from any of SEQ ID Nos 46 to 57 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto. With respect to the sequences being at least 80% identical to SEQ ID NO: 21 or any of SEQ ID Nos 46 to 57 it is to be understood that these sequences retain the capability of SEQ ID NO: 21 or any of SEQ ID Nos 46 to 57 to induce translational frameshifting.

[0024] In some implementations, (see Fig. 2 for an example based on HIV-i derived VLPs), the second fusion polypeptide may further comprise one or more of a protease cleavage site, a synthetic transframe polypeptide, preferably comprising 70% or more of amino acids with small and / or polar non-charged side chains, and a protease regulation domain, such as a retroviral p6pol polypeptide, optionally an HIV-i p6pol polypeptide. Herein, the HIV-i p6pol polypeptide preferably comprises or consists of SEQ ID NO: 22 (“FLREDLAFPQGKAREFSSEQTRANSPTRRELQVWGRDNNSLSEAGADRQGTVSFSF” ) or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto. With respect to the sequences being at least 80% identical to SEQ ID NO: 22 it is to be understood that they retain the capability of SEQ ID NO: 22 to be a HIV-i p6pol protease fusion protein.

[0025] Such a synthetic transframe polypeptide (STF) is the result of artificially moving the translational frameshift element to the end of the Gag open reading frame and creating a single nucleotide sequence expressing both Gag fused to an adaptor domain and Gag fused to a viral protease. Sequentially, the coding region for the adaptor domain is closer to the frameshift element and its amino acid sequence needs to be preserved fully in order to ensure adaptor integrity. The p6pol and viral protease domains are further away from the frameshifting element and encoded in frame -1 respective of the adaptor domain. The -1 coding region overlapping with the adaptor domain needs to be overcome without random stop codons in order to allow p6pol and viral protease expression upon ribosomal frameshifting. This overlapping area was optimized to eliminate these undesired stop codons and to favor small, polar, uncharged amino acids wherever possible. Thus, when a ribosomal frameshifting happens, the resulting Gag construct has an unordered STF peptide right at its C-terminus, functioning like a linker region before the functional p6pol and protease domains.

[0026] As conclusively shown by the experiments discussed below with reference to Fig. 4 to Fig 6, such a system allows for efficient, robust and specific cargo protein packaging into maturing VLPs without significantly obstructing VLP formation. Cotransfection of nucleic acids comprising the nucleotide sequences encoding wild type (WT) Gag or Gag-Pol polypeptides is not necessary to achieve VLP formation. Contrary to prior art (WO 2020 / 102709 Al), where Gag has been modified with functionaldomains totaling an additional 1505 amino acids and VLPs from this polypeptide sequence cannot assemble on their own, our Gag constructs described herein (e.g., polypeptides shown in Fig. 2 Gag-Adp and Gag-FS-Adp, see also SEQ ID NO: 2 and SEQ ID NO: 4, respectively) are able to generate high yield VLP productions with their smaller and engineered Gag modifications of under 60 amino acids in length. This is described in more detail in reference to Gag-FS-Adp, wherein the additional amino acid number may amount to of 55.

[0027] In some preferred implementations, the translational frameshift element is arranged in the first nucleotide sequence between a first region encoding the retroviral gag polypeptide of the VLP and between a second region encoding the protease polypeptide or between a first region encoding the retroviral gag polypeptide of the VLP and second region encoding the adaptor polypeptide. This allows Gag-FS-Adp and Gag-FS-PR expression from the same nucleic acid (presumably in the naturally optimal ratio for VLP maturation of 5-10% Gag-FS-PR). Further, it may yield particles with enhanced functionality when compared to transPR VLPs. Additionally, this moving of the frameshift region might be used beyond the scope of this patent and protein delivery purposes. For example, to generate Gag VLPs with modified Gag on the C-terminus for other purposes, while simultaneously expressing the Gag-Protease from the same DNA sequence to generate mature particles.

[0028] In some implementations the VLP may be an HIV-1 derived VLP, and / or the protease cleavage site may be selected from the group consisting of wild-type HIV-i protease cleavage sites and engineered derivatives thereof, and / or the protease polypeptide may comprise a viral protease, preferably an HIV-i protease. Further, the adaptor polypeptide of the first kind may be at least in part complementary to the adaptor polypeptide of the second kind or at least in part self-complementary - as discussed in more detail above in connection with the first system. Further, the adaptor polypeptide of the first kind and / or the adaptor polypeptide of the second kind may comprise at least partially complementary coiled-coil domains. As also discussed in more detail above, the adaptor polypeptide of the first kind and / or the adaptor polypeptide of the second kind may comprise at least 15, preferably at least 20, and more preferably at least 25 amino acids, and / or the adaptor polypeptide of the first kind and / or the adaptor polypeptide of the second kind may comprise at most 200, preferably at most 100, more preferably at most 50, and even more preferably at most30 amino acids. Also contemplated herein are the ranges that can be formed by the afore-described minimum and maximum lengths of the adaptor polypeptide of the first kind and / or the adaptor polypeptide of the second kind. Examples of the ranges are with increasing preference 15 to 100 amino acids, 20 to 50 amino acids, and 25 to 30 amino acids.

[0029] In some implementations, the system may comprise a third nucleic acid comprising a third nucleotide sequence encoding a first surface protein of the VLP adapted to enable fusion of the VLP with a target cell, wherein the first surface protein may be further adapted to enable binding of the VLP to the target cell, and / or wherein the first surface protein may comprise VSV-G or derivatives thereof or other viral glycoproteins known to be pseudotyped with lentiviral particles.. Further, the third nucleotide sequence or a fourth nucleotide sequence of a fourth nucleic acid may further encode a second surface protein of the VLP adapted to enable binding of the VLP to one or more selected target cell types, wherein the first surface protein may not be adapted to enable binding of the VLP to the target cell, and / or wherein the second surface protein of the VLP may comprise one or more cell-type specific antibody fragments. It is of note that the above-described fourth and fifth nucleic acid of the first system corresponds to the third and fourth nucleic acid of the second system. Hence, what is described herein above for the fourth and fifth nucleic acid of the first system applies mutatis mutandis to the third and fourth nucleic acid of the second system.

[0030] In this manner, cell-type specific protein delivery can be achieved for a wide range of cargo proteins and target cell types enabling new therapy paradigms for a wide range of prevalent diseases. For example, in a first class of therapeutic applications, the functional or therapeutic polypeptide maybe selected from the group consisting of a bacterial toxin, a cytotoxic protein, and a cell death-inducing protein adapted for killing of cell lines in vitro or killing of cancerous cells in vivo.

[0031] In a second class of applications, the functional or therapeutic polypeptide may be selected from the group consisting of: an enzyme for intracellular catalysis of chemical reactions, an immunomodulatory protein for the downregulation or the upregulation of immune function, a protein that influences the degradation of specific proteins in the target cells to modulate protein levels, designer nucleases orrecombinases for genomic editing of cell lines in vitro or gene therapy purposes in vivo, and a fluorescent or light emitting protein.

[0032] In a third class of applications the functional or therapeutic polypeptide may be selected from the group consisting of a transcription factor to induce or repress gene expression, an RNA-binding protein complexed with RNA molecules to influence gene expression in target cells, a signaling cascade component to trigger cellular responses, a vaccination agent, a tumor suppressor protein, and an anti-aging or lifespan-enhancing protein.

[0033] In some implementations, the second fusion polypeptide may further comprise a detector polypeptide that may be arranged between the adaptor polypeptide of the first kind or the second kind and the protease cleavage site or that maybe arranged between the protease cleavage site and the functional or therapeutic polypeptide or that maybe arranged at an end of the adaptor polypeptide of the first kind or the second kind or at an end of the functional or therapeutic polypeptide. As discussed in more detail below, such a detector polypeptide may be used for quantification and characterization of obtained and functional VLPs. For example, the detector polypeptide may comprise an epitope tag capable of producing a bioluminescent signal and / or an (entire) luciferase producing a bioluminescent signal.

[0034] The present disclosure also provides a virus-like particle (VLP) packaging cell, comprising a system (i.e. the first or second system) as described above, as well as a corresponding method of obtaining or for producing such a virus-like particle (VLP) comprising a functional or therapeutic polypeptide, comprising: introducing, preferably transfecting the system above into a virus-like particle (VLP) packaging cell, and obtaining the VLPs produced by the VLP packaging cell based on the introduced system.

[0035] Thus, the present disclosure also provides a virus-like particle (VLP) obtained or being obtainable by such a method. The present disclosure furthermore provides a virus-like particle (VLP) comprising or packaging (i) a first fusion polypeptide that comprises a retroviral gag polypeptide of the VLP and an adaptor polypeptide of a first kind; and a second fusion polypeptide that comprises an adaptor polypeptide of the first kind or of a second kind, and a functional or therapeutic polypeptide; wherein the adaptor polypeptides of the first and / or the second kindinteract with each other within the VLP; or (ii) a first fusion polypeptide comprising a retroviral gag polypeptide of the VLP and an adaptor polypeptide of a first kind; a second fusion polypeptide comprising the retroviral gag polypeptide of the VLP, and a protease polypeptide that is adapted to cleave a protease cleavage site; and a third fusion polypeptide that comprises an adaptor polypeptide of the first kind or of a second kind, and a functional or therapeutic polypeptide; wherein the adaptor polypeptides of the first and / or the second kind interact with each other within the VLP. For example, via choosing suitable cargo proteins and surface proteins / glycoproteins as discussed above and with reference to Fig. 2 below such a VLP may be engineered and obtained for use in the treatment of cancer, for use in the treatment of an autoimmune disease, for use in gene therapy, for use in the treatment of a neurologic disorder, and / or for use in the treatment of an infectious disease.

[0036] The present disclosure also provides one or more vectors (e.g. one or more transfection plasmids) encoding polypeptides derived from the first nucleic acid and the second nucleic acid, and, if present, the third nucleic acid, fourth nucleic acid, and / or fifth nucleic acid of the system (i.e. the first or second system) of the invention.

[0037] Yet further the present invention relates to the use of the above-described VLP of the invention for the infection of an ex vivo or in vitro cell.

[0038] Further details of the aspects described generally above are discussed in the following with reference to exemplary implementations illustrated by the drawings. The foregoing broadly outlines the features and technical advantages of examples in accordance with the present disclosure in order that the detailed description that follows may be better understood. Additional features and advantages will be described hereinafter. The conception and specific examples disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present disclosure. Characteristics of the concepts disclosed herein, both their organization and method of operation, together with associated advantages will be better understood from the following description when considered in connection with the accompanying drawings. Each of the drawings is provided for the purposes of illustration and description, and not as a definition of the limits of the claims.

[0039] In contrast to prior art technology using an HIV-i-based viral particle mediated protein delivery, the VLPs obtainable via the systems and methods disclosed herein may not contain a full-length Gag-Pol sequence to enable VLP assembly andcargo protein packaging as well as, in some cases, may allow eliminating other viral components such as the reverse transcriptase and integrase enzymes to reduce potentially adverse side effects for therapeutic use. Moreover, direct fusion of Gag with the cargo protein can be avoided using the adaptor domains disclosed herein enabling non-covalent interaction-based cargo protein loading making co-expression of wildtype Gag-Pol no longer necessary. The Gag-adaptor constructs disclosed herein lead to viable VLP formation on their own - as illustrated by the exemplary experimental results discussed below with reference to Figs. 4 to 6.BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Fig. 1 illustrates an exemplary overview of several aspects of a typical implementation of a protein delivery system and related methods disclosed herein. Fig.1 was created with Biorender.com.

[0041] Fig. 2 illustrates a juxtaposition of exemplary VLPs and exemplary components thereof. Fig. 2 was created with Biorender.com.

[0042] Fig. 3 illustrates an exemplary method for obtaining the VLPs as per the present invention. Fig. 3 was created with Biorender.com.

[0043] Fig. 4 shows results of a detector assay experiment with VLPs based on HIV-i as shown in Fig. 2 illustrating the quantification of cargo protein levels with the example of Vpx as the cargo protein.

[0044] Fig. 5 illustrates functionality of the same VLPs shown in Fig.4 in terms of the ability of the packaged cargo protein Vpx to downregulate SAMHD1 levels upon fusion with THP-1 cells.

[0045] Fig. 6 shows SDS-PAGE and Western blot analysis of VLPs.

[0046] Fig. 7 shows the superiority of the system of the invention (CCp / CCq adaptor pair) to package Vpx into VLPs compared to coiled coils previously reported in the context of VLP assembly (P3 / P4 pair from An et al. (2024), Biotechnol., 42,11526-1537 and W02023102538A1). Panel a illustrates the differences in charge distribution on the coiled coils for successful interaction. Fig. 7a was created with Biorender.com Panel b represents AlphaFold structures of the coiled coil pairs, highlighting how CCp and CCq have a net charge attraction with total peptide charges of -4 and +1, respectively, while the P3 / P4 pair has a net repulsion with total peptide charges of -4 and -1, respectively. Panel c shows Detector levels in the supernatant from VLP production in HEK293T cells. There is improved Vpx packaging when the CCp / CCq pair is used over the P3 / P4 pair, both in the transPR and the cisPR VLP system. Panel d displays Western blot images from this VLP production process, demonstrating undesired premature processing of Gag for transPR VLPs with the P3 / P4 pair in the cell lysates, resulting in lower VLP yields in the supernatant. Additionally, Vpx with CCq has lower levels in the cell lysate and higher levels in the purified VLPs compared to Pq-Vpx, indicating superior enrichment ability of CCp / CCq in the VLPs.

[0047] Fig. 8 shows the optimization process using different natural HIV-1 protease cleavage sites to release the adaptor (CCq) and the Detector (Det) domains from the cargo protein Vpx. Panel a shows the 4 different natural HIV-1 cleavage sites tested with their respective flanking Gag domains and their precise amino acid sequences. Fig. 8a was created with Biorender.com Panel b illustrates the VLP production process in HEK293T cells with Vpx as cargo protein with the different cleavage sites both the transPR and cisPR VLP versions. Overall, the choice of cleavage site in the cleavage site does not influence incorporation much. Panel c demonstrates that Vpx- VLPs with cleavage site 2 to process the cargo protein have the highest SAMHDi degradation ability when the VLPs are applied to THP-1 cells. SAMHD1 levels are determined here by flow cytometry.

[0048] Fig. 9 shows the versatility of our cisPR VLP system to specifically package proteins of interest other than Vpx. Panel a illustrates how cargo proteins lacking the adaptor domain (CCq) cannot bind to Gag and do not get enriched in the VLPs during the production process in HEK293T cells. Fig. 9a was created with Biorender.com Panel b displays a Western blot with VLPs produced in HEK293T cells and loaded with different cargo proteins. While the structural protein Gag exhibits similar levels throughout the cargo protein panel, the different cargo proteins exhibit varied enrichment levels. Cargo proteins without CCq do not get enriched at all, or atmuch lower levels than their CCq-containing counterparts. Panels c and d show cargo protein VLP packaging data quantified via the bioluminescent detector, reflecting the greatly improved packaging for those peptides containing the CCq adaptor. Panel e demonstrates the versatility of our VLP system, since there is no correlation of packaging efficiency with protein mass in kDa or protein net charge (isoelectric point value; pl) with the panel of n different cargo proteins tested. (Legend of cargo protein abbreviations - mEGFP: monomeric enhanced green fluorescent protein; BlaM: betalactamase; mSTING A l - 133: murine stimulator of interferon genes lacking the first 133 amino acids with the transmembrane domain; DT-A G52E: diphtheria toxin subunit A mutant G52E rendering the toxin inactive; NES-NLS-Casq: Casq with nuclear export signals and nuclear localization signals; CD95K intracellular domain of CD95; SuperTEV: catalytically enhanced version of the tobacco etch virus protease.)

[0049] Fig. 10 schematically represents the switch from the Gag-FS-CCp to the Gag-FS-PR open reading frames at the ribosomal shift area. This construct encoded in one single DNA sequence yields both structural components of cisPR VLPs. Fig. 10 was created with Biorender.com.

[0050] Fig. 11 panel a shows flow cytometry data with mean fluorescence intensity of Jurkat cells treated with VLPs containing either mEGFP, Sumire, or VpxQ 6A. The fluorescent proteins mEGFP and Sumire lead to a dose-dependent increase of fluorescence only in their respective flow cytometry channels FITC and DAPI. Treatment with the non-fluorescent control VpxQ 6A does not increase fluorescence in any flow cytometry channel. Panel b displays flow cytometry data with the percentage of Jurkat cells successfully transduced with BlaM- VLPs. Intracellular delivery of beta-lactamase (BlaM) is commonly used in virology for investigating viral fusion. In the cytoplasm, BlaM can catalyze a dye conversion measurable by flow cytometry in the V450 channel. Control VLPs with VpxQ 6A do not result in any V450 signal. Panel c represents the production of Casq-VLPs, which do not only load the nuclease protein, but also the co-expressed gRNA against the B2M gene in this case. Casq-VLPs can then fuse with Jurkat cells, for example, and cause double strand (ds) breaks in these target cells. Fig. 11c was created with Biorender.com Panel d shows the percentage of B2M-negative Jurkat cells when treated with different amounts of Cas9-gRNA(B2M)-VLPs. As comparison, the recently developed system by Hamilton et al.2024 (Nat Biotechnol., 42:1684-1692), has been tested side by side with no statistically significant differences in terms of performance. The VLPs developed by Hamilton et al.2024 are also HJV-i-based, but unlike our VLPs, still contain potentially pathogenic components of the virus like the reverse transcriptase and integrase enzymes.Additionally, the particles by Hamilton et al. 2024 rely on fusing Gag and Cas9 together for incorporation. This fusion results in particles not forming on their own unless wild type unmodified Gag is co-expressed in the producer cells. In our VLP system, expression of the transPR or cisPR structural protein leads to VLP assembly on its own without the need to co-express WT Gag.

[0051] Fig. 12 panel a depicts the mode of action of the toxic cargo proteins delivered with VLPs. Fig. 12a was created with Biorender.com Panel b shows the percentage of Zombie violet dye positive and negative Jurkat cells after being treated with VLPs loaded with different cargo proteins and analyzed via flow cytometry. Both the intracellular domain of CD95 (CD95i) and the diphtheria toxin subunit A wild type version (DT-A WT) increase the Zombie violet positive dye cell population in a dosedependent manner indicating toxicity in the Jurkat cells. Panel c shows a related experiment to panel b conducted with the murine T cell line EL4 and treated with different cargo proteins. DT-A WT delivered in the VLPs also increases the Zombie violet positive population in EL4 cells. The murine stimulator of interferon genes (mSTING) WT version and N153S constitutively active mutant increased the Zombie violet dye population as well. AWestern blot analysis of these cells confirms that the toxic effect is mediated via phosphorylation of the STING downstream effector TBK1 and activation of caspase 3 (panel d). Overall, we show here, that our VLP system can be used for mammalian cells other than human cells. Panel e demonstrates in more detail the cytotoxic and dose-dependent effect of DT-A and the intracellular domain of CD95, as well as its isolated death domain, in THP-i and SupTi cells using a resazurin-based viability assay.

[0052] Fig. 13 demonstrates that our VLP system can also be used to study enzyme activity in vitro once the cargo protein of interest has been enriched in the VLPs and the particles have been purified. Panel a depicts the principle of the assay used turning coumberone to fluorescent coumberol to measure enzyme activity in the reductase AKR1C family. Fig. 13a was created with Biorender.com Panel b shows datawith the increase in coumberol over time when io pM coumberone solutions are treated with cargo proteins released from VLPs by lysis with a 0.5 % CHAPS detergent solution. AKR1C3 extracted from VLPs successfully catalyzes coumberone reduction allowing for kinetic parameter determinations.

[0053] Fig. 14 demonstrates that the VLPs can be engineered to fuse with cells expressing specific surface markers only. This targeting strategy has been adapted from: Dobson et al. 2022, Nat. Methods. 19:449-460. Panel a depicts how the broadly tropic VSV-G leading to unspecific fusion of VLPs can be mutated and co-expressed with scFv antibody fragments on the VLP surface to modulate the fusion specificity. Fig. 14a was created with Biorender.com Panel b shows that VLPs pseudotyped with VSV-G WT can transduce 5 different cell lines very efficiently, while VSV-G K47Q R354A only has limited fusogenicity or no fusogenicity in some cell lines. The addition of Gemtuzumab (anti-CD33) scFv in conjunction with the mutated VSV-G restores the fusogenicity in CD33-positive cells only.DETAILED DESCRIPTION

[0054] Various aspects of the present disclosure are described in more detail hereinafter with reference to the accompanying drawings. The present disclosure may, however, be implemented in many different forms and should not be construed as limited to any specific structure or function presented herein. Rather, these aspects are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the present disclosure to those skilled in the art. Based on the teachings herein one skilled in the art should appreciate that the scope of the present disclosure is intended to cover any aspect of the present disclosure disclosed herein, whether implemented independently of or combined with any other aspect of the present disclosure. For example, an apparatus, a device or a system maybe implemented, or a method may be practiced using any number of the aspects set forth herein. In addition, the scope of the present disclosure is intended to cover such a device, apparatus, system or method which is practiced using other structure, functionality, or structure and functionality in addition to or other than the various aspects of the present disclosure set forth herein. Any aspect of the present disclosure disclosed herein may be implemented by one or more elements of a claim. While specific feature combinationsare described in the following with respect to certain aspects of the present disclosure, it is to be understood that not all features of the discussed examples must be present for realizing the technical advantages of the devices, apparatuses, systems, methods and computer programs disclosed herein. Disclosed aspects may be modified by combining certain features of one aspect with one or more features of other aspects. A skilled person will understand that features, steps, components and / or functional elements of one aspect can be combined with compatible features, steps, components and / or functional elements of any other aspect of the present disclosure.

[0055] Several aspects of VLPs their composition and production are presented herein with reference to example implementations based on VLPs derived from HIV-1 viruses. Other implementations, lying within the scope of the appended claims are also covered by the present disclosure. As mentioned above, the present disclosure provides an intracellular protein delivery platform, which utilizes engineered structural viral proteins to enrich intracellular cargo proteins of interest in the VLPs and deliver them to target cells in a nearly unmodified state to elicit a wide range of functional or therapeutic effects, depending on the type of packaged protein and target cell type. The VLPs described herein can potentially be used both in research and to deliver proteins to cells in vivo. Research purposes in vitro (e.g., in cell culture) or in vivo (e.g., in a patient’s body) to deliver therapeutically relevant proteins and treat diseases.

[0056] As illustrated in the example of Fig. 1, VLPs may be obtained, e.g., via the intracellular protein delivery platform described herein.

[0057] As shown in Fig. 1, a transfection step maybe performed. Herein, transfection may relate to a process of introducing one or more (e.g., different) foreign nucleic acids (such as DNA and / or RNA) into the VLP packaging cell(s), e.g., the first, second, third, etc. nucleic acid(s) described herein. This may enable the packaging cell(s) to produce the necessary components for VLP formation, including, e.g., the second fusion polypeptide. The transfection process may be achieved through various methods, such as chemical transfection, electroporation, and / or viral vectors, to ensure that the genetic material is successfully delivered and expressed within the packaging cell(s).

[0058] The packaging cell may be a type of cell that may be configured to produce and / or assemble the components necessary for the formation of VLPs, e.g., at the surface of the (VLP) packaging cell. The packaging cell(s) may typically be configured to express the elements described herein required for VLP assembly, including the second fusion polypeptide. Packaging cells provide the cellular machineiy and environment needed for the production of VLPs, which can then be harvested in the subsequent process described herein. In the examples herein below, HEK293T cells are used for packaging that provide all of the components being required for packaging VLPs from plasmids. It is one of the advantages of the present invention as compared to the prior art that the amount of pol required to obtain mature particles is reduced. Only p6pol and the protease are required in the system herein, and, for example, not the reverse transcriptase or the integrase as in the prior art.

[0059] In the example of Fig. 1, the packaging cell assembles VLPs comprising an adaptor polypeptide, a detector polypeptide, a protease cleavage site, and a functional or therapeutic polypeptide (e.g., a cargo protein). Said assembly is performed during the assembly process in the VLP packaging cell (e.g., at the cell surface of the packaging cell).

[0060] The assembly of one or more VLPs during the assembly process in the VLP packaging cell of the packaging cell may involve one or more of the following steps: (i) Once transfected, e.g., as described herein, the introduced nucleic acids maybe transcribed and / or translated to produce the polypeptide(s) and / or protein(s) described herein. Importantly, the polypeptide(s) may comprise the functional or therapeutic polypeptide (e.g., a so-called cargo protein) described herein to provide the desired functionality to the VLP. (ii) The newly synthesized polypeptide(s) and / or protein(s) may be transported to specific locations within the cell. For VLPs that assemble (e.g., at the cell surface), the polypeptide(s) and / or protein(s) maybe directed to the plasma membrane. This trafficking may be mediated by cellular machinery of the packaging cell, (iii) At the cell surface, the polypeptide(s) and / or protein(s) may begin to interact and / or self-assemble into the VLP(s). This process may be driven by the inherent properties of the polypeptide(s), which maybe configured to form the desired structure through polypeptide-polypeptide interaction. The surface protein(s), e.g.,VSV-G or derivatives thereof, maybe integrated into the surface of the VLP and / or attached thereto. In detail, the surface protein may be integrated into the VLP membrane, such that it may be on the surface of the final VLP. (iv) As the VLPs assemble at the plasma membrane, the VLPs may begin to bud off from the cell surface. This so-called budding process may involve the curvature of the membrane around the assembling VLP, eventually pinching off to release the VLP into the extracellular space. The cellular machinery, including the ESCRT (endosomal sorting complexes required for transport) pathway, maybe involved in facilitating this budding process, (v) The fully assembled VLPs may accordingly be released from the packaging cell into the surrounding medium.

[0061] The cargo protein(s) are assembled as followed: The adaptor polypeptide is placed at the N-terminus of the assembled sequence. The detector polypeptide is arranged between the adaptor polypeptide and the protease cleavage site. The protease cleavage site is arranged between the detector polypeptide and the functional or therapeutic polypeptide. The functional or therapeutic polypeptide is placed at the C-terminus. In other examples, detector polypeptide and the cleavage site may swap positions, such that the detector polypeptide is arranged between the protease cleavage site and the functional or therapeutic polypeptide.

[0062] The VLP may further undergo a virion maturation step and / or a cleavage step which may be separate processes or a fully or partly combined process. The virion maturation may comprise an initially immature VLP undergoing structural and / or biochemical changes to become a fully functional VLP, e.g., comprising proteolytic cleavage, conformational change(s), post-translational modification(s) such as glycosylation, phosphorylation, and / or acylation, and / or stabilization. The cleavage step may comprise the cleavage of the polypeptide(s) assembled as described herein at at least one cleavage site, e.g., a protease cleavage site as described herein. The cleavage may be performed by a protease, preferably a HIV-i protease. The use of the HIV-i protease yields the advantage of being suitable for cleaving all possible cleavage sites that maybe used in HIV-i-based platforms. The same may apply to other viruses than HIV-i and the respective cleavage sites. Herein, the HIV-i protease preferably comprises or consists of SEQ ID NO: 17GPOITLWORPLVTII< IGGOLI< EALLDTGADDTVLEEMNLPGRWI< PI< MIGGIGGFII< VRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNF”) or a sequence being at least 8o%, preferably at least 90% and most preferably at least 95% identical thereto, or SEQ ID NO: 18 (“PQITLWQRPLVTIKIGGQLKEALLDSGADDTVLEEMNLPGRWKPKMIGGIGGFIKVR QYDQILIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNF”) or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto. With respect to the sequences being at least 80% identical to SEQ ID NOs 17 and 18 it is to be understood that they retain the capability of SEQ ID NOs 17 and 18 to be a HIV-i protease.

[0063] Fig. 1 further illustrates the purification of the VLPs. The purification may, e.g., as illustrated, be performed after the maturation and / or cleavage step. The purification may refer to a process of harvesting and / or concentrating the VLPs from the culture medium and / or removing any contaminants, such as cellular debris, proteins, and / or other impurities. This process is essential to ensure that the final VLP preparation is of high purity and suitable for its intended functionality and application. The purification may comprise a clarification (e.g., through filtration and / or (low-speed) centrifugation), a concentration (e.g., through ultrafiltration and / or other concentration techniques), chromatography (e.g., size-exclusion chromatography, ionexchange chromatography, and / or affinity chromatography) to separate VLPs according to size, charge, and / or specific binding properties, density gradient centrifugation (e.g., comprising layering the VLP-containing solution onto a density gradient (e.g., sucrose or cesium chloride) and / or centrifuging it at high speed such that the VLPs may band at a specific density, allowing for their separation from contaminants), polishing (e.g., comprising further purification steps as further chromatography or filtration), and / or sterilization. One or more of these purification approaches may be performed in the described or another order.

[0064] Further, the functional or therapeutic polypeptide (i.e., in this example the cargo proteins) may be quantized via the detector polypeptide in a quantification step. The quantification step may comprise inducing a bioluminescent reaction and detecting the bioluminescence such that the concentration and / or number of functional or therapeutic polypeptides may be determined based on the detected bioluminescence intensity.

[0065] In some examples, detector polypeptides may be used, and coiled coil domains may serve as adaptor domains. Further examples of detector polypeptides may include: Epitope peptides like HA or FLAG, enzymes with a measurable reaction rate like P-Galactosidase or Alkaline Phosphatase, or more specifically to detect cargo proteins in a bioluminescence reaction; luciferease enzymes and split luciferase systems (e.g., HiBiT).

[0066] Fig. 2 illustrates key concepts of the present disclosure using an HIV-1 based implementation for illustration and verification. Experimental results for this example are discussed with reference to Fig.4 to Fig 6 below. Panel A of Fig. 2 shows that an engineered versions of the HIV-1 structural protein Gag (pink; Gag (HIV-1)) that can be fused to an adjacent adaptor polypeptide (green; adaptor 1) such as a coiled coil (CC) domain or similar forming a first type of fusion polypeptide (Gag-Adp). Any desired cargo protein (also called functional or therapeutic polypeptide herein) (purple; Cargo protein (here Vpx)) can also be fused to the same (e.g. homodimerizing domains) or a similar adaptor polypeptide (green; adaptor 2) forming a second kind of polypeptide that can be packaged into the VLP during VLP assembly using an attractive, non-covalent interaction between the adaptor domains as illustrated in panel C of Fig.2.

[0067] The top left portion of Panel A of Fig. 2 shows exemplary (structural) polypeptides of so-called transVLPs. In detail, Panel A (under the label “Gag-Adp”) shows the first nucleotide sequence encoding a first fusion polypeptide that comprises the retroviral gag polypeptide of the VLP and the adaptor polypeptide of the first kind. The retroviral gag polypeptide is placed at the N-terminus (amino acids 1-500) and the adaptor polypeptide of the first kind is placed at the C-terminus (amino acids 515-542). Panel A (under the label “Adp-PR”) further shows the third fusion polypeptide that comprises the adaptor polypeptide of the first kind or of the second kind, and the protease polypeptide that is adapted to cleave the protease cleavage site. The adaptor polypeptide of the first kind or of the second kind is placed at the N-terminus (amino acids 4-31) and the protease polypeptide is placed at the C-terminus (amino acids 46-144). An integrated cleavage site is placed at amino acids 41-50.[oo68] The bottom left portion of Panel A shows an exemplary cargo protein polypeptide. In detail, it shows (under the label “Adp-Det-CS-CP”) the second fusion polypeptide that comprises an adaptor polypeptide of the first kind or of the second kind (amino acids 4-31) placed at the N-terminus, and the functional or therapeutic polypeptide (amino acids 71-182) placed at the C-terminus. In between, the exemplary second fusion polypeptide comprises the (optional) detector polypeptide (amino acids 40-50) and the (optional) cleavage site (amino acids 56-65).

[0069] The top right portion of Panel A shows structural polypeptides of cisVLPs. These may originate from the same nucleotide sequence with (ribosomal) frameshift or without (ribosomal frameshift). In the example of HIV-1, the (ribosomal) frameshift typically occurs with a probability of approximately 5-10%. The polypeptide (labelled as “Gag-FS-PR”) originates from the case with (ribosomal) frameshift. It comprises the second fusion polypeptide comprising the retroviral gag polypeptide of the VLP (amino acids 1-500), placed at the N-terminus, and the protease polypeptide (amino acids 614-712) placed at the C-terminus. The exemplary second fusion polypeptide further comprises the (optional) protease cleavage site (amino acids 506-515) arranged between the retroviral gag polypeptide and the protease polypeptide, the synthetic transframe polypeptide (amino acids 526-556), preferably comprising 50%, 60%, 70% or more of amino acids preferably comprising small and / or polar non-charged side chains (wherever possible because of the overlapping reading frame with the adaptor sequence in the non-(ribosomal) frameshift polypeptide product) acting as a flexible linker peptide, and the p6pol polypeptide, (in this example a HIV-i p6pol polypeptide; amino acids 558-613) to modulate the activity of the protease polypeptide at its C-terminus. In detail, the STF peptide, if present, may have 23 out of 31 amino acids which have small and / or polar non-charged side chains (74 %). The rest may have either charged or big hydrophobic side chains.

[0070] The polypeptide (labelled as “Gag-FS-Adp”) comprises the first fusion polypeptide comprising the retroviral gag polypeptide of the VLP (amino acids 1-500) placed at the N-terminus and the adaptor polypeptide of the first kind (amino acids 528-555) placed at the C-terminus. The first fusion polypeptide further comprises a cleavage site (amino acids 506-512).

[0071] The bottom right portion of Panel A shows two different options for the surface protein described herein: First, the VSV-G (VSV glycoprotein) surface protein is configured for unspecified delivery and / or fusion with target and non-target cells. Second, the VSV-Gmut (a mutated version of VSV-G) may be configured to provide the fusion capability of the unmutated VSV-G and provided along with a further surface protein, e.g., comprising a target ligand, configured to allow for targeted fusion and / or delivery to target cells, and, as determined by the VSV-Gmut (mutant VSV glycoprotein), to fuse (only) with said target cells. This is also shown in Fig. 1. For this purpose, a fourth nucleic acid comprising a fourth nucleotide sequence encoding a first surface protein of the VLP adapted to enable fusion of the VLP with a target cell may be provided. Herein, the VSV-G (VSV glycoprotein) surface protein preferably is encoded by SEQ ID NO: 66 or a sequence being at least 8o%, preferably at least 90% and most preferably at least 95% identical thereto; or comprise of consists of SEQ ID NO: 67 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto. The VSV-Gmut (a mutated version of VSV-G) surface protein preferably is encoded by SEQ ID NO: 68 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto; or comprise of consists of SEQ ID NO: 69 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto.

[0072] Further preferred examples for (viral) surface proteins (e.g., for fusion) may include: Rabies Virus Glycoproteins (RV-G, preferably the sequence of SEQ ID NO: 74 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto), Ebola Virus Glycoprotein (EBOV-GP, preferably the sequence of SEQ ID NO: 75 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto), SARS-CoV and SARS-C0V-2 Spike (S) Protein (preferably the sequence of SEQ ID NO: 76 or 77 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto), Measles Virus Hemagglutinin and Fusion Protein (MV-H / F; preferably the sequence of SEQ ID NO: 78 or 79, respectively, or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto).

[0073] Panel B shows two exemplary VLPs. In detail, the VLP shown on the left may be obtained by use of the transPR structural components and the VLP shown on the right maybe obtained by use of the cisPR structural components. The “trans” may relate to different nucleic acids (e.g., plasmids) being used for encoding the Gag- and protease-coding nucleic acids. Accordingly, the “cis” in cisVLPs may relate to a common nucleic acid (e.g., plasmids) being used for encodingthese structural components.

[0074] In detail, the left VLP comprises Gag-Adp, Adp-PR, and Adp-Det-CS-CP. The right VLP comprises Gag-FS-PR / Gag-FS-Adp and Adp-Det-CS-CP. Both VLPs may equally be equipped with the VSV-G and / or VSV-Gmut, as described herein.

[0075] In some aspects, the genetic information encoding such fusion polypeptides can be part of an engineered plasmid (see blue plasmids in Fig. 1 and Fig.3) or similar nucleic acid adapted for transfection of a suitable VLP packaging cell (see Fig. 1; top left) such as a HEK293T cell or any other suitable eukaryotic cell that can be used for VLP production via ribosomal translation of the genetic information in a cell culture.

[0076] Thus, the present disclosure provides in some aspects a system for obtaining a virus-like particle (VLP) comprising a first nucleic acid comprising a first nucleotide sequence encoding a first fusion polypeptide (e.g., Gag-Adp in Fig. 2) that comprises a retroviral gag polypeptide of the VLP and an adaptor polypeptide of a first kind. The system may further comprise a second nucleic acid comprising a second nucleotide sequence encoding a second fusion polypeptide that comprises an adaptor polypeptide of the first kind or of a second kind and a functional or therapeutic polypeptide, wherein the adaptor polypeptides of the first and / or the second kind may be configured to interact (e.g., via direct or indirect electro-static attraction; see Fig. 2, Panel C for an example) with each other such that during formation of the VLP at the cell surface of a VLP packaging cell (illustrated in Fig. 1) the second fusion polypeptide is packaged into the VLP. In contrast to prior art system, this allows to package the second fusion polypeptide and thereby a broad variety of functional or therapeutical cargo proteins into the VLP without forming a covalent bond between the Gag polypeptide and the cargo polypeptide, thereby significantly improving efficiency,specificity and robustness of cargo polypeptide packaging into the VLPs (see experimental results discussed in Fig.4 to Fig. 6 below).

[0077] As discussed above, a detector polypeptide / protein tag for precise quantification of packaged proteins and an HIV-i protease cleavage site for adaptor domain cleavage during VLP maturation can thus also be included via transfecting a suitable packaging cell with appropriately engineered nucleic acids. As mentioned above the VLPs disclosed herein can be engineered to be not replication competent and, in contrast to other previously described HIV-1 protein delivery particles, can be made devoid of viral components such as the HIV-i reverse transcriptase and integrase enzymes, which could have additional adverse effects in some application scenarios. For example, suitable stop codons can be included to prevent translation of such enzymes during VLP production or their encoding nucleic acids can be removed completely. As also mentioned above, certain surface proteins can be used to target and fuse to cells expressing specific surface markers (see US 2020 / 0371088 Al for an instructive example).

[0078] Thus, as discussed above, in a first aspect, a first system for obtaining a VLP may comprise three pieces of nucleic acid (e.g., in form of three plasmids) that can be inserted in a suitable packaging cell. The first nucleic acid may encode a viral hull protein such as Gag fused with an adaptor domain. A second nucleic acid may encode a cargo protein construct engineered to interact with the adaptor domain of the hull protein to enable packaging of the cargo protein in the VLPs when the VLP is formed from multiple hull proteins. A third nucleic acid may encode application of specific surface proteins / glycoproteins for target cell binding and fusion. Optional features and elements are discussed above and are the subject matter of the embodiments 2 to 19 of the “further embodiments” disclosed below.

[0079] For completeness, the various polypeptides shown in Fig. 2 are now discussed in more detail with reference to the attached amino acid and nucleotide sequences. The structural protein Gag from HIV-i (amino acids 1 - 500) may comprise the following additions: a linker region (amino acids 501 - 514) and an adaptor domainfor binding and recruiting cargo proteins through a corresponding adaptor domain into the VLPs (amino acids 515 - 542) (SEQ ID NO: 1; SEQ ID NO: 2).

[0080] Fig. 2 illustrates a further system that may allow to use less nucleic acids (e.g., less plasmids) making use of the ribosomal frameshifting mechanism evolved in certain wild type viruses such as HIV-1. Using this novel technique, a combined Gag and viral protease expression construct permits simultaneous expression of both proteins from the same plasmid to create mature virion particles. On DNA level an exemplary construct may incorporates an HIV-1 ribosomal frameshift region (nucleotides 1516 - 1573 in SEQ ID NO: 3). Translation of the RNA resulting from transcription of this DNA sequence without ribosomal shift generates a Gag protein (amino acids 1 - 500) fused to a linker region (amino acids 501 - 505), the protein product of the frameshift element including an HIV-i protease cleavage site (amino acids 506 - 524), a linker region (amino acids 525 - 527), and an adaptor (e.g., a coiled coil P9S domain) for binding and recruiting cargo proteins through their respective adaptor (e.g., a coiled coil Pios domain) into the virions (amino acids 528 - 555) (SEQ ID NO: 4). Translation of the RNA resulting from transcription of this DNA sequence with ribosomal shift generates a Gag protein (amino acids 1 - 500) fused to a linker region (amino acids 501 - 505), the -1 frame protein product of the frameshift region including an HIV-i protease cleavage site (amino acids 506 - 515), a linker region (amino acids 516 - 525), a synthetic transframe peptide in -1 frame overlapping with the DNA sequence of the adaptor (e.g. coiled coil P9S) domain and codon optimized to favor small, polar, uncharged amino acids wherever possible (amino acids 526 - 556), HIV-i’s p6pol peptide (amino acids 558 - 613), and the HIV-i protease (amino acids 614 - 712) (SEQ ID NO: 5).

[0081] Naturally, this idea, illustrated with HIV-i derived VLPs shown in Fig. 2 can be employed much more generally. Thus, in a further aspect, the present disclosure provides a system for obtaining a virus-like particle (VLP) comprising: a first nucleic acid comprising a first nucleotide sequence comprising a translational frameshift element; wherein, without translational frameshifting, the first nucleotide sequence encodes a first fusion polypeptide comprising a retroviral gag polypeptide of the VLP and an adaptor polypeptide of a first kind; wherein, with translational frameshifting,the first nucleotide sequence encodes a second fusion polypeptide comprising the retroviral gag polypeptide of the VLP, and a protease polypeptide that is adapted to cleave a protease cleavage site; and a second nucleic acid comprising a second nucleotide sequence encoding a third fusion polypeptide that comprises an adaptor polypeptide of the first kind or of a second kind, and a functional or therapeutic polypeptide; wherein the adaptor polypeptides of the first and / or the second kind are configured to interact with each other such that during formation of the VLP on a cell surface of a VLP packaging cell the second fusion polypeptide is packaged into the VLP.

[0082] To date, using a viral (e.g., HIV-i’s) ribosomal frameshift sequence outside of the natural context to generate a modified and truncated Gag-Pol construct with additional protein domains in overlapping coding regions and still yield functional particles has not been described in the field. The ribosomal frameshift sequence is naturally present in the HIV-1 genome within the Gag open reading frame before translation of the p6 domain. The inventors found that it is possible to artificially move it to the end of the Gag open reading frame, to add an adaptor sequence thereafter and optimized the -1 frame over this adaptor sequence to favor small, polar, uncharged amino acids wherever possible. Thus, when a ribosomal shift happens, the resulting Gag construct has an unordered synthetic transframe (STF) peptide right at its C-terminus, functioning like a linker region before the functional p6pol and protease domains (see Fig. 2; Gag-FS-PR for an example). In the illustrated example, HIV-i’s pol gene was truncated after the protease encoding region (e.g., by including a stop codon and eliminating the coding regions past the protease coding region) to eliminate the presence of the reverse transcriptase and integrase enzymes in our particles.

[0083] Aspects of the present disclosure thus allow for intracellular protein delivery in immature particles using SEQ ID NO: 2 as the structural component, or the intracellular protein delivery in mature particles using SEQ ID NO: 2 + SEQ ID NO: 9 / 11 or SEQ ID NO: 4 + SEQ ID NO: 5.

[0084] Exemplary cargo proteins, hereby exemplified by the Vpx protein from the Simian Immunodeficiency Virus (SIV) (amino acids 71 - 182), maybe part of a fusion polypeptide comprising an adaptor domain for binding to Gag’s adaptor domain(amino acids 4 - 31), a linker sequence (amino acids 32 - 39), a detector polypeptide for cargo protein detection and quantification (amino acids 40 - 50), a linker sequence (amino acids 51 - 55), an protease cleavage site for adaptor domain and detector polypeptide removal during virion maturation (amino acids 56 - 65), and a linker sequence (amino acids 66 - 70) (SEQ ID NO: 6; SEQ ID NO: 7). In the provided example of a cargo protein, cleavage of the adaptor domain and the detector polypeptide through the viral protease results in a 10 amino acid scar at the N-terminus of Vpx (cleavage between amino acids 60 and 61; N-terminal scar comprises amino acids 61 - 70).

[0085] Further, HIV-i’s protease (amino acids 46 - 144) modified with the addition of an adaptor domain (amino acids 4 -31) interacting with the adaptor domain on Gag enables virion incorporation of the viral protease and virion maturation for enhanced particle function and cleavage of adaptor and detector domain from cargo proteins (Wild type protease - SEQ ID NO: 8; SEQ ID NO: 9) (T26S catalytically attenuated protease - SEQ ID NO: 10; SEQ ID NO: 11).

[0086] In summary, the SEQ ID NOs may describe the following elements (as described herein): SEQ ID NO: 1 describes the Gag-Adp DNA, SEQ ID NO: 2 describes the Gag-Adp protein, SEQ ID NO: 3 describes the Gag-FS-Adp / Gag-FS-PR DNA, SEQ ID NO: 4 describes the Gag-FS-Adp protein, SEQ ID NO: 5 describes the Gag-FS-PR protein, SEQ ID NO: 6 describes the Adp-Det-CS-CP DNA, SEQ ID NO: 7 describes the Adp-Det-CS-CP protein, SEQ ID NO: 8 describes the Adp-PR DNA (wild type), SEQ ID NO: 9 describes the Adp-PR protein (wild type), SEQ ID NO: 10 describes the Adp-PR DNA (T26S catalytically attenuated protease), and SEQ ID NO: 11 describes the Adp-PR protein (T26S catalytically attenuated protease).

[0087] Linker regions and cleavage protease sites may be modified for easier protease access, enhanced protease cleavage rate, or minimization of amino acid residue scar. Coiled coil domains might be exchanged by other interacting protein domains to achieve Gag - Cargo protein interaction and enrichment in the VLPs.

[0088] Examples of other interacting domains include: Leucine zippers (preferably the sequence pair of SEQ ID NOs 58 and 59 or a pair of sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto), the FKBP-FRB Rapamycin-Inducible Dimerization System (preferably the sequence pair of SEQ ID NOs 60 and 61 or a pair of sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto), synthetic peptides designed to interact with each other like coiled coil domains (preferably the sequence pair of SEQ ID NOs 62 and 63 or a pair of sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto), or PDZ domains binding to PDZ-binding motifs preferably the sequence pair of SEQ ID NOs 64 and 65 (“ETQV”) or a pair of sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto). It is to be understood that the pairs of interacting domains with the indicated sequence identity retain the ability to act as pairs of interacting domains.

[0089] Fig. 3 illustrates a typical experimental sequence to obtain the VLPs disclosed herein based on the disclosed systems. In a first step, cultured VLP packaging cells, such as HEK239T cells, are transfected with the nucleic acids discussed above. After a long enough time period (e.g., 24 - 72 h) produced VLPs can be harvested from the supernatant and concentrated, e.g., via a sucrose cushion. Next, resulting VLP pellets can be resuspended in a suitable medium such as PBS (e.g., for an effective concentration of 50 - 200X).

[0090] In more detail, Fig. 3 illustrates an exemplary method for obtaining the VLPs as per the present invention for use and / or quantification as described herein. In detail, the method comprises the steps of (1) plasmid transfection, (2) harvesting of VLPs, (3) resuspension of VLPs, and (4) treatment with VLPs.

[0091] Step (1) comprises the plasmid transfection. Therein, several key components are provided together with one another. These components one or more of the following: (i) the nucleic acid(s) (e.g., plasmids) comprising nucleotide sequence(s) encoding polypeptide(s) and / or protein(s) described herein; (ii) a transfection reagent (e.g., a chemical and / or biological agent that facilitates the delivery of the nucleic acid(s) into the packaging cells. Common transfection reagents include lipofectamine,polyethylenimine (PEI), calcium phosphate, and / or cationic lipids); (iii) the packaging cell(s) (e.g., HEK293T cells, CHO cells, and / or Vero cells); and (iv) a buffer and / or medium (e.g., for maintaining viability and optimal conditions for packaging during the transfection process; e.g., standard cell culture media).

[0092] Step (2), the harvesting from the supernatant may be performed 48h - 72h post transfection (step (1)) and / or may comprise concentration of the harvested VLPs over a sucrose cushion.

[0093] Subsequently, the as-received VLP (pellet) may be resuspended in a buffer solution, like, e.g., phosphate-buffered saline (PBS), in step (3). This may, e.g., yield an effective concentration of 50-200X.

[0094] In step (4), the VLP resuspension treated with a detergent-containing solution for VLP lysis (total detector sample, as shown in Fig.3) and / or dilution in PBS (free detector sample) may be performed for a (split) luciferase assay. Accordingly, a (split) luciferase maybe added to the total and free detector samples equally. The split luciferase assay is based on the principle of providing two separate, non-functional fragments (here the split luciferase and the detector polypeptides). The two (separately non-functional) fragments may emit light when they interact with one another. In detail, when brought into mutual proximity, the fragments may fuse and form a functional luciferase enzyme. Thus, the luminescence intensity of the split luciferase assay may scale with the amount of detector polypeptides that reacts with the added split luciferase. The lysis may result in the release of the detector polypeptides into the solution that surrounds the VLPs. Therefore, the according (total detector sample) measurement may detect a luminescence intensity that provides a measure for the total amount of detector polypeptides that are present in the VLPs and outside the VLPs (and thus the functional or therapeutic polypeptide present in the VLPs and outside the VLPs). In the free detector sample, however, only the unpackaged detector polypeptides are accessible to the split luciferase. Therefore, the according (free detector sample) measurement may detect a luminescence intensity that provides a measure for the amount of detector polypeptides that are present outside the VLPs (and thus the functional or therapeutic polypeptide present outside the VLPs). The cargo protein quantification may be performed as described herein, as a part of saidstep, via the detector polypeptide. In this example, subtracting the measured signal of the free detector sample from the measured signal of the total detector sample yields a measure for the total amount of detector polypeptides that are present in the VLPs (and thus the functional or therapeutic polypeptide present in the VLPs), which is typically the measure of interest, as only this packaged amount of cargo functional or therapeutic polypeptide will be available for delivery upon fusion of the VLPs with the target cells.

[0095] Fig. 4 to Fig. 6 illustrate experimental results obtained for several exemplary implementations of the systems disclosed herein. In these experiments, as a model for typical cargo protein incorporation and VLP functional testing, the protein Vpx was incorporated into the VLPs as described above. This protein is derived from the simian immunodeficiency virus (SIV) and is known to degrade SAMHD1. As a model cell line to show functionality the acute myeloid leukemia cell line THP-1 with high expression levels of SAMHD1 was used. For further details see:Hrecka K, Hao C, Gierszewska M, Swanson SK, Kesik-Brodacka M, Srivastava S, et al. Nature. 2011;474(7353):658-61.Laguette N, Sobhian B, Casartelli N, Ringeard M, Chable-Bessia C, Segeral E, et al. Nature. 2011;474(7353):654-7.Schneider C, Oellerich T, Baldauf HM, Schwarz SM, Thomas D, Flick R, Bohnenberger H, Kaderali L, Stegmann L, Cremer A, Martin M, Lohmeyer J, Michaelis M, Hornung V, Schliemann C, Berdel WE, Hartmann W, Wardelmann E, Comoglio F, Hansmann ML, Yakunin AF, Geisslinger G, Strobel P, Ferreiros N, Serve H, Keppler OT, Cinatl J Jr. Nat Med. 2017 Feb;23(2):250- 255-

[0096] Fig. 4 shows results of a detector assay experiment with VLPs based on HIV-i as shown in Fig. 2. Concentration of the functional or therapeutic polypeptide (also referred to as cargo protein) as Free, Total, or Packaged expressed as nM detector protein equivalents. Fig.5 shows SAMHD1 protein levels in THP-1 cells after treatment with low doses of VLPs. SAMHD1 protein levels were determined by flowcytometry. Untreated THP-1 wildtype and THP-1 SAMHD1 knock-out cells served as controls and were set to 100 % and 0 %, respectively.

[0097] The detector assay shows that in the absence of an Adp domain on Gag in the “WT Gag + Adp-Det-CS-Vpx VLPs” sample, there is very little incorporation of Vpx into the VLPs. These particles also fail to induce SAMHD1 degradation when tested in THP-i cells. The Western blot analysis shows that these particles remain immature, as there is no p24 capsid band visible at around 24 kDa.

[0098] The sample “Gag -Adp + Adp-Det-CS-Vpx VLPs” effectively incorporates Vpx into the VLPs as seen with the detector assay of Fig.4 but does not result in SAMHDi degradation in THP-i cells due to the absence of the protease. This results in immature VLPs and lack of processing of the cargo protein to separate Vpx from the adaptor and the detector domains. The cargo protein lacks an HIV-i protease cleavage site in sample “transPR + Adp-Det-Vpx VLPs” and therefore, although there is cargo protein enrichment according to the detector assay and Western blot analyses, these VLPs also fail to degrade SAMHDi in THP-1 cells.

[0099] The sample “transPR + Adp-Det-CS-Vpx VLPs” also effectively incorporates the cargo protein and leads to around 25 % SAMHDi degradation under suboptimal conditions, while the “cisPR + Adp-Det-CS-Vpx VLPs” with similar packaged detector signal values are twice as effective and lead to around 50 % SAMHDi degradation.

[0100] For comparative purposes, previously described SIV-based Vpx VLPs described in the literature were included in the panel. These VLPs are derived from natural lentiviruses and therefore do not contain the detector polypeptide, acting as a negative control in the detector assay. In terms of SAMHDi degradation, these SIV VLPs perform similarly to the “transPR + Adp-Det-CS-Vpx VLPs” in the low VLP dose treatments study with around 25 % SAMHDi degradation. SIV VLPs are for instance described as “1st generation VLPs” in: Nair, R., et al. (2024). " Novel Vpx virus-like particles to improve cytarabine treatment response against acute myeloid leukemia." Clin Exp Med 24(1): 155 and originally used in: Schneider C, Oellerich T, Baldauf HM, Schwarz SM, Thomas D, Flick R, Bohnenberger H, Kaderali L, Stegmann L, Cremer A,Martin M, Lohmeyer J, Michaelis M, Hornung V, Schliemann C, Berdel WE, Hartmann W, Wardelmann E, Comoglio F, Hansmann ML, Yakunin AF, Geisslinger G, Strobel P, Ferreiros N, Serve H, Keppler OT, Cinatl J Jr. Nat Med. 2017 Feb;23(2):250-255.

[0101] Fig. 6 shows SDS-PAGE and Western blot analysis of VLPs. CS1-4 correspond to four different HIV-i protease cleavage sites tested on the cargo protein construct Adp-Det-CSx-Vpx.

[0102] The upper panel shows the SDS-PAGE analysis of VLPs in which it becomes apparent that both, the transPR VLPs and the cisPR VLPs mature equally well as both (in contrast to the wild type Gag (WT Gag)) show a clear band at ca. 24 kDa. This corresponds to the molecular weight of the p24 capsid protein, which is a major liberated structural component of mature HIV-1 virions and thus provides an indicator for successful maturation (e.g., by cleavage as described herein).

[0103] The lower panel shows a Western blot with the same samples as in the SDS-PAGE. In detail, the blot has been developed with “anti-Capsid” antibodies that detect the capsid domain in Gag and the “anti-Detector” antibodies that detect the cargo protein / the detector polypeptide. In result, the bottom panel shows that also the antibody-specific detection confirms the SDS-PAGE analysis: The decisive p24 capsid band appears in all transPR VLPs and the cisPR VLPs while they are absent in the WT Gag control sample. Additionally, the detector blot reveals that all transPR VLPs and cisPR VLPs successfully enrich the cargo protein, contrary to WT Gag lacking the adaptor domain and no band is visible in the SIV Vpx VLPs because the wild type Vpx here lacks the detector domain. The additional bands seen in the Western blot result from the intermediate cleavage products of Gag where p24 capsid is still present.

[0104] Combined, the experimental results of Fig. 4 to Fig. 6 clearly show that the novel systems and methods disclosed herein allow for efficient, specific and robust cargo protein packaging in VLPs and functional maturation. The systems, methods and VLPs disclosed herein can thus readily be used for(i) delivery of bacterial toxins, cytotoxic proteins, or cell death-inducing protein domains for the killing of cell lines in vitro or the killing of cancerous cells in vivo(ii) Delivery of enzymes for intracellular catalysis of chemical reactions.(iii) Delivery of immunomodulatory proteins for the downregulation of immune function in diseases like autoimmune diseases or the upregulation of immune function in diseases like cancer.(iv) Delivery of proteins that influence the degradation of specific proteins in the target cells to modulate protein levels.(v) Delivery of designer nucleases, base editors, or recombinases for genomic editing of cell lines in vitro or gene therapy purposes in vivo.(vi) Delivery of fluorescent or light emitting proteins as reporter systems.

[0105] Further use cases of the systems, methods and VLPs disclosed herein include:(i) Delivery of transcription factors to induce or repress gene expression.(ii) Delivery of RNA-binding proteins complexed with RNA molecules to influence gene expression in target cells.(iii) Delivery of signaling cascade components to trigger cellular responses.(iv) Delivery of cargo proteins as vaccination agents.(v) Delivery of tumor suppressor proteins for cancer treatment.(vi) Delivery of anti-aging or lifespan-enhancing proteins.

[0106] The foregoing disclosure provides illustration and description but is not intended to be exhaustive or to limit the aspects to the precise form disclosed.Modifications and variations may be made in light of the above disclosure or may be acquired from practice of the aspects.

[0107] Even though particular combinations of features are recited in the claims and / or disclosed in the specification, these combinations are not intended to limit the disclosure of various aspects. In fact, many of these features may be combined in ways not specifically recited in the claims and / or dis-closed in the specification. Although each dependent claim listed below may directly depend on only one claim, the disclosure of various aspects includes each dependent claim in combination with eveiy other claim in the claim set. A phrase referring to “at least one of’ a list of items refers to any combination of those items, including single members. As an example, “at least one of: a, b, or c” is intended to cover a, b, c, a-b, a-c, b-c, and a-b-c, as well as any combination with multiples of the same element (e.g., a-a, a-a-a, a-a-b, a-a-c, a-b-b, a-c-c, b-b, b-b-b, b-b-c, c-c, and c-c-c or any other ordering of a, b, and c).

[0108] No element, act, or instruction used herein should be construed as critical or essential unless explicitly described as such. Also, as used herein, the articles “a” and “an” are intended to include one or more items, and may be used interchangeably with “one or more.” Furthermore, as used herein, the terms “set” and “group” are intended to include one or more items (e.g., related items, unrelated items, a combination of related and unrelated items, and / or the like), and maybe used interchange-ably with “one or more.” Where only one item is intended, the phrase “only one” or similar language is used. Also, as used herein, the terms “has,” “have,” “having,” and / or the like are intended to be open-ended terms.

[0109] As used herein, the phrase “based on” shall not be construed as a reference to a closed set of information, one or more conditions, one or more factors, or the like. In other words, the phrase “based on A” (where “A” may be information, a condition, a factor, or the like) shall be construed as “based at least on A” unless specifically recited differently.

[0110] As used herein, the term “or” is an inclusive “or” unless limiting language is used relative to the alternatives listed. For example, reference to “X being based on A or B” shall be construed as including within its scope X being based on A, X being based on B, and X being based on A and B. In this regard, reference to “X being based on A or B” refers to “at least one of A or B” or “one or more of A or B” due to “or” being inclusive. Similarly, reference to “X being based on A, B, or C” shall be construed as including within its scope X being based on A, X being based on B, X being based on C, X being based on A and B, X being based on A and C, X being based on B and C, and X being based on A, B, and C. In this regard, reference to “X being based on A, B, or C” refers to “at least one of A, B, or C” or “one or more of A, B, or C” due to “or” being inclusive. As an example of limiting language, reference to “X being based on only one of A or B” shall be construed as including within its scope X being based on A as well as X being based on B, but not X being based on A and B.

[0111] For the ease of reference, the SEQ IDs are also represented below:

[0112] SEQ ID NO: 1 (Name: Gag-Adp, Type: DNA, Sequence:“ATGGGTGCTAGAGCATCTGTTCTGTCTGGCGGCGAGCTGGACAGATGGGAGAAGAT CAGACTCAGACCTGGCGGCAAGAAGAAGTACAAGCTGAAGCACATCGTGTGGGCCAG CAGAGAGCTGGAAAGATTCGCCGTGAATCCCGGCCTGCTGGAAACCTCTGAGGGCTG CAGACAGATCCTGGGACAGCTGCAGCCTTCTCTGCAGACCGGAAGCGAGGAACTGCG GAGCCTGTACAATACCGTGGCCACACTGTACTGCGTGCACCAGCGGATCGAGATCAA GGACACCAAAGAGGCCCTGGACAAGATCGAGGAAGAACAGAACAAGTCTAAGAAGAA GGCCCAGCAGGCTGCCGCCGATACAGGCCACTCTAATCAGGTGTCCCAGAACTACCCC ATCGTGCAGAACATCCAGGGCCAGATGGTGCATCAGGCCATCTCTCCCAGAACACTGA ACGCCTGGGTCAAAGTGGTGGAAGAGAAGGCATTCAGCCCCGAAGTGATCCCCATGT TCAGCGCCCTTTCTGAGGGCGCCACACCTCAGGACCTGAACACCATGCTGAATACCGT TGGCGGACACCAGGCCGCCATGCAGATGCTGAAAGAGACAATCAACGAAGAGGCCGC CGAGTGGGACAGAGTGCATCCTGTTCATGCCGGACCTATCGCTCCCGGCCAGATGAG AGAACCTAGAGGCTCTGATATCGCCGGCACCACCAGCACACTGCAAGAGCAGATCGG CTGGATGACCCACAATCCTCCAATTCCAGTGGGCGAGATCTACAAGCGGTGGATCATC CTGGGCCTGAACAAGATTGTGCGGATGTACAGCCCCACCAGCATCCTGGATATCCGG CAGGGACCCAAAGAGCCTTTCCGGGACTACGTGGACCGGTTCTACAAGACCCTGAGA GCTGAGCAGGCCAGCCAAGAAGTGAAGAACTGGATGACAGAGACACTGCTGGTGCAGAACGCCAATCCTGACTGCAAGACCATCCTGAAGGCCCTCGGACCTGGCGCTACACTGG AAGAGATGATGACCGCCTGTCAAGGCGTTGGAGGACCTGGACACAAAGCCAGAGTGC TGGCCGAGGCCATGAGCCAAGTGACCAATCCTGCCACCATCATGATCCAGAAGGGCA ACTTCCGGAACCAGAGAAAGACCGTGAAGTGCTTCAACTGCGGCAAAGAGGGCCACA TTGCCAAGAACTGCAGAGCCCCTCGGAAGAAAGGCTGCTGGAAGTGTGGAAAAGAGG GGCACCAGATGAAGGACTGCACCGAGAGACAGGCCAACTTCCTGGGCAAGATCTGGC CTAGCCACAAGGGCAGACCCGGCAATTTCCTGCAGAGCAGACCTGAGCCTACCGCTCC TCCTGAAGAGAGCTTCAGATTCGGCGAGGAAACCACCACACCTAGCCAGAAGCAAGA GCCCATCGACAAAGAGCTGTACCCTCTGGCCAGCCTGAGAAGCCTGTTTGGCAGCGA TCCTAGCTCTCAAGGCGGGTCGACTGCATCTGGCGGATCTACAGCTAGCGGCGGAGA GAATCAGCAGCTGGAACAGAAGAACAGCCAGCTCAAGCAAGAGATCAGCCAGCTGGA ACAAGAAATCTCCCAGCTCGAATACTGA”)

[0113] SEQ ID NO: 2 (Name: Gag-Adp, Type: Protein, Sequence:“MGARASVLSGGELDRWEKIRLRPGGKKKYKLKHIVWASRELERFAVNPG LLETSEGCRQILGQLQPSLQTGSEELRSLYNTVATLYCVHQRIEIKDTKEALD KIEEEQNKSKKKAQQAAADTGHSNQVSQNYPIVQNIQGQMVHQAISPRTLN AWVKWEEKAFSPEVIPMFSALSEGATPQDLNTMLNTVGGHQAAMQMLKE TINEEAAEWDRVHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMTHN PPIPVGEIYKRWIILGLNKIVRMYSPTSILDIRQGPKEPFRDYVDRFYKTLRA EQASQEVKNWMTETLLVQNANPDCKTILKALGPGATLEEMMTACQGVGGP GHKARVLAEAMSQVTNPATIMIQKGNFRNQRKTVKCFNCGKEGHIAKNCR APRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSHKGRPGNFLQSRPEP TAPPEESFRFGEETTTPSQKQEPIDKELYPLASLRSLFGSDPSSQGGSTASGGS TASGGENQQLEQKNSQLKQEISQLEQEISQLEY*”) Gag (HIV-1) - Linker - Adaptor 1

[0114] SEQ ID NO: 3 (Name: Gag-FS-Adp / Gag-FS-PR, Type: DNA, Sequence: “ATGGGTGCTAGAGCATCTGTTCTGTCTGGCGGCGAGCTGGACAGATGGGAGAAGAT CAGACTCAGACCTGGCGGCAAGAAGAAGTACAAGCTGAAGCACATCGTGTGGGCCAG CAGAGAGCTGGAAAGATTCGCCGTGAATCCCGGCCTGCTGGAAACCTCTGAGGGCTG CAGACAGATCCTGGGACAGCTGCAGCCTTCTCTGCAGACCGGAAGCGAGGAACTGCG GAGCCTGTACAATACCGTGGCCACACTGTACTGCGTGCACCAGCGGATCGAGATCAA GGACACCAAAGAGGCCCTGGACAAGATCGAGGAAGAACAGAACAAGTCTAAGAAGAAGGCCCAGCAGGCTGCCGCCGATACAGGCCACTCTAATCAGGTGTCCCAGAACTACCCC ATCGTGCAGAACATCCAGGGCCAGATGGTGCATCAGGCCATCTCTCCCAGAACACTGA ACGCCTGGGTCAAAGTGGTGGAAGAGAAGGCATTCAGCCCCGAAGTGATCCCCATGT TCAGCGCCCTTTCTGAGGGCGCCACACCTCAGGACCTGAACACCATGCTGAATACCGT TGGCGGACACCAGGCCGCCATGCAGATGCTGAAAGAGACAATCAACGAAGAGGCCGC CGAGTGGGACAGAGTGCATCCTGTTCATGCCGGACCTATCGCTCCCGGCCAGATGAG AGAACCTAGAGGCTCTGATATCGCCGGCACCACCAGCACACTGCAAGAGCAGATCGG CTGGATGACCCACAATCCTCCAATTCCAGTGGGCGAGATCTACAAGCGGTGGATCATC CTGGGCCTGAACAAGATTGTGCGGATGTACAGCCCCACCAGCATCCTGGATATCCGG CAGGGACCCAAAGAGCCTTTCCGGGACTACGTGGACCGGTTCTACAAGACCCTGAGA GCTGAGCAGGCCAGCCAAGAAGTGAAGAACTGGATGACAGAGACACTGCTGGTGCAG AACGCCAATCCTGACTGCAAGACCATCCTGAAGGCCCTCGGACCTGGCGCTACACTGG AAGAGATGATGACCGCCTGTCAAGGCGTTGGAGGACCTGGACACAAAGCCAGAGTGC TGGCCGAGGCCATGAGCCAAGTGACCAATCCTGCCACCATCATGATCCAGAAGGGCA ACTTCCGGAACCAGAGAAAGACCGTGAAGTGCTTCAACTGCGGCAAAGAGGGCCACA TTGCCAAGAACTGCAGAGCCCCTCGGAAGAAAGGCTGCTGGAAGTGTGGAAAAGAGG GGCACCAGATGAAGGACTGCACCGAGAGACAGGCCAACTTCCTGGGCAAGATCTGGC CTAGCCACAAGGGCAGACCCGGCAATTTCCTGCAGAGCAGACCTGAGCCTACCGCTCC TCCTGAAGAGAGCTTCAGATTCGGCGAGGAAACCACCACACCTAGCCAGAAGCAAGA GCCCATCGACAAAGAGCTGTACCCTCTGGCCAGCCTGAGAAGCCTGTTTGGCAGCGA TCCTAGCTCTCAAGGATCTACAGCTAGCgagagacaggctaattttttagggaagatctggccttcccacaaATCTACAGCTAGCgagagacaggctaattttttagggaagatctggccttcccacaa gggaaggccagggaGCTCGGGGGAGAATCAGCAGCTGGAACAAAAGAACAGCCAGCTCAA GCAGGAGATCAGCCAGCTGGAACAGGAAATCTCCCAGCTGGAGTACTAAACTTTCTG CGTGAAGACCTCGCATTTCCCCAGGGCAAAGCTCGCGAGTTCTCATCTGAACAAACTA GGGCTAATTCTCCTACACGGCGGGAACTGCAAGTGTGGGGTCGCGATAATAATAGCT TGTCTGAGGCCGGCGCTGACCGACAGGGGACGGTCAGCTTCTCCTTTCCACAAATTAC CCTGTGGCAAAGGCCACTTGTGACCATCAAAATCGGCGGCCAGCTGAAAGAGGCGCT GCTGGACACCGGCGCGGACGACACTGTCCTGGAAGAGATGAACCTGCCCGGAAGGTG GAAGCCCAAGATGATCGGTGGCATAGGCGGCTTCATTAAAGTCCGCCAATACGACCA AATTCTGATTGAGATATGTGGGCACAAGGCCATCGGTACCGTGTTGGTTGGTCCGAC CCCCGTTAATATCATCGGGCGTAACTTACTTACACAAATCGGATGTACACTTAACTTCT AA”) (underlined = Ribosomal frameshift region)

[0115] SEQ ID NO: 4 (Name: Gag-FS-Adp, Type: Protein, Sequence:“MGARASVLSGGELDRWEKIRLRPGGKKKYKLKHIVWASRELERFAVNPG LLETSEGCRQILGQLQPSLQTGSEELRSLYNTVATLYCVHQRIEIKDTKEALD KIEEEQNKSKKKAQQAAADTGHSNQVSQNYPIVQNIQGQMVHQAISPRTLN AWVKWEEKAFSPEVIPMFSALSEGATPQDLNTMLNTVGGHQAAMQMLKE TINEEAAEWDRVHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMTHN PPIPVGEIYKRWIILGLNKIVRMYSPTSILDIRQGPKEPFRDYVDRFYKTLRA EQASQEVKNWMTETLLVQNANPDCKTILKALGPGATLEEMMTACQGVGGP GHKARVLAEAMSQVTNPATIMIQKGNFRNQRKTVKCFNCGKEGHIAKNCR APRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSHKGRPGNFLQSRPEP TAPPEESFRFGEETTTPSQKQEPIDKELYPLASLRSLFGSDPSSQGSTASERQA NFLG I< IWPSHI< GRPGSSGENOOLEOI< NSOLI< QEISO LEOEISOLEY'”) Gag (HIV-1) - Linker - Protease cleavage site fwith ribosomal frameshift on RNA levell -Translated peptide from the rest of the ribosomal frameshift on RNA level - Linker -Adaptor 1

[0116] SEQ ID NO: 5 (Name: Gag-FS-PR, Type: Protein, Sequence:“MGARASVLSGGELDRWEKIRLRPGGKKKYKLKHIVWASRELERFAVNPG LLETSEGCRQILGQLQPSLQTGSEELRSLYNTVATLYCVHQRIEIKDTKEALD KIEEEQNKSKKKAQQAAADTGHSNQVSQNYPIVQNIQGQMVHQAISPRTLN AWVKWEEKAFSPEVIPMFSALSEGATPQDLNTMLNTVGGHQAAMQMLKE TINEEAAEWDRVHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMTHN PPIPVGEIYKRWIILGLNKIVRMYSPTSILDIRQGPKEPFRDYVDRFYKTLRA EQASQEVKNWMTETLLVQNANPDCKTILKALGPGATLEEMMTACQGVGGP GHKARVLAEAMSQVTNPATIMIQKGNFRNQRKTVKCFNCGKEGHIAKNCR APRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSHKGRPGNFLQSRPEP TAPPEESFRFGEETTTPSQKQEPIDKELYPLASLRSLFGSDPSSQGSTASERQA NFLREDLAFPQGKAREFSSQTRANSPTRRELNYWGRDNNSLSEAGADRQGTVSFSF PQITLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMNLPGRWKPKMIGGIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNF*) Gag (HIV-1) - Linker - Protease cleavage site (with ribosomal frameshift on RNA level) - Translated peptide from the rest of the ribosomal frameshift on RNA level - Synthetic Transframe Peptide - Linker - p6pol - Protease (HIV-1 )

[0117] SEQ ID NO: 6 (Name: Adp-Det-CS-CP, Type: DNA, Sequence:“ATGGCGGGGAAGAATAGTCAATTAAAGGAAGAAAATTCTCAACTCGAAGAAAAGAT ACAACAACTGAAGGAAAAGATCCAACAGCTCAAATATGGTGGCTCGACTGCTTCAGG CGGGGTGTCAGGGTGGAGACTCTTTAAGAAAATATCTGGTGGGTCCACAGCCAGTGC CACAATTATGATGCAGAGAGGCAACACTGCTAGCGGCGGGATGAGCGACCCACGCGA AAGGATTCCGCCCGGAAATTCCGGGGAAGAGACCATTGGCGAGGCTTTTGAATGGTT GAATAGGACAGTTGAGGAAATTAATCGGGAAGCGGTCAATCACTTGCCAAGGGAATT AATTTTCCAGGTTTGGCAACGTAGCTGGGAATACTGGCATGACGAACAAGGCATGAG TCAATCCTATGTAAAATATCGTTATCTCTGTrTGATGCAAAAGGCTCTTTTCATGCATT GTAAGAAGGGATGTCGGTGTCTGGGTGAGGGTCACGGAGCCGGCGGGTGGAGACCA GGCCCACCACCACCCCCTCCTCCCGGCCTGGCATGA”)

[0118] SEQ ID NO: 7 (Name: Adp-Det-CS-CP, Type: Protein, Sequence:“MAGKNSQLKEENSQLEEKIQQLKEKIQQLKYGGSTASGGVSGPVRLFKKTSGGST ASATIMMORGNTASGGMSDPRERIPPGNSGEETIGEAFEWLNRTVEEINREAVNHLP RELIFQVWQRSWEYWHDEQGMSQSYVKYRYLCLMQKALFMHCKKGCRCLGEGHGA GGWRPGPPPPPPPGLA*”! MAG - Adaptor 2 - Linker - Detector - Linker -Protease cleavage site - Cargo protein (here exemplarily Vpx from SIVmac251)

[0119] SEQ ID NO: 8 (Name: Adp-PR, Type: DNA, Sequence:“ATGGCGGGGAAGAATAGTCAATTAAAGGAAGAAAATTCTCAACTCGAAGAAAAGAT ACAACAACTGAAGGAAAAGATCCAACAGCTCAAATATGGGGGCTCCACTGCTAGCGG CGGGACCGTGAGCTTCAGCTTTCCTCAAATTACCCTGTGGCAAAGACCATTGGTTACC ATCAAAATCGGCGGCCAATTGAAAGAGGCCCTGCTGGACACAGGGGCCGACGACACC GTCCTCGAAGAGATGAACCTGCCCGGCCGCTGGAAGCCCAAGATGATCGGCGGCATA GGCGGGTTCATTAAAGTCCGCCAATACGACCAAATCCTGATTGAGATTTGTGGTCACA AGGCAATCGGGACGGTGCTCGTGGGCCCCACCCCCGTTAATATCATCGGCAGGAACC TTCTCACACAAATCGGGTGTACCTTGAACTTCTGA”)

[0120] SEQ ID NO: 9 (Name: Adp-PR, Type: Protein, Sequence:“MAGKNSOLKEENSOLEEKIQOLKEKIQOLKYGGSTASGGTVNFSFPQITLWQRP LVTIKIGGQLKEALLDTGADDTVLEEMNLPGRWKPKMIGGIGGFIKVRQYDQILIEICG HKAIGTVLVGPTPVNIIGRNLLTQIGCTLNF*”) MAG - Adaptor 2 - Linker - N-terminal side of protease cleavage site - Protease (HIV-i)

[0121] SEQ ID NO: 10 (Name: Adp-PR (T26S), Type: DNA, Sequence:“ATGGCGGGGAAGAATAGTCAATTAAAGGAAGAAAATTCTCAACTCGAAGAAAAGAT ACAACAACTGAAGGAAAAGATCCAACAGCTCAAATATGGGGGCTCCACTGCTAGCGG CGGGACCGTGAGCTTCAGCTTTCCTCAAATTACCCTGTGGCAAAGACCATTGGTTACC ATCAAAATCGGCGGCCAATTGAAAGAGGCCCTGCTGGACAGTGGGGCCGACGACACC GTCCTCGAAGAGATGAACCTGCCCGGCCGCTGGAAGCCCAAGATGATCGGCGGCATA GGCGGGTTCATTAAAGTCCGCCAATACGACCAAATCCTGATTGAGATTTGTGGTCACA AGGCAATCGGGACGGTGCTCGTGGGCCCCACCCCCGTTAATATCATCGGCAGGAACC TTCTCACACAAATCGGGTGTACCTTGAACTTCTGA”)

[0122] SEQ ID NO: 11 (Name: Adp-PR (T26S), Type: Protein, Sequence:“MAGKNSOLKEENSOLEEKIOOLKEKIOQLKYGGSTASGGTVSESFPQITLWQRP LVTIKIGGOLKEALLDSGADDTVLEEMNLPGRWKPKMIGGIGGFIKVROYDQILIEICG HKAIGTVLVGPTPVNIIGRNLLTQIGCTLNF*”) MAG - Adaptor 2 - Linker - N- terminal side of protease cleavage site - Protease (HIV-i; T26S mutant)

[0123] SEQ ID NO: 72 (Name: IgK-scFvGemtuzumab-HA-IgGq-PDFGRb), Type: Protein, Sequence:MDMRVPAOLLGLLLLWLRGARCGSGEVOLVOSGAEVKKPGSSVKVSCK ASGYTI TDSNIHWVRQAPGQSLEWIGYIYPYNGGTDYNQKFKNRATLTVDNPTNTAYMELSSL RSEDTAFYYCVNGNPWLAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSTLS ASVGDRVTITCRASESLDNYGIRFLTWFQQKPGKAPKLLMYAASNQGSGVPSRFSGSG SGTEFTLTISSLQPDDFATYYCQQTKEVPWSFGQGTKVEVKRTVAAGSTASGYPYDVPD YAGSTASGESKYGPPCPSCPAPEFLGGPRELPPTLLGNSSEEESQLETNVTYWEEEQEF[sequence from image - garbled OCR of protein sequence continuation]IgK signal peptide - Linker - scFV Gemtuzumab - Linker - HA-tag - Linker - IgGq hinge - PDFGRb stalk

[0124]

[0125] SEQ ID NO: 73 (Name: IgK-scFvGemtuzumab-HA-CD8a), Type:Protein, Sequence:MDMRVPAOLLGLLLLWLRGARCGSGEVOLVOSGAEVKKPGSSVKVSCK ASGYTI TDSNIHWVRQAPGQSLEWIGYIYPYNGGTDYNQKFKNRATLTVDNPTNTAYMELSSLRSEDTAFYYCVNGNPWLAYWGOGTLVTVSSGGGGSGGGGSGGGGSDIOLTOSPSTLS ASVGDRVTITCRASESLDNYGIRFLTWFOOKPGKAPKLLMYAASNOGSGVPSRFSGSG SGTEFTLTISSLOPDDFATYYCOOTKEVPWSFGOGTKVEVKRTVAAGSTASGypyDVPDIgK signal peptide - Linker - scFV Gemtuzumab - Linker - HA-tag - Linker -CD8a stalk + transmembrane + cytosolic domains

[0126] The present invention also related to the following items:The above definitions and preferred embodiments as described above apply mutatis mutandis to the below items as far as being combinable therewith.1. A system or kit for obtaining a virus-like particle (VLP) comprising:a first nucleic acid comprising a first nucleotide sequence encoding a first fusion polypeptide that comprises a retroviral gag polypeptide of the VLP and an adaptor polypeptide of a first kind; anda second nucleic acid comprising a second nucleotide sequence encoding a second fusion polypeptide that comprises an adaptor polypeptide of the first kind or of a second kind, and a functional or therapeutic polypeptide;wherein the adaptor polypeptides of the first and / or the second kind are configured to interact with each other such that during formation of the VLP in a VLP packaging cell the second fusion polypeptide is packaged into the VLP.2. The system or kit of embodiment 1,wherein the second fusion polypeptide further comprises a protease cleavage site arranged in between the adaptor polypeptide of the first kind or of the second kind and the functional or therapeutic polypeptide, wherein the protease cleavage site preferably comprises or consists of SEQ ID NO: 14 (“SATIMM0RGN1 or a sequence being at least 80%, and preferably at least 90% identical thereto, or SEQ ID NO 15 (“ERQANFLGKI”)=or a sequence being at least 80%, and preferably at least 90% identical thereto.3. The system or kit of embodiment 1 or 2,wherein the VLP is an HIV-1 derived VLP, preferably an HIV-1 derived Gag, and most preferably an HIV-1 derived Gag comprising or consisting of SEQ ID NO: 16 (“MGARASVLSGGELDRWEKIRLRPGGKKKYKLKHIVWASRELERFAVNPGLLETSEG CRQILGQLQPSLQTGSEELRSLYNTVATLYCVHQRIEIKDTKEALDKIEEEQNKSKKKA QQAAADTGHSNQVSQNYPIVQNIQGQMVHQAISPRTLNAWVKWEEKAFSPEVIPMF SALSEGATPQDLNTMLNTVGGHQAAMQMLKETINEEAAEWDRVHPVHAGPIAPGQ MREPRGSDIAGTTSTLQEQIGWMTHNPPIPVGEIYKRWIILGLNKIVRMYSPTSILDIR QGPKEPFRDYVDRFYKTLRAEQASQEVKNWMTETLLVQNANPDCKTILKALGPGATL EEMMTACQGVGGPGHKARVLAEAMSQVTNPATIMIQKGNFRNQRKTVKCFNCGKEG HIAKNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSHKGRPGNFLQSRPEP TAPPEESFRFGEETTTPSQKQEPIDKELYPLASLRSLFGSDPSSQ”) or a sequence being at least 8o% identical, preferably at least 90% identical, and most preferred at least 95% thereto; and / orwherein the protease cleavage site is selected from the group consisting of wildtype HIV-i protease cleavage sites and engineered derivatives thereof, and preferably comprises or consists of SEQ ID NO: 14 (“SATIMMQRGN)_or a sequence being at least 80%, and preferably at least 90% identical thereto.4. The system or kit of any of embodiments 1 to 3,wherein the adaptor polypeptide of the first kind is at least in part complementary to the adaptor polypeptide of the second kind or at least in part self-complementary.5. The system or kit of embodiment 4,wherein the adaptor polypeptide of the first kind and / or the adaptor polypeptide of the second kind comprises at least partially complementary coiled-coil domains, wherein the coiled-coil domains are preferably dimeric coiled-coil (CC) domains.6. The system or kit of any of embodiments 1 to 5, wherein the adaptor polypeptide of the first kind comprises or consists of SEQ ID NO: 12 (“ENQQLEQKNSQLKQEISQLEQEISQLEY”) or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto, and the adaptor polypeptide of the second kind SEQ ID NO: 13 (“KNSQLKEENSQLEEKIQQLKEKIQQLKY”) or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto.7. The system or kit of any one of embodiments 1 to 6,wherein the adaptor polypeptide of the first kind and / or the adaptor polypeptide of the second kind comprise or consist of at least 15, preferably at least 20, and more preferably at least 25 amino acids; and / orwherein the adaptor polypeptide of the first kind and / or the adaptor polypeptide of the second kind comprise or consist of at most 200, preferably at most 100, more preferably at most 50, and even more preferably at most 30 amino acids. 8. The system or kit of any one of embodiments 1 to 7, wherein the adaptor polypeptide of the first kind and / or the adaptor polypeptide of the second kind comprise or consist of 15 to 100 amino acids, preferably 20 to 50 amino acids, and most preferably 25 to 30 amino acids.9. The system or kit of any of embodiments 1 to 8, further comprising:a third nucleic acid comprising a third nucleotide sequence encoding a third fusion polypeptide that comprises an adaptor polypeptide of the first kind or of the second kind, and a protease polypeptide that is adapted to cleave the protease cleavage site.10. The system or kit of any of embodiment 7, wherein the protease polypeptide comprises a viral protease, preferably an HIV-i protease, wherein the HIV-i protease preferably comprises or consists of SEQ ID NO: 17 (“PQITLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMNLPGRWKPKMIGGIGGFIKVR QYDQILIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNF”) or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto, or SEQ ID NO: 18 (“PQITLWQRPLVTIKIGGQLKEALLDSGADDTVLEEMNLPGRWKPKMIGGIGGFIKVR QYDQILIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNF”) or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto.11. The system or kit of embodiment 9 or 10,wherein the protease polypeptide of the third fusion polypeptide comprises a natural protease cleavage site arranged at the N-terminus of the protease polypeptide, wherein the natural protease cleavage site preferably comprises or consist of SEQ ID NO: 19 (“SFSFPQIT”) or a sequence being least 75%, preferably 87.5% identical thereto. (In this connection it is of note that the complete cleavage sequence is SFSFPQIT, but that the PQIT part is itself already part of the protease (see start of the above SEQ ID NOs 17 and 18)).12. The system or kit of any of embodiments 1 to 9, further comprising:a fourth nucleic acid comprising a fourth nucleotide sequence encoding a first surface protein of the VLP adapted to enable fusion of the VLP with a target cell.13. The system or kit of embodiment 12,wherein the first surface protein is further adapted to enable binding of the VLP to the target cell; and / orwherein the first surface protein comprises VSV-G or derivatives thereof or other viral glycoproteins known to be pseudotyped with lentiviral particles..14. The system or kit of embodiment 12 or 13, wherein the fourth nucleotide sequence or a fifth nucleotide sequence of a fifth nucleic acid further encodes a second surface protein of the VLP adapted to enable binding of the VLP to one or more selected target cell types.15. The system or kit of embodiment 14, wherein the first surface protein is not adapted to enable binding of the VLP to the target cell and / or comprises a mutated VSV-G; and / orwherein the second surface protein of the VLP comprises one or more cell-type specific antibody fragments.16. The system or kit of any of embodiments 1 to 15 wherein the functional or therapeutic polypeptide is selected from the group consisting of:a bacterial toxin, a cytotoxic protein, and a cell death-inducing protein adapted for killing of cell lines in vitro or killing of cancerous cells in vivo.17. The system or kit of any of embodiments 1 to 15 wherein the functional or therapeutic polypeptide is selected from the group consisting of:an enzyme for intracellular catalysis of chemical reactions, an immunomodulatory protein for the downregulation or the upregulation of immune function, a protein that influences the degradation of specific proteins in the target cells to modulate protein levels, designer nucleases or recombinases for genomic editing of cell lines in vitro or gene therapy purposes in vivo, and a fluorescent or light emitting protein.18. The system or kit of any of embodiments 1 to 15, wherein the functional or therapeutic polypeptide is selected from the group consisting of:a transcription factor to induce or repress gene expression, an RNA-binding protein complexed with RNA molecules to influence gene expression in target cells, asignaling cascade component to trigger cellular responses, a vaccination agent, a tumor suppressor protein, and an anti-aging or lifespan-enhancing protein.19. The system or kit of any of embodiments 1 to 18,wherein the second fusion polypeptide further comprises a detector polypeptide that is arranged between the adaptor polypeptide of the first kind or the second kind and the protease cleavage site or that is arranged between the protease cleavage site and the functional or therapeutic polypeptide or that is arranged at an end of the adaptor polypeptide of the first kind or the second kind or at an end of the functional or therapeutic polypeptide, wherein the detector polypeptide preferably comprises of consist of SEQ ID NO: 20 (“VSGWRLFKKIS”) or a sequence being at least 80%, and preferably at least 90% identical thereto.20. The system or kit of embodiment 19, wherein the detector polypeptide comprises an epitope tag capable of producing a bioluminescent signal and / or an entire luciferase producing a bioluminescent signal.21. The system or kit of any of embodiments 1 to 20, wherein the first, the second, the third, the fourth and / or the fifths nucleic acids are part of a respective transfection plasmid.22. A system or kit for obtaining a virus-like particle (VLP) comprising:a first nucleic acid comprising a first nucleotide sequence comprising a translational frameshift element;wherein, without translational frameshifting, the first nucleotide sequence encodes a first fusion polypeptide comprising a retroviral gag polypeptide of the VLP and an adaptor polypeptide of a first kind;wherein, with translational frameshifting, the first nucleotide sequence encodes a second fusion polypeptide comprising the retroviral gag polypeptide of the VLP, and a protease polypeptide that is adapted to cleave a protease cleavage site; and a second nucleic acid comprising a second nucleotide sequence encoding a third fusion polypeptide that comprises an adaptor polypeptide of the first kind or of a second kind, and a functional or therapeutic polypeptide;wherein the adaptor polypeptides of the first and / or the second kind are configured to interact with each other such that during formation of the VLP on a cell surface of a VLP packaging cell the third fusion polypeptide is packaged into the VLP.23. The system or kit of any of embodiment 20, wherein the first nucleic acid comprises a translational frameshifting element, wherein the translationalframeshifting element is preferably a ribosomal frameshift region, wherein the ribosomal frameshift region preferably comprises of consists of SEQ ID NO: 21 f”agagacaggctaattttttagggaagatctggccttcccacaagggaaggccaggga”) or a sequence being at least 8o%, preferably at least 90% and most preferably at least 95% identical thereto, wherein the translational frameshifting element is within the first nucleic acid C-terminal of the nucleic acid sequence encoding the retroviral gag polypeptide of the VLP and N-terminal of the nucleic acid sequences encoding the adaptor polypeptide of a first kind and the protease polypeptide that is adapted to cleave a protease cleavage site.24. The system or kit of embodiment 22 or 23, wherein the second fusion polypeptide further comprises one or more of:a protease cleavage site, wherein the protease cleavage site preferably comprises or consists of SEQ ID NO: 14 (“SATIMMQRGN) or sequence being at least 80%, and preferably at least 90% identical thereto, or SEQ ID NO: 15 (“ERQANFLGKI”) or sequence being at least 80%, and preferably at least 90% identical thereto;a synthetic transframe polypeptide, preferably comprising 70% or more of amino acids with small and / or polar non-charged side chains;a protease regulation domain, preferably a retroviral p6pol polypeptide; and most preferably an HIV-i p6pol polypeptide, wherein the HIV-i p6pol polypeptide preferably comprises or consists of SEQ ID NO: 22 (“FLREDLAFPQGKAREFSSEQTRANSPTRRELQVWGRDNNSLSEAGADRQGTVSFSF” ) or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto.25. The system or kit of any of embodiments 22 to 24,wherein the translational frameshift element is arranged in the first nucleotide sequence between a first region encoding the gag polypeptide of the VLP and between a second region encoding the protease polypeptide or between a first region encoding the gag polypeptide of the VLP and second region encoding the adaptor polypeptide, wherein the translational frameshifting element is preferably a ribosomal frameshift region, wherein the ribosomal frameshift region preferably comprises or consists of SEQ ID NO: 21 C’agagacaggctaattttttagggaagatctggccttcccacaagggaaggccaggga”) or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto.26. The system or kit of any of embodiments 22 to 25,wherein the VLP is an HIV-1 derived VLP, preferably an HIV-1 derived Gag, and most preferably an HIV-1 derived Gag comprising or consisting of SEQ ID NO: 16 (“MGARASVLSGGELDRWEKIRLRPGGKKKYKLKHIVWASRELERFAVNPGLLETSEG CRQILGQLQPSLQTGSEELRSLYNTVATLYCVHQRIEIKDTKEALDKIEEEQNKSKKKA QQAAADTGHSNQVSQNYPIVQNIQGQMVHQAISPRTLNAWVKWEEKAFSPEVIPMF SALSEGATPQDLNTMLNTVGGHQAAMQMLKETINEEAAEWDRVHPVHAGPIAPGQ MREPRGSDIAGTTSTLQEQIGWMTHNPPIPVGEIYKRWIILGLNKIVRMYSPTSILDIR QGPKEPFRDYVDRFYKTLRAEQASQEVKNWMTETLLVQNANPDCKTILKALGPGATL EEMMTACQGVGGPGHKARVLAEAMSQVTNPATIMIQKGNFRNQRKTVKCFNCGKEG HIAKNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSHKGRPGNFLQSRPEP TAPPEESFRFGEETTTPSQKQEPIDKELYPLASLRSLFGSDPSSQ”) or a sequence being at least 80% identical, preferably at least 90% identical, and most preferred at least 95% thereto; and / orwherein the protease cleavage site is selected from the group consisting of wildtype HIV-i protease cleavage sites and engineered derivatives thereof, wherein the HIV-i protease cleavage site preferably comprises or consists of SEQ ID NO: 15 (“ERQANFLGKI”) or a sequence being at least 80%, and preferably at least 90% identical thereto; and / orwherein the protease polypeptide comprises a viral protease, preferably an HIV-1 protease, wherein the HIV-i protease preferably comprises or consists of SEQ ID NO: 17 (“PQITLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMNLPGRWKPKMIGGIGGFIKVR QYDQILIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNF”) or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto, or SEQ ID NO: 18 (“PQITLWQRPLVTIKIGGQLKEALLDSGADDTVLEEMNLPGRWKPKMIGGIGGFIKVR QYDQILIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNF”) or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto.27. The system or kit of any of embodiments 22 to 26,wherein the adaptor polypeptide of the first kind is at least in part complementary to the adaptor polypeptide of the second kind or at least in part self-complementary.28. The system or kit of embodiment 27, wherein the adaptor polypeptide of the first kind and / or the adaptor polypeptide of the second kind comprises at least partially complementary coiled-coil domains, wherein the coiled-coil domains are preferably dimeric coiled-coil (CC) domains.29. The system or kit of any one of embodiments 22 or 28,wherein the adaptor polypeptide of the first kind and / or the adaptor polypeptide of the second kind comprise at least 15, preferably at least 20, and more preferably at least 25 amino acids; and / orwherein the adaptor polypeptide of the first kind and / or the adaptor polypeptide of the second kind comprise at most 200, preferably at most 100, more preferably at most 50, and even more preferably at most 30 amino acids.30. The system or kit of any one of embodiments 22 to 29, wherein the adaptor polypeptide of the first kind and / or the adaptor polypeptide of the second kind comprise or consist of 15 to 100 amino acids, preferably 20 to 50 amino acids, and most preferably 25 to 30 amino acids.31. The system or kit of any of embodiments 22 to 30, further comprising:a third nucleic acid comprising a third nucleotide sequence encoding a first surface protein of the VLP adapted to enable fusion of the VLP with a target cell.32. The system or kit of embodiment 31,wherein the first surface protein is further adapted to enable binding of the VLP to the target cell; and / orwherein the first surface protein comprises VSV-G or derivatives thereof.33. The system or kit of embodiment 31 or 32, wherein the third nucleotide sequence or a fourth nucleotide sequence of a fourth nucleic acid further encodes a second surface protein of the VLP adapted to enable binding of the VLP to one or more selected target cell types.34. The system or kit of embodiment 33, wherein the first surface protein is not adapted to enable binding of the VLP to the target cell and / or comprises a mutated VSV-G; and / orwherein the second surface protein of the VLP comprises one or more cell-type specific antibody fragments.35. The system or kit of any of embodiments 22 to 34 wherein the functional or therapeutic polypeptide is selected from the group consisting of:a bacterial toxin, a cytotoxic protein, and a cell death-inducing protein adapted for killing of cell lines in vitro or killing of cancerous cells in vivo.36. The system or kit of any of embodiments 22 to 34 wherein the functional or therapeutic polypeptide is selected from the group consisting of:an enzyme for intracellular catalysis of chemical reactions, an immunomodulatory protein for the downregulation or the upregulation of immune function, a protein that influence the degradation of specific proteins in the target cells to modulate protein levels, a designer nucleases or recombinases for genomic editing of cell lines in vitro or gene therapy purposes in vivo, and a fluorescent or light emitting protein.37. The system or kit of any of embodiments 22 to34, wherein the functional or therapeutic polypeptide is selected from the group consisting of:a transcription factor to induce or repress gene expression, an RNA-binding protein complexed with RNA molecules to influence gene expression in target cells, a signaling cascade component to trigger cellular responses, a vaccination agent, a tumor suppressor protein, and an anti-aging or lifespan-enhancing protein.38. A virus-like particle (VLP) packaging cell, comprising the system or kit of any one of the preceding embodiments 1 to 21 or the system or kit of any of embodiments 22 to 37.39. A method of obtaining or for producing a virus-like particle (VLP) comprising a functional or therapeutic polypeptide, the method comprising:Introducing, preferably transfecting the system of any one of embodiments 1 to 21 or the system of any of embodiments 22 to 37 into a virus-like particle (VLP) packaging cell; andObtaining, preferably isolating the VLPs produced by the VLP packaging cell based on the introduced system.0. A virus-like particle (VLP) obtained or obtainable by the method of embodiment 39- 1. A virus-like particle (VLP) comprising or packaging(i)a first fusion polypeptide that comprises a retroviral gag polypeptide of the VLP and an adaptor polypeptide of a first kind; anda second fusion polypeptide that comprises an adaptor polypeptide of the first kind or of a second kind, and a functional or therapeutic polypeptide;wherein the adaptor polypeptides of the first and / or the second kind interact with each other within the VLP; or(ii)a first fusion polypeptide comprising a retroviral gag polypeptide of the VLP and an adaptor polypeptide of a first kind;a second fusion polypeptide comprising the retroviral gag polypeptide of the VLP, and a protease polypeptide that is adapted to cleave a protease cleavage site; and a third fusion polypeptide that comprises an adaptor polypeptide of the first kind or of a second kind, and a functional or therapeutic polypeptide;wherein the adaptor polypeptides of the first and / or the second kind interact with each other within the VLP.42. The VLP of embodiment 40 or 41 for use in treatment of cancer.43. The VLP of embodiment 40 or 41 for use in treatment of an autoimmune disease.44. The VLP of embodiment 40 or 41 for use in gene therapy.45. The VLP of embodiment 40 or 41 for use in treatment of a neurologic disorder.46. The VLP of embodiment 40 or 41 for use in treatment of an infectious disease.47. One or more vectors (e.g. one or more transfection plasmids) encoding polypeptides derived from the first nucleic acid and the second nucleic acid, and, if present, the third nucleic acid, fourth nucleic acid, and / or fifth nucleic acid of the system or kit of any one of the preceding embodiments 1 to 19 or the system or kit of any of embodiments 20 to 33.48. Use of the VLP of embodiment 36 or 37 for the infection of an ex vivo or in vitro cell.

Claims

Claims1. A system for obtaining a virus-like particle (VLP) comprising:a first nucleic acid comprising a first nucleotide sequence comprising a translational frameshift element;wherein, without translational frameshifting, the first nucleotide sequence encodes a first fusion polypeptide comprising a retroviral gag polypeptide of the VLP and an adaptor polypeptide of a first kind;wherein, with translational frameshifting, the first nucleotide sequence encodes a second fusion polypeptide comprising the retroviral gag polypeptide of the VLP, and a protease polypeptide that is adapted to cleave a protease cleavage site; and a second nucleic acid comprising a second nucleotide sequence encoding a third fusion polypeptide that comprises an adaptor polypeptide of the first kind or of a second kind, and a functional or therapeutic polypeptide;wherein the adaptor polypeptides of the first and / or the second kind are configured to interact with each other such that during formation of the VLP on a cell surface of a VLP packaging cell the third fusion polypeptide is packaged into the VLP.

2. The system of claim 1, wherein the second fusion polypeptide further comprises one or more of:a protease cleavage site;a synthetic transframe polypeptide, preferably comprising 70% or more of amino acids with small and / or polar non-charged side chains;a protease regulation domain, such as a retroviral p6pol polypeptide; optionally an HIV-i p6pol polypeptide.

3. The system of claim 1 or 2, wherein the third fusion polypeptide further comprises a detector polypeptide that is arranged between the adaptor polypeptide of the first kind or the second kind and the protease cleavage site or that is arranged between the protease cleavage site and the functional or therapeutic polypeptide or that is arranged at an end of the adaptor polypeptide of the first kind or the second kind or at an end of the functional or therapeutic polypeptide.4- The system of any of claims 1 to 3,wherein the translational frameshift element is arranged in the first nucleotide sequence between a first region encoding the gag polypeptide of the VLP and between a second region encoding the protease polypeptide or between a first region encoding the gag polypeptide of the VLP and second region encoding the adaptor polypeptide; and / orwherein the adaptor polypeptide of the first kind is at least in part complementary to the adaptor polypeptide of the second kind or at least in part self-complementary, and optionally,wherein the adaptor polypeptide of the first kind and / or the adaptor polypeptide of the second kind comprises at least partially complementary coiled-coil domains.

5. The system of any of claims 1 to 4, further comprising:a third nucleic acid comprising a third nucleotide sequence encoding a first surface protein of the VLP adapted to enable fusion of the VLP with a target cell and, optionally,wherein the first surface protein is further adapted to enable binding of the VLP to the target cell; and / orwherein the first surface protein comprises VSV-G or derivatives thereof.

6. The system of claim 4 or 5, wherein the third nucleotide sequence or a fourth nucleotide sequence of a fourth nucleic acid further encodes a second surface protein of the VLP adapted to enable binding of the VLP to one or more selected target cell types; and, optionally,wherein the first surface protein is not adapted to enable binding of the VLP to the target cell; and / or wherein the second surface protein of the VLP comprises one or more cell-type specific antibody fragments.

7. A system for obtaining a virus-like particle (VLP) comprising:a first nucleic acid comprising a first nucleotide sequence encoding a first fusion polypeptide that comprises a retroviral gag polypeptide of the VLP and an adaptor polypeptide of a first kind; anda second nucleic acid comprising a second nucleotide sequence encoding a second fusion polypeptide that comprises an adaptor polypeptide of the first kind or of a second kind, and a functional or therapeutic polypeptide;wherein the adaptor polypeptides of the first and / or the second kind are configured to interact with each other such that during formation of the VLP in a VLP packaging cell the second fusion polypeptide is packaged into the VLP.

8. The system of claim 7,wherein the second fusion polypeptide further comprises a protease cleavage site arranged in between the adaptor polypeptide of the first kind or of the second kind and the functional or therapeutic polypeptide; and / orwherein the adaptor polypeptide of the first kind is at least in part complementary to the adaptor polypeptide of the second kind or at least in part self-complementary; and, optionally,wherein the adaptor polypeptide of the first kind and / or the adaptor polypeptide of the second kind comprises at least partially complementary coiled-coil domains.

9. The system of any of claims 7 to 8, further comprising:a third nucleic acid comprising a third nucleotide sequence encoding a third fusion polypeptide that comprises an adaptor polypeptide of the first kind or of the second kind, and a protease polypeptide that is adapted to cleave the protease cleavage site; and, optionally,wherein the protease polypeptide comprises a viral protease, preferably an HIV-1 protease, and optionally wherein the protease polypeptide of the third fusion polypeptide comprises a natural protease cleavage site arranged at the N-terminus of the protease polypeptide.

10. The system of any of claim 7 to 9, further comprising:a fourth nucleic acid comprising a fourth nucleotide sequence encoding a first surface protein of the VLP adapted to enable fusion of the VLP with a target cell; and, optionally,wherein the first surface protein is further adapted to enable binding of the VLP to the target cell; and / orwherein the first surface protein comprises VSV-G or derivatives thereof; and, optionally, wherein the fourth nucleotide sequence or a fifth nucleotide sequence of a fifth nucleic acid further encodes a second surface protein of the VLP adapted to enable binding of the VLP to one or more selected target cell types; and, optionally, wherein the first surface protein is not adapted to enable binding of the VLP to the target cell; and / or wherein the second surface protein of the VLP comprises one or more cell-type specific antibody fragments.

11. The system of any of claims 7 to 10,wherein the second fusion polypeptide further comprises a detector polypeptide that is arranged between the adaptor polypeptide of the first kind or the second kind and the protease cleavage site or that is arranged between the protease cleavage site and the functional or therapeutic polypeptide or that is arranged at an end of the adaptor polypeptide of the first kind or the second kind or at an end of the functional or therapeutic polypeptide.

12. The system of any of claims 1 to 6 or 7 to 11 wherein the functional or therapeutic polypeptide is selected from the group consisting of:a bacterial toxin, a cytotoxic protein, and a cell death-inducing protein adapted for killing of cell lines in vitro or killing of cancerous cells in vivo, an enzyme for intracellular catalysis of chemical reactions, an immunomodulatory protein for the downregulation or the upregulation of immune function, a protein that influences the degradation of specific proteins in the target cells to modulate protein levels, designer nucleases or recombinases for genomic editing of cell lines in vitro or gene therapy purposes in vivo, and a fluorescent or light emitting protein, a transcription factor to induce or repress gene expression, an RNA-binding protein complexed with RNA molecules to influence gene expression in target cells, a signaling cascade component to trigger cellular responses, a vaccination agent, a tumor suppressor protein, and an antiaging or lifespan-enhancing protein.

13. A virus-like particle (VLP) packaging cell, comprising the system of any one of the preceding claims 1 to 12.14- A method of obtaining a virus-like particle (VLP) comprising a functional or therapeutic polypeptide, the method comprising:introducing the system of any one of claims 1 to 12 into a virus-like particle (VLP) packaging cell; andobtaining the VLPs produced by the VLP packaging cell based on the introduced system.

15. A virus-like particle (VLP) obtained by the method of claim 14, and being configured for one of: for use in treatment of cancer, for use in treatment of an autoimmune disease, for use in gene therapy, for use in treatment of a neurologic disorder, and for use in treatment of an infectious disease.

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