Method of producing leukocytes expressing artificial antigen receptors

Abstract

The present invention relates to a lipid nanoparticle (LNP) suitable for transfecting a leukocyte, which nanoparticle comprises an mRNA encoding for a transposase enzyme, and a DNA encoding for an antigen receptor.

Description

Method of producing leukocytes expressing artificial antigen receptorsREFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT XML 1.0 FORMAT FILE (.xml)Pursuant to the EFS-Web legal framework and 37 CFR §§ 1.821-825 (see MPEP § 2442.03(a)), Rule 30 EPC, and § 11 PatV, an electronic sequence listing compliant with WIPO standard ST.26 in the form of an XML 1.0 format file is submitted concurrently with the instant application, and the entire contents of the sequence listing are incorporated herein by reference. For the avoidance of doubt, if discrepancies exist between the sequences mentioned in the specification and the electronic sequence listing, the sequences in the specification shall be deemed to be the correct ones.FIELD OF THE INVENTIONThe present application relates to the production of leukocytes expressing artificial antigen receptors.BACKGROUNDCAR-T cells are a proven therapeutic modality representing one of the most effective treatment modalities for certain types of cancers, especially leukemias and lymphomas. A single administration of autologous CAR-T cells can cure the majority of leukemia and lymphoma patients as well as myeloma patients, as demonstrated in published clinical trials.Therefore, there is a high interest in improving manufacturing efficiencies for autologous CAR- T therapies with the objective to increase supply and to lower the manufacturing costs. Currently, due to very complex centralized CAR-T manufacturing using y-retroviral or lentiviral vectors, which themselves are difficult to produce at scale, all marketed CAR-T products approved by the FDA for the therapy of human disease, are very expensive with reimbursement costs in the US ranging between USS 350-475’000 per CAR-T dose. In addition, the complexity of centralized CAR-T manufacturing employing y-retroviral or lentiviral vectors is also suffering from scalability issues. As a consequence, effective CAR-T cell therapies are not broadly accessible even in G7 countries and hardly accessible to cancer patients globally.Recent successes of CAR-T therapies also in treating severe autoimmune conditions (Mackensen et al, 2022) and in solid tumors (Uslu and June, 2024) will even demand higher numbers of CAR-T doses to be manufactured for patients in the future, which will be a significant challenge with current y-retroviral and lentiviral vector technologies.All FDA-approved autologous CAR-T cells to date are manufactured by a highly complex, centralized manufacturing process employing lentiviral vectors, which is associated with a number of issues, of which the most relevant are: (i) very high cost per manufactured CAR-T batch, (ii) limited supply of CAR-T therapies (abou 9'000 CAR-T doses per year, globally in 2023, due to limited viral vector supply, (iii) long manufacturing timelines, during which patients require bridging therapy, often not effective enough to keep the patients alive for CAR- T therapy until the completion of the manufacturing process, and (iv) cell expansion procedures in the manufacturing process, which can lead to undesired differentiation and / or exhaustion of T cells lowering their effectiveness against tumor cells.In recognition of these above-mentioned challenges, alternative technologies for CAR-T manufacturing have been developed. These alternative technologies involve mostly two non- viral CAR-T manufacturing approaches: First, DNA-transposon technologies, or, second, the use of mRNA-Lipid-Nano-Particles (mRNA-LNPs) (Moretti et al., 2022).DNA-Transposon-based technologies are the most established alternative to viral CAR-T cell manufacturing, because, as opposed to mRNA-based CAR-T technologies, that only enable transient CAR expression, DNA transposition delivers CAR-T cells stably expressing ectopic CARs, similar to y-retroviral or lentiviral vectors. Several academic and commercial groups arecurrently evaluating transposon-generated CAR-T cells in clinical trials (Magnani et al., 2020). The manufacturing of CAR-T cells by DNA transposon technology involves the transfer of either DNA or mRNA / DNA vectors into cells by electroporation, often preceded and followed by cell expansion, in order to compensate for the loss of cells during harsh electroporation conditions (Lock et al., 2022). In fact, electroporation in combination with cell expansion of primary patients T cells for weeks, bears the risk of T cell differentiation and T cell exhaustion, rendering such cells less effective for anti-cancer treatment (Watanabe et al., 2022).More recently, and based on establishing mRNA-LNPs for the development of vaccines, mRNA-LNP technology has become the second major alternative to complex lentiviral CAR- T manufacturing, in which mRNA encoding CAR mRNA vectors are transfected into primary T-lymphocytes to generate CAR-T cells in several settings (Moretti et al., 2022). While the mRNA-LNP technology has been shown to work quite efficiently for gene transfer and CAR expression, this technology can only confer transient CAR expression due to the instability of mRNA in the cytosol of cells. As a consequence, durable responses in cancer treatment with mRNA-LNP generated CAR-T cells cannot be achieved and are not expected. This challenge may be addressed with repeat dosing of mRNA-LNP generated CAR T cells, but (i) in the autologous setting, autologous CAR-T cells are only available in limited quantity, and (ii) repeat dosing is associated with increased toxicity and immunogenicity lowering the effectiveness of the mRNA-LNP approach in cancer treatment.Although gene transfer to mammalian cells with Lipid-Nano-Particles (LNPs) loaded with mRNA (mRNA-LNPs) has been known for over 25 years (Malone et al., 1989), mRNA-LNP technology reached center stage only recently, due to the SARS-Cov-2 (COVID-19) pandemic. The first and most effective SARS-Cov-2 vaccines were mRNA-LNPs encoding viral SARS- Cov2 spike proteins. Vaccination with these mRNA-LNPs leads to a transient expression of SARS-Cov2 spike proteins primarily in human muscle cells in situ, but also their shedding into the tissue locally, where the viral proteins are taken up and processed by tissue-resident professional antigen-presenting cells (APCs). This in turn elicits a SARS-Cov2 specific immune response involving the activation of B and T-lymphocytes that eventually results in the generation of a strong SARS-Cov2 spike-protein-specific, humoral antibody response and cellular response with the formation of memory B and T cells protecting the vaccinated individuals from infection with a real SARS-Cov-2 virus, or at least attenuating the course of a SARS-Cov2 infection (Bettini et al., 2021).The broad SARS-Cov2 vaccination campaigns with mRNA-LNPs have demonstrated the feasibility of large-scale and cost-effective manufacturing of mRNA-LNPs and expression of ectopic proteins in human cells in situ. This has triggered a surge of research and development activities and a high interest to also utilize this technology in therapeutic settings, including e.g. enzyme-replacement strategies, gene editing approaches, but also for the expression of chimeric antigen receptors (CARs) in T-lymphocytes for the treatment of cancer (Kiaie et al., 2022).However, due to the short half-life of mRNA after LNP-mediated delivery of mRNA into the cytosol of cells, this technology may be well suited for vaccination, enzyme-replacement or gene editing strategies, but it does not allow stable expression of CARs, which is essential for achieving durable responses in cancer treatment. Stable expression of chimeric antigen receptors in CAR-T cells has been shown to be critical for long durability of responses or complete responses in cancer patients and the persistance of CAR-T positive cells in patients for many years, in particular CAR-T cells with a central memory phenotype has been associated with long durability of responses (Melenhorst et al., 2022). Targeting mRNA LNPs effectively to patient’s primary T cells has been a challenge, which recently has been ameliorated by conjugating targeting T cell targeting moieties to the surface of mRNA-LNPs, like anti-CD4 or anti-CD8 binders (Tombacz et al, 2021), which then direct the mRNA-LNPs to the respective CD4 or CD8 T cell subpopulation for mRNA vector delivery.In addition, recently, mRNA transfection of pan-T cell populations using mRNA-LNPs were demonstrated using mRNA-LNPs decorated with CD3 and / or CD28 targeting moieties. W02020080475 (EP 3 868 889) discloses LNPs decorated with a CD3 and a CD28 binder for targeting RNA cargos to T cells. WO2022081699 also discloses the use of targeted LNPs to transfect T cells with mRNA encoded CARs. In contrast to W02020080475 (EP 3 868 889), WO 2022081699 discloses the use of two delivery vehicles conjugated with a T cell targeting domain, wherein each targeting domain targets a different T cell antigen. These inventions disclose the conjugation of fragments of CD3 and CD28 binding antibody fragments to maleimide linker functionalized LNPs via reduced disulphide interchain bonds of the antibody fragments (Metzloff et al., 2024). While this method appears to be straightforward, chemical conjugation can lead to side reactions in case of partial reduction of also intra-immunoglobulin disulphide bridges and other contaminations with free cysteines in the reaction. In addition, maleimide linker conjugated LNPs are subject to a so-called retro-Michael reaction, when freecysteines are available, which is e.g. the case for in vivo targeting of T cells, where such maleimide thioether bonds can react with cysteine-34 of human serum albumin, inadvertently leading to an exchange of the targeting moiety with the abundant human serum albumin (Yu et al., 2022). WO2023148276 discloses the use of a bispecific CD3 VHH X NbAlfa (Nb = nanobody) construct to couple a CD3 binder to alpha-peptide-tagged lipids on LNPs comprising a RNA cargo. However, such a non-covalent binding of the targeting ligand to the LNP surface bears the risk of losing the targeting moiety especially when such non-covalently decorated LNPs are used for in vivo targeting of T cells.As mentioned above, the conventional technology of effecting stable ectopic expression of natural or synthetic antigen receptors (e.g. CARs or TCRs) in T lymphocytes, either involves the manufacturing by means of y-retroviral or lentiviral vectors, which is complex in an industrial setting and results in high cost and limited availability due to hardly scalable viral vector manufacturing. In addition, due to the risk of viral vector cross-contamination and the need to work under biosafety level 2 conditions, this manufacturing technology is difficult to deploy in a de-centralized, hospital internal, point-of-care manufacturing setting (Levine et al., 2017)As a result, there is a high need for less complex, less costly and more scalable technologies, which simultaneously effects stable CAR-expression in leukocytes, preferably in T cells, generating an alternative to the lenti- or y-retrovirus-based manufacturing of antigen receptor and CAR expressing leukocytes, including, but not limited to the generation of CAR-T, CAR- NK and TCR-T cells.These and other objects are solved by the features of the independent claims. The dependent claims disclose embodiments of the invention which may be preferred under particular circumstances. Likewise, the specification discloses further embodiments of the invention which may be preferred under particular circumstances.BRIEF DESCRIPTION OF THE FIGURESFig. 1 : Overview of the design of a leucocyte-targeting and -activating mRNA-DNA transposon-LNP according to an embodiment of the invention. The (i) mRNA encodes at least one transposase enzyme and (ii) the DNA encodes at least one transposable antigen receptorconstruct for a disease target, preferably a cancer cell. Furthermore, the resulting LNPs are decorated with (iii) at least one cell targeting moiety for a selected leukocyte subset, and in addition, optionally, with (iv) at least one, but preferably two moieties (one of which activates leukocytes and the other one stimulates them), ensuring more selective and more efficient stable genetic modification, respectively, of primary leukocytes, preferably primary T cells. In the example provided in Fig. 1, LNP tethered IL15 (IL15) is used as a T cell activating and an anti- CD28 binder as a T cell stimulatory moiety, in conjunction with an anti-CD3 binder as a T cell targeting moiety.Fig. 2: Schematic Overview of a CD 19 CAR expression vector SEQ ID NO: 1 as a plasmid map, including annotations of relevant functional regions in the DNA vector.Fig. 3: Schematic Overview of a pBS-KSII-based Sleeping Beauty lOOx expression vector provided in SEQ ID No. 2 as a plasmid map, including annotations of relevant functional regions in the DNA vector (including the ORF of Sleeping Beauty lOOx transposase, whose amino acid sequence is provided in SEQ ID No.3). Note that while this sequence is provided as a DNA sequence it is within the routine of the skilled person to in vitro transcribe said DNA into an mRNA encoding for the transposase, e.g. by means of a T7 RNA polymerase (mRNA sequence that is derived by T7 RNA polymerase transciption is provided in SEQ ID No 5).Fig. 4: Overview of the generation of glycine-tagged mRNA-DNA-transposon LNP intermediates for conjugation of leukocyte-targeting, -stimulating and -activating moieties. In Step 1 ionizable cationic lipids cholesterol, normal lipid and / or wax components, as well as PEGylated lipids and PEGylated-lipides containing at least 2 glycine residues with a free amino-terminus are dissolved in a defined ratio in Ethanol, while mRNA and DNA vectors are dissolved in a defined ratio in aqueous phase. These separate solutions are then mixed together by either microfluidic or flow devices and settings known in the art to create homogenous lipid- nanoparticle-spheres (LNP spheres in which mRNA-DNA molecules are co-encapsulated at high efficieny (preferably with greater than 80 encapsulation efficiency for each nucleic acid component). The glycine-tagged lipids may contain n>l gly cine-residues with a free amino(NH2)-terminus to serve as a substrate for sortase enzyme-mediated covalent coupling, preferably with two glycines as depicted here.Fig. 5: Schematic overview of the covalent decoration of a mRNA-DNA-LNP with three different moieties by Sortase enzyme-mediated bio-conjugation. For this the binding moieties (here an IL 15 -cytokine, an anti-CD3-scFv and an anti-CD28-scFv) need to be provided as recombinant proteins with a C-terminal LPXTG-peptide tag. The glycine-residues with a free amino (NH2) terminus on the surface of the glycine-tagged mRNA-DNA-LNP -intermediate are taken as one substrate by a recombinant sortase enzyme as a catalyst, e.g. a sortase A enzyme of Staph, aureus, together with the LPETG-tags of the recombinant proteins as the other substrates and the sortase enzyme catalyzes under physiologic conditions the covalent coupling of the LPETG-tagged proteins to the glycines of the Glycine-coated LNP.Fig 6: SDS page analysis of sortase conjugation reaction A) LNPl(Negative control): Lane 1 : Marker; Lane 2: Sortase A, Lane 3: Naked LNP, Lane 4: Mixture of negative control reach on(add EDTA) , Lane 5~7: Samples from the permeate, Lane 8: DF product of negative control (after filtration); B) LNP2 (anti-CD3 conjugated LNP): Lane 1 : Marker, Lane 2: anti- CD3 scFv, Lane 3: Sortase A, Lane 4: Naked LNP, Lane 5: Mixture of negative control reaction(add EDTA) , Lane 6: DF product of negative control (after filtration) , Lane 7: Mixture of anti-CD3 conjugated reactionadd EDTA) , Lane 8—10: Samples from the permeate, Lane 11 : DF product of anti-CD3 conjugated LNP(after filtration); C) LNP3 (anti-CD28 conjugated LNP): Lane 1 : Marker, Lane 2: anti-CD28 VHH, Lane 3 : Sortase A, Lane 4: Naked LNP, Lane 5: Mixture of anti-CD28 conjugated reaction (add EDTA) , Lane 6~8: Samples from the permeate, Lane 9: DF product of anti-CD28 conjugated reaction (after filtration), D) LNP4(IL- 15 conjugated LNP) Lane 1 : Marker, Lane 2: mb-ILl 5, Lane 3 : Sortase A, Lane 4: Naked LNP, Lane 5: Mixture of IL- 15 conjugated reaction (add EDTA) , Lane 6~8: Samples from the permeate, Lane 9: DF product of IL-15 conjugated reaction (after filtration); E) LNP7(anti- CD28 / IL-15 conjugated LNP): Lane 1 : Marker, Lane 2: anti-CD28 VHH, Lane 3: mb-IL15, Lane 4: Sortase A, Lane 5: Naked LNP, Lane 6: Mixture of anti-CD28 / IL-15 conjugated reach on(add EDTA) , Lane 7~9: Samples from the permeate, Lane 10: DF product of anti- CD28 / IL- 15 conjugated reach on(after filtration).Fig. 7: CD 19 CAR gene transfer using mRNA / DNA transposon LNPs. Positivity of CAR transgene was assessed by EGFRt transduction marker transcriptionally coupled to the CAR- expression. Stimulated CD4+ T cells were treated with Apolipoprotein E4 (ApoE4) and undecorated mRNA / DNA transposon LNPs containing a CD19-CAR transgene (MC-DNA) and SB100X encoding mRNA. Transgene delivery was judged at d6 after gene transfer andafter an optional magnetic enrichment step. Depicted are representative flow cytometry plots for untreated control T cells (Left), and LNP -modified T cells before (Middle) and after magnetic sort (Right). This experiments provides evidence that mRNA / DNA transposon LNPs can stably transfer DNA constructs stably into the nucleus of primary human T cells.Fig. 8. Activation of T cells using decorated LNPs. Positivity for expression of CD25 and / or CD69. Unstimulated CD4+ and CD8+ T cells were treated with Apolipoprotein E4 (ApoE4) and undecorated LNPs, or LNPs decorated with anti-CD3 and anti-CD28 antibodies or IL-15 or with combinations of these as indicated. Expression was judged at d3 after LNP addition. Depicted are representative flow cytometry dot-plots (A), the percentage of CD25+ and / or CD69+ cells (B), and overlays of CD25 expression, as a marker for T cell activation after stimulation with three different serial dilutions (2pl, 0.2 pl and 0.02 pl) of mRNA / DNA transposon LNPs either decorated with CD3 / CD28 targeting and stimulatory moiety (top row), or decorated with CD3 / CD28 / IL15 targeting, stimulatory and activating moiety (bottom row). The CD25 activation marker was compared to T cells only treated with undecorated mRNA / DNA transposon LNPs which is comprised as the control histogram in all overlays. The experiment shows that optimal activation of T cells occurs with the combination of all three moieties, as evidenced particularly when the decorated LNPs are serially diluted (C).DETAILED DESCRIPTION OF EMBODIMENTSBefore the invention is described in detail, it is to be understood that this invention is not limited to the particular component parts of the devices described or process steps of the methods described, as such devices and methods may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It must be noted that, as used in the specification and the appended claims, wherein the singular forms "a", "an", and "the" include singular and / or plural referents unless the context clearly dictates otherwise. It is moreover to be understood that, in case parameter ranges are given which are delimited by numeric values, the ranges are deemed to include these limitation values.It is further to be understood that embodiments disclosed herein are not meant to be understood as individual embodiments which would not relate to one another. Features discussed with one embodiment are meant to be disclosed also in connection with other embodiments shownherein. If, in one case, a specific feature is not disclosed with one embodiment, but with another, the skilled person would understand that does not necessarily mean that said feature is not meant to be disclosed with said other embodiment. The skilled person would understand that it is the gist of this application to disclose said feature also for the other embodiment, but that just for purposes of clarity and to keep the specification in a manageable volume this has not been done.Furthermore, the content of the prior art documents referred to herein is incorporated by reference. This refers, particularly, for prior art documents that disclose standard or routine methods. In that case, the incorporation by reference has mainly the purpose to provide sufficient enabling disclosure, and avoid lengthy repetitions.According to an aspect of the invention, a lipid nanoparticle (LNP), preferably suitable for transfecting a leukocyte, is provided, which nanoparticle a) is decorated with(i) at least one targeting moiety that binds to and optionally signals via a leukocyte surface antigen selected from, CD3, TCR alpha, beta, gamma or delta chain, CD2, CD4, CD5, CD8, CCR5, CCR7, CD27, CD127, 77CD45RA, CD58, CXCR3, CD122, CD7, CD56, CD161, CD57 (HNK-1, LEU-7 or L2), CD94 (NKG2D), CD19, CD20, CD15, CDl lb / CD18 or CD66;(ii) at least one activating moiety that is a gamma chain cytokine, preferably selected from the group consisting of IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21, or IL-12, or a functionally active fragment thereof, and b) further comprises an mRNA encoding for at least one transposase enzyme, and a DNA encoding for at least one antigen receptor.According to embodiments, the LNP is further decorated with least one stimulatory moiety that binds to and optionally interacts with at least one costimulatory molecule expressed on leukocytes.According to embodiments, the stimulatory moiety interacts with, and / or binds to, at least one costimulatory molecule on the leukocyte, including, but not limited to NK cells, macrophages and B lymphocytes, but preferably on T cells, selected from the group consisting of• CD2, CD28, 4-1BB / CD137, OX-40 / CD134, or CD40L / CD154, ICOS, CD27, DR3, LIGHT / TNFS14, HVEM / TNFRSF14, GITR,• FcyRIII / CD 16, FcyRI I / CD32, FcyRIa / CD64, or• Ig-co-receptors, CD79a (mb-1) or CD79b (B21).According to another aspect of the invention, a lipid nanoparticle (LNP), preferably suitable for transfecting a leukocyte, is provided, which nanoparticle a) is decorated with(i) at least one targeting moiety comprising means for binding, and optionally signalling via, a surface antigen selected from, CD3, TCR alpha, beta, gamma or delta chain, CD2, CD4, CD5, CD8, CCR5, CCR7, CD27, CD127, 77CD45RA, CD58, CXCR3, CD122, CD7, CD56, CD161, CD57 (HNK-1, LEU-7 or L2), CD94 (NKG2D), CD19, CD20, CD15, CDl lb / CD18 or CD66;(ii) at least one activating moiety that is a gamma chain cytokine, preferably selected from the group consisting of IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21, or IL-12, or a functionally active fragment thereof, and b) further comprises at least one mRNA encoding for a transposase enzyme, and a DNA encoding for at least one antigen receptor.Exemplary amino acid sequences for these cytokines are given in SEQ ID NOs: 38 - 45 herein. The respective sequences may relate to specific isotypes, splice variants, mutants or wildtypes. The skilled artisan is able to find other isotypes, splice variants, wildtypes or functional based on this disclosure. These variants are also encompassed by the above language, and hence by the present application.According to embodiments, the LNP is further decorated with least one stimulatory moiety comprising means for binding, and / or interacting with, at least one costimulatory molecule expressed on leukocytes, including, but not limited to NK cells, macrophages and B lymphocytes, but preferably on T cells.According to embodiments, the costimulatory molecule on the leukocyte including, but not limited to NK cells, macrophages and B lymphocytes, but preferably on T cells, to which the stimulatory moiety binds and / or with which it interacts is selected from the group consisting of• CD2, CD28, 4-1BB / CD137, OX-40 / CD134, or CD40L / CD154, ICOS, CD27, DR3, LIGHT / TNFS14, HVEM / TNFRSF14, GITR,• FcyRIII / CD 16, FcyRI I / CD32, FcyRIa / CD64, or• Ig-co-receptors, CD79a (mb-1) or CD79b (B21).The skilled artisan can find these costimulatory molecules, and their sequences, in the public databases, and is hence enabled to generate a respective moiety that bind thereto, like an antibody or fragment, or an antibody mimetic.According to embodiments, the stimulatory moiety which decorates the LNP and interacts with the costimulatory molecule on the leukocyte is the soluble, extracellular domain of OX-40L, CD40, 4-1BBL, CD70, GITR ligand (GITRL), LIGHT / TNFS14, HVEM / TNFRSF14, or ICOS Ligand (ICOSL).Exemplary amino acid sequences for these stimulatory moi eties are given in SEQ ID NOs: 52 - 59 herein. The respective sequences may relate to specific isotypes, splice variants, mutants or wildtypes. The skilled artisan is able to find other isotypes, splice variants, wildtypes or functional based on this disclosure. These variants are also encompassed by the above language, and hence by the present application.According to one alternative embodiment, feature b) comprises a DNA encoding for at least one antigen receptor, or a RNA encoding for at least one antigen receptor. In such embodiment, which lacks a transposase enzyme, integration of the antigen receptor into the leukocyte’s genome is accomplished by means of a programmable endonuclease method (CRISPR Cas9, CRISPR Casl2a, CRISPR Cas X, Cas lambda, TALEN, Zinc Finger, Prime Editing, prime editing combined with an integrase or a ligase).According to embodiments, the targeting moiety and / or the stimulatory moiety is selected from the group consisting of a) an antibody, or functional fragment thereof, b) a DARPin or an Affilin.According to embodiments, the functional antibody fragment is a VHH or other single-domain antibody (sdAb), a Fab, a F(ab)2 or an scFv.According to embodiments, the transposase belongs to a transposase family selected from the group consisting of• Tel -mariner super-family• PiggyBac (PB) transposase family, and / or• hAT super-family.According to further embodiments the leukocyte is selected from the group consisting of a T cell, a macrophage or an NK cell.According to an aspect of the invention, a lipid nanoparticle (LNP), preferably suitable for transfecting a leukocyte, including, but not limited to an NK cell, a macrophage and a B lymphocytes, but preferably a T cell, is provided, which nanoparticle comprises a) at least one mRNA sequence encoding at least one transposase enzyme, and / or b) at least one DNA sequence encoding at least one antigen receptor.In a further aspect of the invention, said lipid nanoparticle (LNP). preferably suitable for transfecting a leukocyte, may be a nanoparticle only comprising at least one DNA encoding for both at least one transposase enzyme and at least one antigen receptor, or may be a nanoparticle only comprising RNA encoding for both at least one transposase enzyme and at least one antigen receptor.In a further aspect, said lipid nanoparticle (LNP), preferably suitable for transfecting a leukocyte, may be a nanoparticle only comprising at least one DNA encoding at least one antigen receptor, or may be a nanoparticle only comprising RNA encoding at least one antigen receptor.Most preferably, the LNPs according to the present invention comprise mRNA and DNA vectors co-encapsulated in the same LNP. Preferably, the mRNA encodes at least one transposase enzyme and the DNA encodes at least one antigen receptor gene flanked by transposase recognition sequences allowing transposition and mobilization of the antigen receptor by the at least one transposase enzyme encoded by the co-encapsulated mRNA. Such mRNA-DNA LNPs can serve as efficient synthetic transposon vectors that can deliver stable antigen-receptor expression to cells that can be transfected with such LNPs, which aresynonymously named herein also mRNA / DNA transposon-LNPs. Like mRNA-LNPs known in the art, the manufacturing of all components of such mRNA / DNA transposon LNPs can be achieved at any scale and at low cost, enabling highly scalable and thereby cost-effective manufacturing of CAR-T cells, with a potential even for in vivo generation of CAR-T cells, if such transposon-LNPs can efficiently be targeted to T cells of a patient in vivo.As such, the LNPs according to the present invention help to reduce manufacturing times, which may often be critical for patients in immediate need for therapy. Furthermore, the invention allows manufacturing of transfected leukocytes at the point of care or even in suitable devices that can perform extracorporeal manufacturing of CAR-T cells at the bedside of a patient or in an outpatient setting. Furthermore, upon proper targeting of such LNPs CAR-T cells can be generated in vivo upon direct, systemic administration of the transposon-LNPs, hence, further increasing access and reducing costs for CAR-T therapies.The LNPs according to the present invention furthermore provide an alternative the complex lenti- or y-retrovirus-based manufacturing of antigen receptor and CAR expressing leukocytes, avoiding the respective disadvantages discussed elsewhere herein.The term “transposase”, as used herein, relates to an enzyme capable of binding to defined inverted terminal repeats (ITRs) at the end of a transposon and catalysing its transfer to another part of a genome, typically by a cut-and-paste mechanism or a replicative mechanism, in a process known as transposition. The word "transposase" was first coined by the individuals who cloned the enzyme required for transposition of the Tn3 transposon. Transposases are classified under EC number EC 2.7.7. Genes encoding transposases are widespread in the genomes of most organisms and are the most abundant genes known.The term “antigen receptor”, as used herein, relates to a construct that is suitable to incorporate into the membrane of a cell, preferably a leukocyte, forming an extracellular domain and an intracellular domain. Such antigen receptor comprises at least a) a targeting moiety capable of binding to a surface antigen on a target cell (forming the extracellular domain (ED)) b) a membrane domain or transmembrane domain (TD), and optionally c) an intracellular domain (ID), which may have a signaling or costimulatory function.Examples for such antigen receptor are shown below.The antigen receptor is preferably an ectopic antigen receptor, in the sense that said antigen receptor is not the endogenous antigen receptor of the leukocyte to be transfected or transposed. Its encoding DNA may be comprised in an expression cassette containing a promoter and polyA elements for proper expression in mammalian cells.The term “ectopic receptor is used synonymously herein with the terms “artificial receptor” and “heterologous receptor”.Said expression cassette is, in one embodiment, designed for stable expression of at least one antigen receptor in cells or, preferably leukocytes.The LNPs according to the present invention allow efficient transfer of both mRNA and DNA into leukocytes by endocytosis, allowing efficient endosomal escape and delivery of the mRNA and DNA payloads into the cytosol of leukocytes.The transposase enzyme is transiently expressed from the mRNA, which then effects the stable integration of the DNA from the transposable DNA vector, comprising e.g. a vector harbouring an expression cassette for the antigen receptor, into the host cell genome.In one embodiment, the expression cassette is flanked by inverted terminal repeats (ITRs), recognized by a transposase enzyme including as discussed elsewhere herein. The transposase enzyme is preferably encoded by mRNA, which is translated into a transposase enzyme after transfer of the mRNA into the target cells.However, the invention can also be realized by providing an entirely DNA-based transposon- LNP, if the at least one DNA encodes both a transposase enzyme and the transposable DNA vector, in which situation preferably the transposase enzyme and the transposable antigen receptor expression cassette are comprised on two separate DNA vectors.According to embodiments of the invention, the transposase belongs to a transposase family selected from the groups consisting of• Tel -mariner super-family• PiggyBac (PB) transposase family• hAT super-familyThe following table gives an overview of these families.Table 1. Examples of transposases that can be used in the present inventionThis table shall not be interpreted by way of limitation, because other mutated and / or optimized transposase enzyme variants of these transposases can equally be used to realize the invention. A preferred transposase enzyme for the mRNA-DNA transposon LNPs is the Sleeping Beauty transposase lOOx (disclosed in: W02009003671 A2). However, also wild-type Sleeping Beauty transposase (disclosed in W09840510A1), or the SBIOx (Cui et al., 2002) J. Mol Biol. 318: pp. 1221-1235), or SB 1 IX variant (disclosed in: WO03089618A2) can be used.In one embodiment, the mRNA encoding for a transposase enzyme is comprised in an RNA sequence as set forth in SEQ ID NO: 5, which includes 5’ and 3’ UTRs and a poly-A tail and an ORF encoding the Sleeping Beauty lOOx transposase enzyme (derived by T7 RNA polymerase transcription of DNA template vector provided in SEQ ID NO: 2, as disclosed in detail in EXAMPLE 2, and the DNA comprising an expression cassette for a CD 19 specific chimeric antigen receptor (CAR) with the DNA sequence as set forth in SEQ ID NO: 1.The invention can also be realized with other, potentially less active variants of the sleeping beauty transposase, including but not limited to sleeping beauty SB1 lx (Geurts et al, 2003) andor sleeping beauty lOx (Cui et al., 2002), or even with wildtype sleeping beauty transposase (Ivies et al., 1996).According to embodiments of the invention, the DNA encoding the at least one antigen receptor is provided as a transposable DNA.The term “transposable DNA”, as used herein, relates to a DNA which is flanked by inverted terminal repeats (ITRs). A construct comprising a DNA stretch flanked by such two ITRs is called “transposon” or “transposable element” (TE). The transposase binds at or near the ITRs, excises the transposon and stably integrates it into the target genome.TEs are present in the genome of every organism, and genomic sequencing has revealed that approximately 45% of the human genome is transposon derived. However, as opposed to invertebrates, where functional (or autonomous) TEs have been identified, humans and most higher vertebrates do not contain functional TEs. It has been hypothesized that evolutionary selective pressure against the mutagenic potential of TEs lead to their functional inactivation millions of years ago during evolution.There are two different classes of transposons: class I, or retrotransposons, that mobilize via an RNA intermediate and a “copy-and-paste” mechanism, and class II, or DNA transposons, that mobilize via excision-integration, or a “cut-and-paste” mechanism.Bacterial, lower eukaryotic (e.g. yeast) and invertebrate transposons appear to be largely species specific, and cannot be used for efficient transposition of DNA in vertebrate cells. Only, after a first active transposon had been artificially reconstructed by sequence shuffling of inactive TEs from fish, which was therefore called “Sleeping Beauty” (Ivies et al., 1997), did it become possible to successfully achieve DNA integration by transposition into vertebrate cells, including human cells. Sleeping Beauty is a class II DNA transposon belonging to the Tcl / mariner family of transposons (Ni et al., 2008). In the meantime, additional functional transposons have been identified or reconstructed from different species, including Drosophila, frog and even human genomes, that all have been shown to allow DNA transposition into vertebrate and also human host cell genomes. Each of these transposons, have advantages and disadvantages that are related to transposition efficiency, stability of expression, genetic payload capacity, etc.According to embodiments of the invention, the encoding DNA is provided in at least one moiety selected from the group consisting of linear DNA with open ends, linear DNA with closed ends, and circular DNA, which can be supercoiled or relaxed circular in structure. In one embodiment, the encoding DNA is a minicircle DNA (mcDNA).One example for linear DNA with closed ends is doggybone DNA (dbDNA), which is a minimal, linear, double stranded and covalently closed DNA construct that can encode long, complex, or unstable DNA sequences, eliminates bacterial sequences and has a strong expression profile (Barreira et al., 2023.The circular DNA can be provided as a plasmid, either in supercoiled or in relaxed conformation.Minicircle DNA (mcDNA) are plasmids largely devoid of bacterial sequence information. In addition to their small size, which is advantageous for LNP delivery, they have the advantage of not triggering Toll-like receptors and thereby minimizing immunogenicity (Shankar et al., 2017)In one embodiment, the DNA encoding for at least one antigen receptor (e.g, the mcDNA), and the RNA encoding for at least one transposase enzyme may be comprised in the LNP in a mass ratio of DNA:RNA of about 1 : up to about 10, preferably 1 : up to about 8, more preferably between about 1 :3 to about 1 :6, more preferably about 1 :4 to about 1 :5.As used herein the term “about” with regard to a numerical value relates to the respective value + / -10%, preferably + / -5% but also includes said numerical value without a deviation.According to embodiments of the invention, the leukocyte that is to be transfected is at least one selected from the group consisting of• T Cell• NK cell• B cell, and / or• NeutrophilMacrophageIn one embodiment, the leukocyte is a leukocyte of a mammalian subject. In one embodiment, the leukocyte is a leukocyte of a human subject.In one embodiment, such leukocytes are primary leukocytes. In further selected embodiments, the T cells areT cells expressing an alpha-beta T cell receptor ( PTCR)T cells expressing a gamma-delta T cell receptor (y6TCR)T cells lacking expression of CD4 or CD8 co-receptors (double-negative (DN) T cells) invariant NKT cells (iNKT cells) being characterized by a unique V-alpha24 (Va24) and a J-alphal8 (Jal 8) gene segment rearrangement.According to embodiments of the invention, the antigen receptor for which the DNA encodes is at least one selected from the group consisting of• Chimeric antigen receptor• Engineered T cell receptorExamples for configurations such receptors may have are shown in the following table.Table 2: Examples for configurations the antigen receptor may haveAccording to embodiments of the invention, the targeting moiety that decorates the LNP or the stimulatory moiety that decorates the LNP is for example selected from the group consisting ofantibodies, antibody fragments including (but not limited to) scFv, Fab, F(ab)2, domain antibodies, such as VHH or other single domain VH, in particular from camelid species, nanobodies, or antibody mimetics, including but not limited to DARPins and Affilins.A single-chain variable fragment (scFv) is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short linker peptide of ten to about 25 amino acids. It can be in VH-VL orientation or in VL-VH orientation. (Holliger and Hudson, 2005).The fragment antigen-binding region (Fab region) is a region on an antibody that binds to antigens. It is composed of one constant and one variable domain of each of the heavy and the light chain. The variable domain contains the paratope (the antigen-binding site), comprising a set of complementarity-determining regions, at the amino terminal end of the monomer. Each arm of the Y thus binds an epitope on the antigen. (Flanagan and Jones, 2004)The F(ab)2 fragment, also known as F(ab)2 antibody, is the antigen-binding fragment of an antibody that can be obtained by cleavage with the enzyme pepsin. The F(ab)2 fragment is a protein made up of two Fab fragments. The two Fab fragments are held together by disulphide bridges or alternatively by adhesive domains. In contrast to classic antibodies, such as immunoglobulin G, F(ab)2 fragments do not trigger a cytotoxic reaction via activation of the complement system, as they lack large parts of the Fc fragment (Hudson and Souriau, 2003).A single-domain antibody (sdAb), also known as a Nanobody, is an antibody fragment consisting of a single monomeric variable antibody domain. Like a whole antibody, it is able to bind selectively to a specific antigen. With a molecular weight of only 12-15 kDa, singledomain antibodies are much smaller than common antibodies (150-160 kDa) which are composed of two heavy protein chains and two light chains, and even smaller than Fab fragments (~50 kDa, one light chain and half a heavy chain) and single-chain variable fragments (~25 kDa, two variable domains, one from a light and one from a heavy chain).The first single-domain antibodies were engineered from heavy-chain antibodies found in camelids; these are called VHH fragments. Cartilaginous fishes also have heavy-chain antibodies (IgNAR, 'immunoglobulin new antigen receptor'), from which single-domain antibodies called VNAR fragments can be obtained. An alternative approach is to split the dimeric variabledomains from common immunoglobulin G (IgG) from humans or mice into monomers. Although most research into single-domain antibodies is currently based on heavy chain variable domains, Nanobodies derived from light chains have also been shown to bind specifically to target epitopes.DARPins (Pliickthun et al., 2015) are genetically engineered antibody mimetic proteins typically exhibiting highly specific and high-affinity target protein binding. They are derived from natural ankyrin repeat proteins, one of the most common classes of binding proteins in nature, which are responsible for diverse functions such as cell signaling, regulation and structural integrity of the cell. DARPins consist of at least three, repeat motifs or modules, of which the most N- and the most C-terminal modules are referred to as "caps", since they shield the hydrophobic core of the protein.Affilins (Ebersbach et al., 2007) are artificial proteins designed to selectively bind antigens. Affilin proteins are structurally derived from human ubiquitin (historically also from gamma- B crystallin). Affilin proteins are constructed by modification of surface-exposed amino acids of these proteins and isolated by display techniques such as phage display and screening. They resemble antibodies in their affinity and specificity to antigens but not in structure, which makes them a type of antibody mimetic. Affilins were developed by Scil Proteins GmbH as potential new biopharmaceutical drugs, diagnostics and affinity ligands.According to embodiments, such surface antigen on a target cell is a cancer associated antigen. According to embodiments of the invention, such “cancer-associated antigen” is a receptor, a protein, a protein complex or another macromolecular structure that is present on the surface of a cancer cell. In the context of the present invention, the term “tumor associated antigen (TAA)” is used synonymously therewith.Such “cancer associated antigen” may be characteristic for a cancer cell, for example because it is a) exclusively presented on cancer cells, yet not on healthy cells, b) presented in higher abundancy or surface density on cancer cells than on healthy cells c) presented on cancer cells, yet not on post-embryonic healthy cellsd) presented on cancer cells and healthy cells of reproductive organs (like testis, ovary and trophoblasts), yet not on healthy somatic cells (“cancer-testis antigens”) e) presented on B lymphocytesIn some embodiments, such “cancer-associated antigen” is a complex of a major histocompatibility complex (MHC) and a peptide presented by the latter. Such complex is also abbreviated as pMHC. Preferably, such presented peptide is a tumor associated peptide (TUMAP), or a neoantigen.In some embodiments, such “cancer-associated antigen” is selected from the group consisting of AXL, BCMA, CA9, CD5, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD52, CD56, CD72, CD70, CD79a (mb-1), CD79b (B29), CD117 (also known as c- kit), CD99, CD123 (also known as IL-3R), CD133, CD135 (also known as flt3), CD138, CD 147, CD 174, CD276, CLEC12A, CSPG4, c-Met, CEA, EBV-related antigens, GPC3, GUCY2C, EPCAM, EPHA2, EGFR, EGFRvIII, DLL3, DLL4, FAP, FOLH1, folate receptor alpha, GD2, GPC3, GPRC5D, IL1RAP, HER2, HER3, HER4, HPV-related antigens, KDR, L1CAM, LILRA3, LILRB4, MAGE antigens, Nectin-4, Mesothelin, MS4A1, MUC-1, NCAM- 1, NY-ESO-1, PDCD1, PSCA, PSMA, PTK7, SDC1, SLAMF7 (also known as CS1), SIRP- alpha, SSTR2, TEM1, Tissue Factor, TNFRSF8, TNFRSF17, ULBP1, ULBP2, VEGFR2, WT1, Claudin 1, Claudin 6, Claudin 18.2, ROR-1 or R0R2. This list is not limiting though.According to another embodiment, such surface antigen on a target cell is an antigen associated with a non-cancer disease, including, but not limited to, autoimmune diseases, for instance, but not limited to systemic lupus erythematosus (SLE) caused by overproduction of autoantibodies by autoreactive B lymphocytes. In some embodiments, such non-cancer antigen is selected from the group consisting of CD10, CD19, CD20, CD22, CD52, CD79a, and / or CD79b. This list is not limiting though.The skilled artisan can find these cancer-associated antigens or non-cancer antigens, and their sequences, in the public databases, and is hence enabled to generate a respective moiety that bind thereto, like an antibody or fragment, or an antibody mimetic.According to embodiments of the invention, the LNP is decorated with at least one targeting moiety that is suitable to direct the LNP to the leukocyte that is to be transfected.As used herein, the term “decorated” refers to the covalent or non-covalent coupling of a component to the surface of the LNP after or during formation thereof.Such targeting moiety facilitates the targeting of specific leukocyte subsets, especially in mixtures of peripheral primary mononuclear cells (PBMNCs) during ex vivo or in vivo generation of artificial antigen receptor expressing leukocytes. Such targeting moiety confers a specific binding of the decorated LNP to a selected leukocyte subpopulation.The functional terms “targeting moiety” or “stimulatory moiety” (oftentimes referring to antibodies binding to a given target) should be understood also in the sense of means-plus- function language in the meaning of 35 U.S.C. § 112 (f). In the following table, a synopsis is given of the respective means-plus-function language deemed to be disclosed herein when the respective functional language is used.Table 3: Functional language and corresponding means-plus-function languageAccording to embodiments of the invention, the targeting moiety, which decorates the LNP, binds to at least one surface antigen of a leukocyte, selected from the group consisting of• a subunit of the T cell receptor, such as (i) CD3, (ii) TCR alpha, (iii) TCR beta, (iv) TCR gamma or (v) TCR delta, CD2, CD4, CD5, CD8, CCR5, CCR7, CD27, CD127, CD45RA, CD58, CXCR3, CD 122 or CD7,• CD56, CD161 (NK1.1), CD57 (HNK-1, LEU-7 or L2), or CD94 (NKG2D),• CD 19, CD20, CD22, or• CD15, CDl lb / CD18 or CD66b.According to embodiments, the targeting moiety which decorates the LNP is at least one selected from the group consisting of a) an antibody, or functional fragment thereof, or b) an antibody mimetic, including DARPin or an Affilin.The cell surface receptor may be a receptor having an intracellular immunoreceptor tyrosinebased activation motif (ITAM).According to embodiments, the targeting moiety is an anti-CD3 antibody or functional fragment thereof, or an antibody mimetic, including DARPin or an Affilin. Exemplary amino acid sequences for targeting moi eties targeting CD3 are given in SEQ ID NOs: 6 - 16 herein. These moieties encompass antibodies, scFv, VHH and DARPins.According to embodiments, the CD3 to which the targeting moiety which decorates the LNP binds is at least one selected from CD3-gamma, CD3-delta, and / or CD3-epsilon. In a preferred embodiment the targeting moiety binds to CD3 -epsilon.According to embodiments, the targeting moiety is an anti-CD56 antibody or functional fragment thereof, or an antibody mimetic, including DARPin or an Affilin. Exemplary amino acid sequences for stimulatory moieties interacting with CD56 are given in SEQ ID NOs: 21 - 22 herein.According to embodiments, the stimulatory moiety interacting with a costimulatory molecule is an anti-CDl lb antibody or functional fragment thereof, or an antibody mimetic, including DARPin or an Affilin. Exemplary amino acid sequences for stimulatory moieties interacting with CD1 lb are given in SEQ ID NOs: 23 - 24 herein.Some surface antigens of leukocytes may exert a first intracellular signal to the cell upon binding of the targeting moiety. For instance, when CD3 on T cells is bound with an agonistic CD3 targeting moiety, this may already lead to a certain degree of T cell activation, which is later complemented by the activity of the stimulatory moiety and the activating moiety. In such embodiment, the surface antigen of the leukocyte hence exerts an intracellular signal upon binding of the targeting moiety.For the case of transfecting leukocytes, and in particular T cells orNK cells, in one embodiment, two additional signals (“activating” and “stimulatory”) delivered by the LNP are used to trigger activation of resting cells to open up their densely packed chromatin, thus allowing stable integration of a transposon DNA vector into the genome of the host cells.According to embodiments of the invention, the LNP is decorated with at least one stimulatory moiety that is suitable to stimulate a leukocyte either alone or via an additional costimulatory moiety. According to embodiments of the invention, the LNP is decorated with at least one stimulatory moiety that interacts with at least one costimulatory molecule on the leukocyte.The terms “stimulatory moiety” and “stimulating moiety” are used interchangeably herein.According to embodiments of the invention, the stimulatory moiety which decorates the LNP interacts with at least one costimulatory molecule selected from the group consisting of• CD2, CD28, 4-1BB / CD137, OX-40 / CD134, or CD40L / CD154, ICOS, CD27, DR3, LIGHT / TNFS14, HVEM / TNFRSF14, GITR,• FcyRIII / CD 16, FcyRI I / CD32, FcyRIa / CD64, or• Ig-co-receptors, CD79a (mb-1) or CD79b (B21).The skilled artisan can find these costimulatory molecules, and their sequences, in the public databases, and is hence enabled to generate a respective moiety that binds thereto, like an antibody or fragment, or an antibody mimetic.According to embodiments, the stimulatory moiety which decorates the LNP and interacts with at least one costimulatory molecule is at least one selected froma) an antibody, or functional fragment thereof, or b) an antibody mimetic, including a DARPin or an Affilin. all of which being able to bind to a costimulatory molecule.According to embodiments, the afore described antibodies, antibody fragments are agonistic antibodies, antibody mimetics or fragments thereof.According to embodiments, the stimulatory moiety interacting with a costimulatory molecule is an agonistic anti-4-lBB antibody such as urelumab (BMS-663513, IgG4, SEQ ID NOs: 29 and 30 shown herein) and utomilumab (PF-05082566, IgG2, SEQ ID NOs: 31 and 32 shown herein).According to embodiments, the stimulatory moiety interacting with a costimulatory molecule is an anti-CD28 antibody or functional fragment thereof, or an antibody mimetic, including DARPin or an Affilin. Preferably, an agonistic anti-CD28 antibody is provided. Exemplary amino acid sequences for stimulatory moi eties interacting with CD28 are given in SEQ ID NOs: 17 - 20 herein. These moieties encompass antibodies, scFv and VHH. In a specific embodiment the anti-CD28 antibody is a VHH.According to embodiments, the stimulatory moiety interacting with a costimulatory molecule is an anti-4-lBB antibody or functional fragment thereof, or an antibody mimetic, including DARPin or an Affilin. Preferably, an agonistic anti -4- IBB antibody is provided. Exemplary amino acid sequences for stimulatory moieties interacting with 4-1BB are given in SEQ ID NOs: 25 - 28 herein.According to embodiments, the stimulatory moiety which decorates the LNP comprises the soluble, extracellular domain of OX-40L, CD40, 4-1BBL, CD70, GITR ligand (GITRL), LIGHT / TNFS14, HVEM / TNFRSF14, ICOS Ligand (ICOSL). In these embodiments, the stimulatory moiety is not an antibody or fragment, nor an antibody mimetic.Exemplary amino acid sequences for these stimulatory moieties are given in SEQ ID NOs: 52 - 59 herein. The respective sequences may relate to specific isotypes, splice variants, mutants or wildtypes. The skilled artisan is able to find other isotypes, splice variants, wildtypes orfunctional based on this disclosure. These variants are also encompassed by the above language, and hence by the present application.According to embodiments of the invention, the LNP is decorated with at least one activating moiety.In this context, it is important to note that while mRNA encoding the transposase enzyme only needs to reach the cytosol of the lymphocytes, in order to be efficiently translated into protein, stable expression of the at least one artificial antigen receptor requires the DNA encoding for the at least one antigen receptor to be stably integrated the genome of the targeted leukocytes, i.e., it needs to enter the nucleus of the targeted cell, wherein the chromatin should be accessible and not tightly packed into a closed chromatin structure as usually the case in resting cells.The role of the activating moiety and / or the stimulatory moiety is therefore to stimulate resting leukocytes to such an extent that they open up their densely packed chromatin structure, as they transition from Go of the cell cycle and enter into Gi, S, M and G2 phases of the cell cycle. The concomitant stimulation of the targeted leukocytes will result in the more efficient stable integration of the transposable vector encoding the antigen receptor into the genome of these cells. An increased transfection efficiency and / or increased stable integration of transposon DNA leading to a durable expression of the antigen receptor may be determined experimentally.According to embodiments of the invention, the activating moiety is a gamma chain cytokine, preferably selected from the group consisting of IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21, or a functionally active fragment thereof. A “functionally active fragment” of one of the recited interleukins is a fragment which is capable of binding to IL-2Ry. Alternatively the activating moiety is IL-12.Exemplary amino acid sequences for these activating moi eties are given in SEQ ID NOs: 38 - 45 herein. The respective sequences may relate to specific isotypes, splice variants, mutants or wildtypes. The skilled artisan is able to find other isotypes, splice variants, wildtypes or functional based on this disclosure. These variants are also encompassed by the above language, and hence by the present application..In an alternative embodiment, the stimulatory moeity is an agonistic anti-cytokine receptor antibody, for example as disclosed by Harris et. al (2021) or a peptide cytokine mimetic, for example as disclosed by Dower et al. (2023).The common cytokine receptor gamma chain (yc) family consists of IL-2, IL-4, IL-7, IL-9, IL- 15 and IL-21, and is so named because the receptors for these cytokines share a common gamma-chain (also known as IL-2Ry and CD 132).The common gamma-chain (yc) is critical for signalling by common gamma-chain (yc) cytokines. All yc family cytokines similarly activates the Janus kinase (JAK)-family protein tyrosine kinases JAK1 and JAK3, with JAK1 binding a unique a or P chain and JAK3 binding the common gamma-chain (yc). They exert their effect through interaction with gamma-chain (yc) cytokine receptor complex, which is composed of a unique receptor chain and the common gamma-chain (yc).In a preferred embodiment the activating moiety is selected from IL-2 or IL-15 or functionally active fragments thereof, most preferably IL- 15 functionally active fragments thereof.The following table summarizes examples of targeting, activating and stimulatory moieties for decoration of LNPs according to the present inventionTable 4: examples of targeting, activating and stimulatory moieties for decoration of LNPsIt should be understood that, in preferred embodiments, the LNP is decorated with at least one targeting moiety, one activating moiety and one stimulatory moiety. As such, all conceivable combinations of individual members from the 3 different moiety classes shown in the above table shall be deemed disclosed.Also, in this context, it should be understood that LIGHT / TNFS14 and HVEM / TNFRSF14 are ligand and receptor to one another. Both can be found on the surface of T cells and act as costimulatory molecules there. As such, LIGHT / TNFS14, or the extracellular domain thereof, can act as a stimulatory moiety for HVEM / TNFRSF14 and HVEM / TNFRSF14 or the extracellular domain thereof, can act as a stimulatory moiety for LIGHT / TNFS14.In one embodiment, the LNP according to the invention is decorated in a molar ratio of targeting moiety: stimulatory moiety of up to about 50: 1 such as for example , about at least 5: 1, about at least 10: 1, about at least 20: 1, about at least 30: 1, about at least 40: 1, and for example a maximal ratio of up to about 60: 1. The LNP may be decorated in a molar ration of targeting moiety: stimulatory moiety of 1 : up to about 50 such as for example, 1 : up to about at least 5, 1 : up to at least 10, 1 : up to about at least 20, 1 : up to about at least 30, 1 : up to about at least 40, and for example a maximal ratio of 1 : up to about 60. In a preferred embodiment the LNP is decorates with an excess of stimulatory moieties, for example molar ration of targeting moiety: stimulatory moiety of 1 : up to about at least 20, 1 : up to about at least 30, 1 : up to about at least 40.Furthermore, the LNP according to the invention is decorated in a molar ration of targeting moiety: activating moiety of up to about 50: 1 such as for example, about at least 5: 1, about at least 10: 1, about at least 20: 1, about at least 30: 1, about at least 40: 1, and for example a maximal ratio of up to about 60: 1. The LNP may be decorated in a molar ration of targeting moiety: activating moiety of 1 : up to about 50 such as for example , 1 : up to about at least 5, 1 : up to about at least 10, 1 : up to about at least 20, 1 : up to about at least 30, 1 : up to about at least 40, and for example a maximal ratio of 1 : up to about 60.Furthermore, the LNP according to the invention is decorated in a molar ration of stimulatory moiety : activating moiety of up to about 50: 1 such as for example , about at least 5: 1, about at least 10: 1, about at least 20: 1, about at least 30: 1, about at least 40: 1, and for example a maximal ratio of up to about 60: 1. The LNP may be decorated in a molar ration of stimulatory moiety : activating moiety of 1 :up to about 50 such as for example , 1 : up to about at least 5, 1 : up to about at least 10, 1 : up to about at least 20, 1 : up to about at least 30, 1 : up to about at least 40, and for example a maximal ratio of 1 : up to about 60.In a further embodiment, the LNP is decorated with targeting moiety, stimulatory moiety and activating moiety in about equimolar ratios.In one preferred embodiment, the leukocytes, and in particular the T cells, are activated by means of two different signals plus one optional signal, namely(i) optionally, one signal via the T cell receptor or the CD3 signaling complex,(ii) one stimulatory signal via a co-stimulatory molecule expressed on the leukocytes, and in particular on the T cells, and(iii) one activating signal via a cytokine receptor expressed on the leukocytes, and in particular on the T cells.Accordingly, in a preferred embodiment, the LNP according to the invention is decorated with(i) at least one targeting moiety that binds and optionally signals via the T cell receptor or the CD3 signalling complex,(ii) optionally at least one stimulatory moiety that interacts with at least one costimulatory molecule expressed on leukocytes, and in particular on the T cells, and(iii) at least one activating signal via a cytokine receptor expressed on a leukocytes, and in particular on the T cells.In another embodiment, the LNP according to the invention is decorated with(i) at least one targeting moiety that binds and optionally signals via the T cell receptor or the CD3 signalling complex, and(ii) at least one activating moiety, preferably a cytokine interacting with cytokine receptor expressed on a leukocytes, and in particular on the T cells, and not decorated with stimulatory moiety that interacts with at least one costimulatory molecule expressed on leukocytes.In another embodiment, the LNP according to the invention is decorated with only(i) at least one targeting moiety that binds and optionally signals via the T cell receptor or the CD3 signalling complex, and(ii) at least one stimulatory moiety binding to a stimulatory molecule expressed on the leukocytes, and in particular on the T cells, for instance, but not limited to CD28, and not decorated with activating interacting with cytokine receptor expressed on a leukocytes, and in particular on the T cells, for instance, but not limited to IL15.The above discussed combination of activating and stimulatory signals is helpful for the shortterm ex vivo generation or the in vivo generation of leukocytes expressing ectopic, heterologous or artificial antigen receptors, because primary leukocytes are mostly resting in Go phase of the cell cycle. Activating them to leave the Go resting stage into Gi, M, S or G2 stages, in which the chromatin structure is opened up, makes them more accessible for transposon-based stable integration of the DNA encoding the antigen receptor. This is particularly beneficial for in vivo applications.According to embodiments of the invention, at least one of the targeting moiety, the stimulatory moiety and the activating moiety is coupled to the LNP either by non-covalent or covalent coupling.According to embodiments of the invention, at least one of the targeting moiety, the activating moiety and / or the stimulatory moiety is associated to the LNP indirectly, by means of an adaptor or linker.Such adaptor can be a binder binding such targeting moiety, activating moiety or stimulatory moiety , like a .g. antibody, antibody fragment including but not limited to scFv, Fab, F(ab)2, domain antibody, such as VHH or other single domain VH, or fragment thereof, nanobody, or antibody mimetic including but not limited to DARPins and Affilins.Such adaptor can also comprise an affinity binder like e.g. a Streptavidin / Biotin combination, or a protein A that binds to the Fc domain of a targeting moiety, activating moiety or stimulatory moiety , if applicable.The at least one targeting moiety, stimulatory moiety and / or activating moiety can covalently be conjugated to the surface of LNPs with standard maleimide linker chemistry provided e.g. in Metzloff et al. (2024). In this approach, maleimide functionalized lipids are incorporated into the outer membrane of a mRNA-LNP, which is then reacted with enzymatically hydrolyzed and TCEP reduced antibody fragments which are then reacted with the maleimide functionalized LNPs. This leads to stable conjugation of the maleimide linker via the reduced thiols of the reduced antibody fragments. However, it is known that maleimide linkers to thiol groups in proteins are not stabile and susceptible to a so-called reverse Michael reaction in the presence of free thiol groups. This is particularly problematic if such maleimide-linker conjugated LNPs are used for in vivo applications, where cysteine-34 of the abundant human Serum Albumin (HSA) protein may break up the maleimide-thiol bond to replace and targeting and other domain with HSA (Yu et al., 2022).The at least one targeting moiety, activating moiety and / or stimulatory moiety may be associated to the LNP by various other conjugation technologies.According to a preferred embodiment of the invention, at least one of the targeting moiety, the activating moiety and / or the stimulatory moiety is bound to the LNP by means of sortase enzyme-mediated covalent bio-conjugation, in such way decorating the LNP therewith, as disclosed in detail in the examples.Sortase enzyme transpeptidation is a highly selective conjugation mechanism and provides stable, covalent peptide-bonds that are also stable in serum of mammalian species, including human subjects.Furthermore, sortase enzyme-mediated peptide bond formation has the advantage that the conjugation can be performed under physiologic and mild conditions. In such way, the structural integrity of LNPs can be preserved and remains unaffected. In fact, sortase enzymatic reactions can be performed at a low temperature range from 16-37°C, at physiologic pH and at physiologic salt concentration.Sortase-mediated transpeptidation has been established as a tool for protein engineering, in particular with sortase A derived from gram-positive bacteria (Parthasarathy et al, 2007). Sortase A mediated transpeptidation has also been established as a tool for site-specific conjugation of small-molecular weight payloads to antibodies for the generation of stable, site- specifically conjugated antibody-drug conjugates, a technology also know as SMAC- technology (SMAC=sortase-mediated antibody conjugation) (see: Beerli et al., 2015 and WO 201414031).Sortase-enzyme mediated bio-comjugation can be catalyzed by many sortases known in the art. For instance, Sortase F is described in W02020089485A1.In order to achieve sortase-mediated transpeptidation, one substrate needs to contain a pentapeptide motif, which -for the most commonly used sortase A enzyme from the gram positive bacteria Staph, aureus- is of the sequence LPXTG (SEQ ID NO: 4), (X being any of the 20 naturally occurring amino acids, sequence read from N to C terminus), whereas the other substrate needs to contain a stretch of > 2 glycine residues with a free N-terminus. Sortase enzymes then catalyze a transpeptidation, by hydrolysis of the peptide bond between the 4thand 5thposition of the pentapeptide motive and formation of a new peptide bond between the 4thamino acid of the pentapeptide motif and the N-terminal glycine of the other substrate. Because one peptide bond is broken and a new peptide bond is formed, the reaction is characterized as a transpeptidation reaction. Concretely this means that the 5thamino acid from the pentapeptide sortase motif (also called sortase-tag) and any additional amino acid appended to it at its C- terminus is removed from the LPXTG substrate after the incoming N-terminal glycine from the other substrate has formed a new peptide bond with the 4thamino acid (i.e. the T-residue in the LPXTG motif.This means that for the mild conjugation of protein moieties to an mRNA-DNA transposon- LNP, the outer membrane of the LNP needs to be functionalized with at least 2 glycine residues with a free N-terminus, while the protein moieties need to be appended with a C-terminal recognition sequence (also called “sortase tag” herein, which in case of sortase A enzyme from Staph, aureus minimally has to be of sequence LPXTG.Sortase A enzyme from Staph, aureus (see W02007108013A2) is one suitable sortase enzyme to realize the invention, preferably an optimized version described by Chen et. al. (2011) However, also other sortase enzymes from other gram-positive bacteria, known in the art, that recognize other pentapeptide motifs disclosed in the following table, can be used to realize the invention.Table 5: Sortases that can be used in the context of the present inventionAmino acid sequences for these recognition sequences are given in SEQ ID NOs: 4 and 46 - 50 herein. The respective sequences may relate to specific isotypes, splice variants, mutants or wildtypes. The skilled artisan is able to find other isotypes, splice variants, wildtypes or functional based on this disclosure. These variants are also encompassed by the above language, and hence by the present application.However, in order to prevent that the sortase tag is not accessible due to potential secondary structures close to the C-terminus of a protein moiety, it is advisable to add a nonstructural amino acid spacer, (e.g., a GGGGS motif) in between the C-terminus of the protein moiety and the C-terminal LPXTG pentapeptide sortase tag. Furthermore, it is advisable to add a fewadditional amino acids, at least one additional amino acid (e.g. another G) to the C-terminus of the sortase tag to allow efficient transpeptidation. While the spacer sequence added in between the C-terminus of the protein substrate and the N-terminus of the sortase tag can be any amino acid stretch of > 1 amino acid, a preferred sequence is GGGGS.Similarly, while the additional > 1 amino acid appended to the C-terminus of the sortase tag can contain any amino acid and any amino acid length, the addition of a 1-5 glycine residues followed by a purification tag, e.g., but not limited to, Strep-tag, Strep-II-tag, MYC-tag, HA- tag, or a His-tag, with 3 glycines followed by a 6x-His-tag being preferred.Therefore, according to embodiments of the invention at least one of the targeting moiety, the stimulatory moiety and / or the activating moieties is labelled, C-terminally, with a sortase tag as set forth above, optionally comprising, an N-terminal linker plus a C terminal tag consisting of e.g. one or more glycine residues plus a 6xHis tag. In one embodiment, at least one of the targeting moiety, the stimulatory moiety and / or the activating moieties is labelled, C terminally with the following construct (GS-linker_sortase-tag_(Gly)n_His-tag): GGGGS-LPXTG- GGGHHHHHH. (SEQ. ID No. 51).Such constructs, even if not explicitly disclosed herein, can comprise any of the other sortase tags shown in the above table. Regarding the C-terminal glycine residues, N can be anything between 1 and 10, with n > 1 and < 3 preferred.Using different sortase tags for e.g, the targeting moiety, the activating moiety and / or the stimulatory moiety allows the use of different sortase enzymes. In such way, the decoration of the LNP with the diffrent moieties can be adjusted and controlled.The ORF of Sortase A from Staphylococcus aureus is published in Genbank and can be found under entry: AF162687.1 Expression of an enzymatically active fragment of recombinant sortase A in E.coli, comprising amino acids 60-205 with 6xHis tag are disclosed in reference W02007108013A2.In one embodiment, at least one lipid component of the LNP is modified with at least one glycine or an oligoglycine, and preferably two or three glycines. The oligoglycine in the modified lipid may be a di-, or tri-glycine, preferably a di-glycine residue. The glycine oroligoglycine modified lipid is also referred as “glycine-lipid” herein. The at least one glycine- lipid may also be a cationic / cationizable lipid, a neutral lipid, a steroid or steroid analogue as disclosed herein, preferably a neutral lipid.In a preferred embodiment, one lipid component of the glycine-lipid and the glycine or an oligoglycine are connected via a linker. The linker may be a chemical, a peptide linker, or a polymer linker. Preferably the linker is a polyethylene glycol (PEGxy), wherein “xy” designated the average molecular weight of the PEG. For example, the polymer linker may be PEG, about 1000 to about 8000, about 3000 to about 7000, about 4000 to about 6000, or 4500 to about 5500, or about 5000. In a preferred embodiment the glycine-lipid is a lipid comprising DSPE, preferably DSPE-PEG(5000), most preferably DSPE-PEG(5000)-di-glycine.PEGylated-lipids are added in LNP formulations to increase their solubility of LNPs and to prevent aggregation of LNPs after nucleic acid encapsulation. However, it is also known that PEG-oligomers are toxic at high concentrations, if administered systemically. Therefore, the LNPs of this invention may also comprise neutral lipids to be incorporated into the outer membrane of a LNP that may also contain other hydrophilic structures providing similar benefits for enhanced solubility and decreased aggregation as PEG, but which are less toxic and more biodegradable, e.g. but not limited to stretches of hydrophilic amino acids, or hyaluronic acid oligomers, or other oligo-saccharides.The sortase-tagged targeting moiety, stimulatory and / or activating moiety can then be conjugated to an oligoglycine on the surface of the LNP by the sortase enzyme activity (see Fig 5). Due to the size difference between the LNP and the sortase enzyme, the catalyzing enzyme can be removed from the final LNP product by diafiltration, tangential flow filtration, dialysis or other ways of separation techniques known in the art (as decribed in detail in the EXAMPLES).To obtain LNPs decorated with targeting, stimulatory and / or activating moiety in specific ratios, the targeting, stimulatory and / or activating moieties are provided in the sortase conjugation reaction in the respective molar ratios to be achieved in the decorated LNP according to the invention.The following schematic shows one exemplary fusion reaction mediated by sortase A (N->C orientation, T / A / S means targeting / activating / stimulatory)GGGHHHHHHIt should be understood that the same principle should be deemed disclosed for the other sortase tags and sortases.Preferably the C-terminus of the targeting, stimulatory and / or activating moiety is conjugated solely by its C-terminus to a lipid, preferably a PEG lipid, comprised in the LNP. In one embodiment, the linker between the targeting, stimulatory and / or activating moiety and the LNP does not comprise any other chemical linker other than the glycine peptide directly linked to a lipid, preferably a PEG lipid.The sortase tagging method facilitates a specific, site directed conjugation of the targeting, stimulatory and / or activating moiety to the LNP solely via the C-terminus of these moieties. Preferably the C-terminus of these moieties of the targeting, stimulatory and / or activating moiety is conjugated solely by its C-terminus to a PEG lipid comprised in the LNP. Preferably, the linker between the targeting, stimulatory and / or activating moiety to the LNP does not comprise any other chemical linker than a peptide directly linked to a lipid, preferably a PEG lipid.According to embodiments of the invention, the mRNA encoding for the transposase comprises a 5' untranslated region (UTR) and / or a 3' UTR as shown in SEQ ID No. 5.According to embodiments of the invention, the mRNA encoding the transposase comprises a modified nucleoside in place of uridine.According to embodiments of the invention, the modified nucleoside is selected from the group consisting of pseudouridine (y), N 1-methyl-pseudouridine (m IT), and 5-methyl-uridine (m5U).According to embodiments of the invention, wherein the mRNA encoding for the transposase comprises a coding sequence which is codon-optimized and / or in which the G / C content is increased and the uridine content is decreased compared to wild type coding sequence, wherein the codon-optimization and / or the increase in the G / C content preferably does not change the sequence of the encoded amino acid sequence.According to a preferred embodiment of the invention, the mRNA encoding for the sleeping beauty lOOx transposase enzyme comprises an RNA of to SEQ ID NO: 5 in vitro transcribed by T7 RNA polymerase from the DNA of SEQ ID NO:2 according to the details provided in EXAMPLE 2.According to embodiments of the invention, the mRNA encoding for the sleeping beauty transposase comprises an RNA integrity of 70% or more.According to embodiments of the invention, the mRNA encoding for the transposase has a capping degree of 70% or more, preferably wherein at least 70%, 80%, or 90% of the mRNA species comprise a Capl structure, or, preferably, an ARC A cap.According to embodiments of the invention, the DNA encoding for at least one antigen receptor encodes for at least one chimeric antigen receptor. According to embodiments of the invention, the DNA comprises an expression cassette for at least one a chimeric antigen receptor (CAR) as disclosed in SEQ ID NO: 1, which may contain any coding region for an antigen binding receptor, including but not limited to a CAR specific for any target of interest.According to further embodiments, the DNA encoding for a chimeric antigen receptor CAR comprises at least a DNA encoding for the amino acid sequence consisting of SEQ ID NOs: 33- 37. Therein, SEQ ID NO: 33 is the anti CD19 scFv antibody FMC63, SEQ ID NO: 34 is the Hinge (CD8), SEQ ID NO: 35 is the TMD (CD8), SEQ ID NO: 36 is the 4-1BB costimulatory domain and SEQ ID NO: 37 is the CD3 domain.In one embodiment, the DNA encoding for a chimeric antigen receptor encodes for an amino acid sequence consisting SEQ ID NOs: 33 - 37 fused directly to one another in N^C direction to make the complete Chimeric antigen receptor.According to embodiments of the invention, the LNP comprises, in its lipid sheath, at least three lipids selected from the group consisting of(i) a cationic / cationizable lipid(ii) a neutral lipid(iii) a steroid or steroid analogue; and(iv) a polymer conjugated lipid, preferably a PEG-lipid.According to embodiments at least one of these lipids comprises at least two glycine residues with a free N-terminus,The term "neutral lipid" refers to any one of a number of lipid species that exist in either an uncharged or neutral zwitterionic form at physiological pH.The term "cationic / cationizable lipid" refers to a lipid that is cationic or becomes cationic (protonated) as the pH is lowered below the pK of the ionizable group of the lipid, but is progressively more neutral at higher pH values. At pH values below the pK, the lipid is then able to associate with negatively charged nucleic acids. In certain embodiments, the cationic lipid comprises a zwitterionic lipid that assumes a positive charge at pH decrease.According to embodiments of the invention, the LNP further comprises, in its lipid sheath, phosphatidylserine, preferably L-a-phosphatidylserine.According to embodiments of the invention, (i) to (v) are in a molar ratio of about 20-60% cationic lipid, 5-25% neutral lipid, 25-55% steroid or steroid analogue, 0.0-15% PEG-lipid, and 0.0-15% glycine-modified PEG-lipid.According to embodiments of the invention, the cationic / cationizable lipid is at least one selected from the group consisting ofc) DOTAP (Dioleoyl-3-trimethylammonium-propane, and / or d) DODMA (l,2-dioleyloxy-3-(dimethylamino)propane f) DODAC (N,N-dioleyl-N,N-dimethylammonium chloride) g) DOTMA (N-(2, 3-di oleyl oxy )propyl)-N,N,N-trimethylammoniumchloride) h) DDAB (N,N-distearyl-N,N-dimethylammonium bromide); i) DC-Chol (3-(N — (N',Ni-dimethylaminocthanc)-carbamoyl)cholesterol), j) DOSPA (N-(l-(2,3-diolcoyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N- dimethylammonium trifluoracetate) k) DOGS (dioctadecylamidoglycyl carboxyspermine) l) DODAP (1,2-di oleoyl -3 -dimethylammonium propane),DMRIE (N-(l,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxy ethyl ammonium bromide)In further embodiment, the cationic / cationizable lipid may be selected from the lipids disclosed WO2021113777A2, especially in para.

[0007] to

[0014] , preferably para

[0010] ; WO2021226597A2, especially in para.

[0007] to

[0020] , preferably para.

[0011] ,

[0013] ,

[0016] , or

[0020] ; WO2023081526A1, especially in para.

[0007] to

[0027] , preferably para.

[0025] or

[0026] ; WO202023141576A1, especially in para.

[0022] to

[0083] , preferably para.

[0074] ,

[0078] ,

[0082] , or

[0083] ; or WO2024035783, especially in para.

[0014] to

[0076] , preferably para.

[0067] ,

[0067] , or compound 1 to compound 35, preferably compound 35 disclosed in EP3868889A1, which are incorporated herein by reference.The ratio (mol%) of the cationic / cationizable lipid to the total lipids present in the LNP is, for example, about 10% to about 80%, preferably about 20% to about 70%, more preferably about 40% to about 60%; however, the ratio is not limited to these.Only one kind of the above-mentioned cationic / cationizable lipid may be used or two or more kinds thereof may be used in combination in the LNP. When multiple cationic / cationizable lipids are used, the ratio of the whole cationic lipid is preferably as mentioned above.According to embodiments of the invention, the polymer conjugated lipid is at least one selected from the group consisting of:wherein n has a mean value of 44 or 45, preferably 1,2-Dimyristoyl-rac-glycero- 3 -methoxypolyethylene glycol-2000 (PEG2000 DMG)wherein n has a mean value of 49 or 45, preferably 2-[(polyethylene glycol)- 2000]-N,N-di tetradecyl acetami de(ALC-0159),PEG substituted polyethylene glycol-lipids, for exampleDMPE-PEG2000, 1,2-dimyristoyl- sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000. Suitable polyethylene glycol-lipids include PEG-modified phosphatidylethanolamine, ceramides, for example PEG-modified phosphatidic acid, PEG-modified ceramidesPEG-CerC14 or PEG- CerC20), PEG-modified dialkylamines, PEG-modifieddiacylglycerols, PEG-modified dialkylglycerols. ,Representative polyethylene glycol-lipids include PEG-c-DOMG, PEG-c-DMA, and PEG- s-DMG. In one embodiment, the polyethylene glycol-lipid is N-[(methoxy poly(ethylene glycol)2000)carbamyl l-l,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA). In one embodiment, the polyethylene glycol-lipid is PEG-c-DOMG).In other embodiments, the LNPs comprise a pegylated diacylglycerol (PEG-DAG) such as l-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG), a pegylated phosphatidylethanoloamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-0-(2',3 '-di(tctradccanoyloxy)propy 1-1 -0-(w-mcthoxy(polycthoxy)cthyl)butancdioatc (PEG-S-DMG), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as w-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or 2,3- di(tetradecanoxy)propyl-N-(w-methoxy(polyethoxy)ethyl)carbamate.The ratio (mol%) of the polymer conjugated lipid to the total lipids present in the LNP of the present invention may be, for example, about 0% to about 20%, preferably about 0.1% to about 5%, more preferably about 0.7% to about 2%.Only one kind of the above-mentioned polymer conjugated lipid may be used or two or more kinds thereof may be used in combination in the LNP. When multiple polymer conjugated lipids are used, the ratio of the whole neutral lipid is preferably as mentioned above.According to embodiments of the invention, the neutral lipid is selected from the group consisting of• l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)• 1 , 2-dioleoyl-.s / / -glycero-3 -phosphocholine (DOPC)• l-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC)• l-stearioyl-2-oleoyl -phosphatidy ethanol amine (SOPE),• dipalmitoylphosphatidylcholine (DPPC),• dioleoylphosphatidylglycerol (DOPG),• dipalmitoylphosphatidylglycerol (DPPG),• dioleoyl-phosphatidylcthanolamine (DOPE),• palmitoylolcoylphosphatidylcholine (POPC),• palmitoyloleoylphosphatidyl glycero• palmitoyloleoyl-phosphatidylethanolamine (POPE),• dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane- 1 - carboxylate (DOPE-mal),• dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE),• 16-0-monom ethyl PE,• 16-0-dimethyl PE,• 18-1 -trans PE, and / or• l,2-dielaidoyl-sn-glycero-3-phophoethanolamine (transDOPE)The ratio (mol%) of the neutral lipid to the total lipids present in the LNP is, for example, 0% to about 90%, preferably about 5% to about 30%, more preferably about 8% to about 15%, however, the ratio is not limited to these.Only one kind of the above-mentioned neutral lipid may be used or two or more kinds thereof may be used in combination in the LNP. When multiple neutral lipids are used, the ratio of the whole neutral lipid is preferably as mentioned above.According to embodiments of the invention, the steroid or steroid analogue is selected from the group consisting of• Cholesterol, and• C-24 alkyl-substituted cholesterol analogues, including Stigmasterol, B-Sitosterol, Stigmastanol, Campesterol, Fucosterol, Brassicasterol, Ergosterol and 9, 11 — Dehydroergosterol, 5a-cholestanol, 513-coprostanol, cholesteryl-(2'-hydroxy)-ethylether, cholesteryl-(4'-hydroxy)-butylether, 6-ketocholestanol, 5 a -cholestane, cholestenone, 5 a -cholestanone, 513-cholestenone, and cholesteryl decanoateThe ratio (mol%) of the steroid or steroid analogue to the total lipids present in the LNP when steroids are present may be, for example, about 10 mol% to about 60 mol%, preferably about 15 mol% to about 55 mol%, more preferably about 40 mol% to about 50 mol%.In one embodiment, the LNP according to the present invention comprises the lipids ALC- 0315 / / DSPC / Cholesterol / DSPE-PEG5000-(Gly)2-NH2 / ALC-0159 in a molar ratio of from about 40 to about 60 : from about 8 to about 15 : from about 30 to about 50 : from about 0.5: to about 1.2 : from about 0.5 to about 1.2, preferably from about 45 to about 50 : from about 9 to about 11 : from about 38 to about 43 : from about 0.8 to about 1 : from about 0.8 to about 1, most preferably about 47.5 : about 10 : about 40.7 : about 0.9 : about 0.9.According to another aspect of the invention, a method for providing an LNP covalently decorated with at least one targeting moiety, activating moiety and / or stimulatory moiety is provided.In a first step of the method, LNPs comprising oligoglycine on the surface are provided. The LNPs comprising an oligoglycine on its surface may be provided by: a. providing a first solution, preferably an organic solution, comprising a glycine-lipid and at least three lipids selected from the group consisting of (i) a cationic / cationizable lipid, (ii) a neutral lipid, (iii) a steroid or steroid analogue; and(iv) a polymer conjugated lipid, preferably a PEG-lipid; b. providing a second solution, preferably an aqueous solution, comprising a polynucleotide, preferably a DNA, encoding an antigen receptor and preferably further comprising an mRNA encoding for a transposase enzyme; c. mixing the first and the second solution to obtain a medium comprising LNPs comprising oligoglycine on its surface; d. optionally subsequently purifying, enriching, or exchanging the medium.In a second step of the method, at least one targeting moiety, activating moiety and / or stimulatory moiety , preferably all three moieties, comprising a sortase recognition tag at their C-termini is conjugated to the LNPs obtained in the first step by a sortase in a suitable mediumto obtain LNPs covalently decorated with at least one targeting moiety, activating moiety and / or stimulatory moiety .In a further optional step, the obtained LNPs may be further purified, enriched or the medium may be exchanged by methods known in the art. Preferably, the sortase is separated from the medium comprising LNPs by diafiltration using a suitable membrane with appropriate MW cutoffIn the method, the lipids, moieties, DNA RNA, and sortase as described above are preferably used.In a further aspect the invention relates to a LNPs covalently decorated with at least one targeting moiety, activating moiety and / or stimulatory moiety obtained by the method.According to another aspect of the invention, an in vitro method of transfecting a leukocyte with an LNP according to the above description is provided.Regarding this and further aspects of the invention, the respective embodiments disclosed above in the context of the LNP shall be deemed disclosed as well, and are not repeated again here for reasons of procedural economy.According to another aspect of the invention, a leukocyte comprising an ectopic antigen receptor is provided, which leukocyte has been produced by transfection with an LNP according to the above description.The transfection may have been performed either in vitro, or in vivo. In vivo transfection maybe carried out in human patients, preferably by intravenous injection.According to another aspect of the invention, the LNP according to the above description or the leukocyte according to the above description is provided for (the manufacture of a medicament for) use in the treatment of a human or animal subj ect being diagnosed for, suffering from orbeing at risk of developing cancer or an autoimmune disease.This language is deemed to encompass both the swiss type claim language accepted in some countries (in this case, brackets are deemed absent) and EPC2000 language (in this case, brackets and content within the brackets is deemed absent).According to another aspect of the invention, a method for treating or preventing a cancer or an autoimmune disease in a human or animal subject is provided, which method comprises administration of the LNP or the leukocyte according to the above description in sufficient doses.According to another aspect of the invention, an in vitro transfection method for transfecting leukocytes is provided. In a first step, a leukocyte population is obtained from a subject, preferably a human subject, e.g. by leukapheresis. Subsequently, an leukocyte population, preferably a T cell population, is obtained (also called “enriched leokocyte / T cell population)”. The leukocyte population may for example be a peripheral blood mononuclear cell (PBMC) population or a CD4+ and / or CD8+ T cell population that may be obtained by methods commonly known in the art. PBMC populations may for example be obtained using a Ficoll gradient. CD4+ and / or CD8+ enriched cell populations are commonly obtained through magnetic bead-based cell sorting. A mixed cell population of CD4+ and CD8+ cells can be isolated through the depletion of non-T cells or by positive selection using a combination of CD4 and CD8-binding microbeads or magnetic antibodies. Alternatively, a population enriched only in CD4+ or CD8+ may be obtained by using CD8-or CD4 binding microbeads. Methods to obtain populations enriched in NK cells, B cell or neutrophils are likewise known in the art. For example, magnetic beads binding to the antigens preferably expressed by these cell types as disclosed above may be used.In a subsequent step, the leukocytes, preferably T cells, of the enriched leukocyte population are transfected with at least one antigen receptor, preferably a CAR, by contacting the leukocytes with at least one LNP as described herein. Optionally, the method may comprise a step of expanding the leukocytes prior or after the transfection step. Optionally, the method maycomprise a step of determining the transfection rate. In one embodiment, the method may comprise a step of isolating leukocytes expressing and / or transfected with an antigen receptor. However, in a preferred embodiment, the method does not comprise a step of isolating leukocytes expressing and / or transfected with an antigen receptor, due to the high transfection rates of the LNPs disclosed herein.In one aspect the invention relates to a transfected leukocyte obtained by the afore described method.In one aspect, the invention relates to a method of treatment of a disease, preferably of cancer or an autoimmune disease, or the use of transfected leukocytes in such method, wherein the leukocytes obtained by the in vitro transfection method disclosed herein are obtained from a subject in need of treatment, preferably a patient suffering from cancer. Said method comprises a step of administering the transfected leukocytes to said patient.In a further embodiment, the invention relates to an in vivo transfection method. The method comprises contacting at least one leukocyte in a subject to at least one LNP according to the invention. Accordin g to embodiments, at least one LNP is administered to the subject, either by intravenous, intraperitoneal, intra-lymph-node or intra-tumor injection. Preferably the LNP is administered intravenously. The LNP may be comprised in a pharmaceutical composition.According to another aspect of the invention, a pharmaceutical composition comprising at least one LNP according to the invention is provided.The pharmaceutical composition comprises at least one LNP according to the invention and at least one pharmaceutically acceptable carrier (including but not limited to excipient, diluent, bulking agent, binder, lubricant, flow aid, disintegrant, surfactant, and the like) and conventional additives, and the like. Preferably the pharmaceutical composition is in liquid or liquid frozen form.Alternatively, the pharmaceutical composition may be in a dry form which is reconstituted with a suitable sterile liquid prior to use. When used in an injectable form, acceptable buffering agent, solubilizing agent, isotonic agent and the like can also be added.The pharmaceutical composition is preferably for use in a method of treating cancer or an autoimmune disease.EXAMPLESWhile the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive; the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.All amino acid sequences disclosed herein are shown from N-terminus to C-terminus; all nucleic acid sequences disclosed herein are shown 5'->3'.EXAMPLESEXAMPLE 1: Generation of a transposable minicircle DNA vector encoding a CD19 CARThe sequence of a 5554 bp long DNA vector encoding a transposable element comprising and encoding a CD19 targeting CAR is provided in SEQ ID NO: 1. This sequence can be generated by gene-synthesis as known in the art. All genetic elements of the construct from the promoter for the expression to the poly-adenylation signal in the expression construct are annotated, in order to enable the invention. A plasmid-map of the 5554bp long pBS-KSII+-based expression vector for a CD19 targeting CAR (SEQ ID NO: 1) is provided in Figure 2.EXAMPLE 2: Generation of mRNA encoding Sleeping Beauty transposaseThe DNA sequence of an 4224bp long pBS-KSII+-based expression vector encoding for Sleeping beauty lOOx transposase enzyme is provided in SEQ ID NO: 2. This sequence can be generated by gene-synthesis as known in the art. Note that while this sequence is provided as a DNA sequence it is within the routine of the skilled person to transcribe said DNA into anmRNA encoding for the transposase, e.g. by means of a T7 RNA polymerase as disclosed in detail in EXAMPLE 2. A respective plasmid-map of the template vector for T7 RNA polymerization is provided in Fig. 3.More specifically, in a first step of the Sleeping Beauty mRNA manufacturing, the 4224bp long pBS-KSII+ -based sleeping beauty expression vector can be linearized by Adel restriction enzyme digestion, which is a unique cutter in the fl ori of the vector backbone for 4h at 37°C. The linearized plasmid can then be purified by magnetic bead purification with carboxylic acid coated magnetic beads as follows.First, NaCl solution is added to the linearized DNA to reach a concentration of 1.0 M. Then, ethanol is added to reach a concentration of 50%. By doing this, the NaCl is diluted to 0.5 M. After that, carboxylic acid coated magnetic beads are added in a ratio of 1 mg beads per 2 mg DNA. Under those conditions high-molecular weight nucleic acids bind to the beads, but proteins stay in solution, making it possible to pull the beads, with the DNA bound to them, contact-free via a permanent magnet to the tube wall and take off the supernatant with all unbound impurities. The bound DNA is first washed with 0.5 M NaCl / 50 % ethanol and after that with 80 % ethanol. After washing the DNA is dissolved in WFI and separated from the beads via a permanent magnet.Next, 4 mg of linearized DNA template is transcribed into long-chain mRNA using 1250 kU recombinant T7 RNA-polymerase in the presence of 0.6 mol of each nucleotide triphosphates ATP, CTP, GTP, UTP and 0.23 mol of the ARCA Cap Analog. During the polymerization the mRNA is capped at the 5’ end with ARCA Cap Analog. At the end of the transcription reaction, the template DNA is hydrolysed by addition of 15.5 kU recombinant DNase I in the presence of calcium chloride. The RNA is purified by magnetic bead purification using a similar approach as for the purification of the linearized plasmid DNA, as follows: NaCl solution is added to the Dnase I treated transcription mixture to reach a concentration of 1.0 M. Then, ethanol is added to reach a concentration of 50%. By doing this, the NaCl is diluted to 0.5 M. After that, magnetic beads are added in a ratio of 1 mg beads per 2 mg RNA. Under those conditions high-molecular weight nucleic acids bind to the beads, but proteins and DNA fragments stay in solution, making it possible to pull the beads, with the RNA bound to these, contact free via a permanent magnet to the tube wall and take off the supernatant with all unbound materials.By adding 11.5 ml 10 mM HEPES / O. lOmM EDTA pH 7.0 the RNA is dissolved and 1.3 ml 0.5 M EDTA is added. Then the RNA gets bound again by adding NaCl and ethanol and supernatant is removed as described above. This step is repeated twice. In three consecutive steps, bound RNA is washed with 32% ethanol / 0.5 M NaCl. The purpose is to remove EDTA and other impurities from the previous step. Bound RNA is washed once with 80% ethanol. The purpose is to prevent carry-over of high levels of NaCl into the RNA solution. Bound RNA is incubated at room temperature under a partial vacuum of 40 mbar absolute pressure to remove ethanol. The evaporation is conducted in 5 steps. Between the steps the RNA is wetted with water for injection to prevent over drying. The RNA is eluted from the beads with 10 mM HEPES / 0.10 mM EDTA pH 7.0. Beads are retained by a magnet and supernatant is pooled in a new container. After determination of RNA concentration, the eluate is diluted to 2 mg / mL using 10 mM HEPES / 0.10 mM EDTA pH 7.0. The diluted RNA solution is filtered through a 0.2 pm filter and aseptically dispensed into appropriate aliquots.Annotations for all genetic elements of the DNA vector provided in SEQ ID NO: 2 are provided in Fig 3, in order to enable the invention. The ORF of the Sleeping Beauty protein from position 728-1747 translates into the amino acid sequence of SEQ ID NO: 3.The mRNA encoding for the same enzyme inclusive of the 5’UTR, 3’UTR and 120-mer poly A Tail is shown as SEQ ID NO: 5. As discussed herein, said sequence can comprise pseudouridine instead of uridine, or can be G / C optimized in order to reduce the U content.EXAMPLE 3: Preparation of mRNA / DNA transposon LNPs3, 1 Preparation of LNPLNPs comprising lipids ALC-0315 / DSPC / Cholesterol / DSPE-PEG5000-(Gly)2-NH2 / ALC- 0159 in a molar ratio of 47.5: 10:40.7:0.9:0.9 and a mRNA conc. / mcDNA cone, ration of 4: 1 were produced by ethanol injection by standard methods as follows using the lipids according to the following table.Table 6: Lipids used in studymcDNA encoding a CD 19 CAR obtainable according to Example 1 and RNA encoding Sleeping Beauty transposase obtainable according to Experiment 2 were used.For preparation of an organic phase the following lipids were dissolved in 15 ml ethanol and heated in 55 °C water bath for 30 min, cooled to RT and filtered using a 0.22pM PES membrane.Table 7: Lipids amountsFor preparation of the aqueous phase, mRNA and mcDNA were diluted into aqueous 100 mM citrate buffer (pH 4.0) to a target concentration: 266 pg / mL mRNA and 66.5 pg / mL mcDNA. The lipid comprising ethanol solution organic phase and the mRNA / mcDNA aqueous phase comprising citrate buffer were mixed in a T Junction mixer at a total flow rate of 40 mL / min and a V(mRNA / mcDNA buffer) : V(LiPid solution) of 3 : 1 and subsequently in-line diluted into 50 mM citrate buffer (pH 4.0). The resulting material was stirred using a magnetic stirrer at 50 rpm for 10 minutes.The diluted mixture was subsequently subjected to tangential flow filtration (TFF) to achieve concentration and buffer exchange. A Pellicon® 3 cassette with Ultracel® membrane, 30kD MWCO, D-Screen and 88 cm2membrane area was applied. The cassette was first rinsed with ultrapure water and the normalised water permeability (NWP) was tested. After passing the test, the TFF system was equilibrated with 50mM Citrate buffer, pH 4.0. The material was concentrated and then buffer-exchanged into 50 mM Citrate buffer pH 4.0 for 3 diafiltration volumes, followed by 50 mM HEPES, 150 mM NaCI, 5 mM CaCh, 8% (w / w) sucrose, pH 7.5 until the pH at the permeation end reached 7.5. The buffer-exchange was completed and the materials was blown out. Finally, the used cassette was cleaned, tested for NWP, rinsed with 0.1 M NaOH and stored.The TFF-treated material was tested using RiboGreen® RNA Reagent and Kit, which showed an mRNA concentration of 223.2 pg / mL and an mRNA encapsulation efficiency (EE%) of 91.7%. Then the mRNA concentration was adjusted to 200 pg / mL using 50 mM HEPES, 150 mMNaCl, 5 mM CaCh, 8% (w / w) sucrose, pH 7.5, followed by filtering using a 0.22 pm sterile needle filter to yield mRNA / mcDNA transposon LNPs.For particle size and PDI detection, the DLS Nanoparticle Sizer ZSU3200 from Malvern Panalytical Limited with a sample cell type ZEN0040 was used. The sample type is Liposome, the dispersant is 8% sucrose, the test type is Size, and the repeated test number is 2. Set the test temperature to 250C and equilibration time to 30 seconds. The results showed that the sample had a mean particle size of 72.5 nm and a particle dispersion index (PDI) of 0.07. mRNA and mcDNA were tested for concentration and EE% using RiboGreen® RNA Reagent and Kit and PicoGreen® dsDNA Reagent and Kit, respectively. Testing was performed using black 96-well plates with a plate reader set to Fluorescence mode, Endpoint read type, excitation at 485 nm, emission at 525 nm. According to the standard curve, the mRNA or mcDNA concentration and EE% corresponding to 485 / 525 nm fluorescence values of the samples were calculated, and the results showed that the mRNA concentration was 179.1 pg / mL, mRNAEE% was 91.4%, mcDNA concentration was 39.0 pg / mL, and mcDNAEE% was 83.8%.Table 8: Characteristics of undecorated LNPThe high encapsulation efficiency of both the mRNA (> 90%) and mcDNA (> 80%) shows that mRNA / mcDNA transposon LNPs can be produced efficiently.3.2 Decoration of LNP with ligands by sortase-mediated transpeptidation6x His-tagged recombinant Sortase A enzyme from Staph, aureus based on publication by Chen et al. (2011) was provided by Genscript USA, Piscataway, New Jersey, 08854, and producedin E.coli BL21 Star™ (DE3). The enzyme was provided at 4mg / mL concentration in 50 mM Tris-HCl, 500 mM NaCl, 10% glycerol, pH8.0.Conjugation of protein ligands to the LNPs was performed with lOOOpl LNPs compositions obtained according to Experiment 3.1 in 10 mL volume. The LNP concentration in the reaction mixture corresponds to a DSPE-PEG(5000)-Gly2 concentration of about: 8.59 pM.RNAse free water for injection, undecorated LNP, LPQTG-tagged protein ligands to yield a final concentration of 10 pM, of 5x sortase buffer (250 mM HEPES, 750 mM NaCl, 25 mM CaC12, 8% Sucrose, pH 7.5) and sortase enzyme (4 mg / ml) to yield a final concentration of 5 pM were added as shown in Table 7 in the respective order with gentle mixing after addition of each component.The following stock solutions were used:2.12mg / ml, anti-CD3 scFv corresponds to ~74pM2.34mg / ml, anti-CD28 VHH corresponds to ~165pM0.98mg / ml, mb-IL15 corresponds to -67 pM4.05mg / ml, sortase A corresponds to -228 pMTable 9: Conjugation reaction mixtures:To remove free anti-CD3, anti-CD28, IL15, and sortase A were subjected to UF / DF. Add 10~15mL of UF / DF buffer comprising 50mM HEPES, 150mM NaCl, 5mM CaC12,8%Sucrose pH7.5 was to the empty ultrafiltration tube, and centrifuged at 4°C, 1500rcf for 5~10min to complete the pre-rinse of the ultrafiltration tube membrane. Subsequently, for UF 10 ml reactant was added to the rinsed ultrafiltration tube and perform UF to ensure that the sample volume at the cut-off end is about 1 mL after centrifugation. Thereafter, DF was performed in a cycle of 10~30min. DF was finished when the concentration of free protein near or less than 10 ng / pL at the permeable end and the pH& conductivity of the permeate same as the UF / DF buffer. At the end of DF, the volume of liquid in the retentive end of the ultrafiltration tube is about 0.2 ~ 0.3 mL. Aspirate all samples into an EP tube. Chasing: Pipette a certain volume of UF / DF buffer into the ultrafiltration tube, and rinse the ultrafiltration tube membrane repeatedly. Mix it with the DF sample from the previous step, and ensure the final volume of ~0.5 mL.Samples from the conjugation reaction mixtures at different reaction steps and subsequent process steps were analysed by SDS page analysis. Results are shown in Figure 6.Furthermore, PDI and particle size of the LNP in samples from the conjugation reaction mixtures at different reaction steps and subsequent process steps were determined. Results are shown in the following table:Table 10: Size and PDI of conjugated LNPsEXAMPLE 4: Functional Analysis of LNP4.1 MaterialsT cells for LNP -modification were isolated from the peripheral blood of healthy donors. All participants provided written informed consent to participate in research protocols approved by the institutional review board of the University of Wurzburg. Peripheral blood mononuclear cells (PBMCs) of healthy donors were purified using Ficoll-hypaque density centrifugation in 50 mL LeukoSep tubes (Greiner Bio One, Frickenhausen, Germany), and CD4+and CD8+T cells were isolated using negative magnetic sorting (CD4+and CD8+T cell Isolation Kits, human, Miltenyi Biotec).T cells were maintained in ImmunoCult-XF T Cell Expansion Medium (StemCell Technologies, Vancouver, BC, Canada) containing 0.5% penicillin / streptomycin (Gibco, Thermo Fisher Scientific, Schwerte, Germany). T cell cultures were supplemented with 100 U / ml IL-2 (Miltenyi Biotec, Bergisch-Gladbach, Germany).4.2 Generation of CAR T cells4.2.1 For experiments with undecorated LNPs of Batch 1 obtained in Experiment 3 , 2.5xl05purified CD4+stimulated with anti-CD3 / CD28 TransAct (Miltenyi Biotec) were seeded in a 48-well plate at a total volume of 250 pL and treated with LNPs (13 pl) and, supplemented with Apolipoprotein E4 (ApoE4; Ipg / mL; PeproTech, Thermo Fisher Scientific) and centrifuged (45 min, 32°C, 800xg, Break 2) prior to incubation. After 4h at 37°C, medium was filled up to 1 mL and supplemented with 50 IU IL-2 (Miltenyi Biotec). Where indicated, T cells were enriched for EGFRt+using the anti-EGFR monoclonal antibody (mAb) Cetuximab (Merck, Darmstadt, Germany), that had been biotinylated in-house (EZ-Link™Sulfo-NHS-SS-Biotin, ThermoFisher Scientific) according to the manufacturer’s instructions) and anti -Biotin Microbeads (Miltenyi Biotec).4.2.2 For experiments with decorated LNPs of Batches 2 to 8 obtained in Experiment 3, 1.25xl05purified CD4+and 1.25xl05purified CD8+T cells were seeded in a total volume of 100 pL in a 96-well flat bottom plate (Sarstedt, Numbrecht, Germany). Cells were than either left untreated or supplemented with Apolipoprotein E4 (ApoE4; 2pg / mL; PeproTech, Thermo Fisher Scientific), followed by the addition of the respective LNPs (0.5 pl) one hour later. Cells were subsequently centrifuged (15 min, 200xg, 32°C, break 5) prior to incubation. After overnight culture at 37°C, medium was filled up to 250 pL and supplemented with 100 IU IL- 2.4.2.3 T cells obtained according to 4.2.1 were analyzed by staining with the anti-EGFR monoclonal antibody Cetuximab (Eli Lilly and Company, Indianapolis, IN, USA) that have been conjugated to AF647 using the Alexa Fluor™ 647 Protein Labeling Kit (Thermo Fisher Scientific) and analysis by flow cytometric analyses. Results of EGFRFACS analysis is shown in Figure 7.T cells obtained according to 4.2.1 and 4.22 were further analyzed by staining with antibodies against CD4 (clone M-T466; VioGreen; Miltenyi Biotec), CD8 (clone REA734; VioBlue; Miltenyi Biotec), CD25 (clone BC96; FITC; BioLegend, San Diego, CA, USA), CD69 (clone FN50; PE-Cy7 or APC-Cy7; BioLegend) as well as 7-AAD (Miltenyi Biotec), to exclude dead cells from analysis were used, and subsequent analysis by flow cytometric analyses.Flow cytometric analyses were performed with either a MACSQuant 10 (Miltenyi Biotec) or CytoFlex LX (Beckman Coulter, Indianapolis, IN, USA), and analyzed using FlowJo software (TreeStar, Ashland, OR, USA).Results of CD25 / CD69 T cell activation marker FACS analysis is shown in Figures 8A and 8B. T cells that were incubated with LNPs decorated with anti-CD3 revealed higher expression of CD25 and CD69. An additional decoration with anti-CD28 or IL- 15 further increased the expression of these activation markers. In contrast, decoration with anti-CD28 or IL-15 led to only minor increase in CD25 and CD69 expression. A triple combination of anti-CD3, anti- CD28 and IL- 15 lead to slightly higher increase in expression of activation markers over a combination of anti-CD3 and IL- 15, which already had reached almost maximal activation.The EXAMPLES disclosed herein in detail and the results provided in the Figures, demonstrate that (i) mRNA and mcDNA can efficiently be co-encapsulated (> 90% mRNA, > 80% mcDNA) into mRNA / DNA transposon LNPs, that (ii) they can be decorated with targeting, and / or stimulatory and / or activating moieties via sortase-emzyme mediated bio-conjugation, that (iii) the decorated mRNA / DNA transposon LNPs can effectively activate and stimulated primary human T lymphocytes, that (iv) mRNA / LNP transposon LNPs can transfect primary human T lypmhocytes, and that (v) the mRNA / LNP transposon LNPs can effect the stable transposition of a transposable expression construct encoded by a mcDNA vector into the nucleus of stimulated primary T cells by the transiently transfected SBIOOx mRNA.Therefore, invention disclosed herein can be used to replace expensive and hardly scalable lentiviral or y-retroviral CAR-T vectors for CAR-T manufacturing with cost-effective and highly scalable mRNA / DNA-transposon LNP vectors for CAR-T manufacturing, but, in contrast to mRNA-LNPs, enabling the same stable expression phenotype of the resulting CAR- T cells, as obtained with lenti / y-retroviral viral vector systems.The absence of any viral components in this system represents an advantage to employ this invention not only in the context of conventional centralized CAR-T manufacturing, but also in the context of point-of-care and bedside CAR-T manufacturing and eventually, also the generation of CAR-T cells in vivo by direct single systemic administration of the mRNA / DNA- transposon LNP vectors into patients.References1. Magnani CF et al. (2020) «Transposon-based CAR-T Cells in Acute Leukemias: Where Are We Going ? Cells 9: 1337.2. Lock D et al. (2022) «Automated, scaled, transposon-based production of CAR T cells” J. Immunother. Cancer 10: e005189.3. Watanabe N et al. (2022) «Impact of Manufacturing Procedures on CAR T Cell Functionality” Front. Immunol. 13: 876339.4. Moretti A et al. (2022) «The Past, Present, and Future of Non- Viral CAR-T cells» Front. Immunol. 13: 867013.5. Malone RW et al. (1989) «Cationic liposome-mediated RNA transfection» Proc. Natl. Acad. Sci. USA 86: 6077-60816. Bettini E et al. (2021) “SARS-CoV-2 mRNA Vaccines: Immunological Mechanism and Beyond” Vaccines 9: 147.7. Kiaie SH et al. (2022) «Recent advances in mRNA-LNP therapeutics: immunological and pharmacological aspects» J. Nanobiotechnol. 20: 276.8. Melenhorst JJ et al. (2022) «Decade-long leukaemia remissions with persistence of CD4+ CAR T cells» Nature 602: 503-509.9. Levine BL et al. (2017) «Global Manufacturing of CAR T Cell Therapy» Molecular Therapy: Methods & Clinical Development 4: 92-101 :10. Dower WJ, Park Al, Bakker AV, Cwirla SE, Pongtornpipat P, Williams BM, Joshi P, Baxter BA, Needels MC, Barrett RW. A mechanistically novel peptide agonist of the IL- 7 receptor that addresses limitations of IL-7 cytokine therapy. PLoS One. 2023 Oct 24;18(10):e0286834.11. Harris, K.E., Lorentsen, K.J., Malik-Chaudhry, H.K. et al. A bispecific antibody agonist of the IL-2 heterodimeric receptor preferentially promotes in vivo expansion of CD8 and NK cells. Sci Rep 11, 10592 (2021).12. Beerli RR, Hell T, Merkel AS, Grawunder U. Sortase Enzyme-Mediated Generation of Site-Specifically Conjugated Antibody Drug Conjugates with High In Vitro and In Vivo Potency. PLoS One. 2015 Jul 1; 10(7):e0131177.13. Sommermeyer, D. et al. Chimeric antigen receptor-modified T cells derived from defined CD8+ and CD4+ subsets confer superior antitumor reactivity in vivo. Leukemia 30, 492- 500 (2016).Wang, X. et al. A transgene-encoded cell surface polypeptide for selection, in vivo tracking, and ablation of engineered cells. Blood 118, 1255-1263 (2011). Zola, H. et al. Preparation and Characterization of a Chimeric-Cdl9 Monoclonal- Antibody. Immunol Cell Biol 69, 411-422 (1991). Monjezi, R. et al. Enhanced CAR T cell engineering using non-viral Sleeping Beauty transposition from minicircle vectors. Leukemia 31, 186-194 (2017). Mackensen, A., Muller, F., Mougiakakos, D. et al. Anti-CD19 CAR T cell therapy for refractory systemic lupus erythematosus. Nat Med 28, 2124-2132 (2022). Uslu, U., June, C.H. Beyond the blood: expanding CAR T cell therapy to solid tumors. Nat Biotechnol (2024). Tombacz I, Laczko D, Shahnawaz H, Muramatsu H, Natesan A, Yadegari A, Papp TE, Alameh MG, Shuvaev V, Mui BL, Tam YK, Muzykantov V, Pardi N, Weissman D, Parhiz H. Highly efficient CD4+ T cell targeting and genetic recombination using engineered CD4+ cell-homing mRNA-LNPs. Mol Ther. 2021 Nov 3;29(11):3293-3304. Metzloff AE, Padilla MS, Gong N, Billingsley MM, Han X, Merolle M, Mai D, Figueroa- Espada CG, Thatte AS, Haley RM, Mukalel AJ, Hamilton AG, Alameh MG, Weissman D, Sheppard NC, June CH, Mitchell MJ. Antigen Presenting Cell Mimetic Lipid Nanoparticles for Rapid mRNA CAR T Cell Cancer Immunotherapy. Adv Mater. 2024 Jun;36(26):e2313226 Yu X, Ruan M, Wang Y, Nguyen A, Xiao W, Ajena Y, Solano LN, Liu R, Lam KS. Site- Specific Albumin-Selective Ligation to Human Serum Albumin under Physiological Conditions. Bioconjug Chem. 2022 Dec 21;33(12):2332-2340 Miskey C, Izsvak Z, Plasterk RH, Ivies Z. The Frog Prince: a reconstructed transposon from Rana pipiens with high transpositional activity in vertebrate cells. Nucleic Acids Res. 2003 Dec 1;31(23):6873-81. Zhao S, Jiang E, Chen S, Gu Y, Shangguan AJ, Lv T, Luo L, Yu Z. PiggyBac transposon vectors: the tools of the human gene encoding. Transl Lung Cancer Res. 2016 Feb;5(l): 120-5. doi: 10.3978 / j.issn.2218-6751.2016.01.05. PMID: 26958506; PMCID: PMC4758974. Arensburger P, Hice RH, Zhou L, Smith RC, Tom AC, Wright JA, Knapp J, O'Brochta DA, Craig NL, Atkinson PW. Phylogenetic and functional characterization of the hAT transposon superfamily. Genetics. 2011 May;188(l):45-57.Geurts AM, Yang Y, Clark KJ, Liu G, Cui Z, Dupuy AJ, Bell JB, Largaespada DA, Hackett PB. Gene transfer into genomes of human cells by the sleeping beauty transposon system. Mol Ther. 2003 Jul;8(l): 108-17. Cui Z, Geurts AM, Liu G, Kaufman CD, Hackett PB. Structure-function analysis of the inverted terminal repeats of the sleeping beauty transposon. J Mol Biol. 2002 May 17;318(5): 1221-35 Ivies Z, Izsvak Z, Minter A, Hackett PB. Identification of functional domains and evolution of Tcl-like transposable elements. Proc Natl Acad Sci U S A. 1996 May 14;93(10):5008-13. Ivies et al. Molecular reconstruction of Sleeping Beauty, a Tcl-like transposon from fish, and its transposition in human cells. Cell 91, 501-510 (1997) Ni et al. Briefings Funct. Genomics Proteomics 7, 444-453 (2008)) P Holliger, PJ Hudson: Engineered antibody fragments and the rise of single domains. In: Nat. Biotechnol. 23. Jahrgang, Nr. 9, September 2005, S. 1126-1136, doi: 10.1038 / nbtl l42, PMID 16151406. Flanagan RJ, Jones AL (2004). "Fab antibody fragments: some applications in clinical toxicology". Drug Saf. 27 (14): 1115-1133. Hudson PJ, Souriau C: Engineered antibodies. In: Nat. Med. 9. Jahrgang, Nr. 1, Januar 2003, S. 129-134 Pliickthun A (2015). "Designed ankyrin repeat proteins (DARPins): binding proteins for research, diagnostics, and therapy". Annu. Rev. Pharmacol. Toxicol. 55 (1): 489-511. Ebersbach, H.; Fiedler, E.; Scheuermann, T.; Fiedler, M.; Stubbs, M. T.; Reimann, C.; Proetzel, G.; Rudolph, R.; Fiedler, U. (2007). "Affilin-Novel Binding Molecules Based on Human y-B-Crystallin, an All P-Sheet Protein". Journal of Molecular Biology. 372 (1): 172-185 Harris KE, Lorentsen KJ, Malik-Chaudhry HK, et al. A bispecific antibody agonist of the IL-2 heterodimeric receptor preferentially promotes in vivo expansion of CD8 and NK cells. Sci Rep. 2021;l l(l) Dower WJ, Park Al, Bakker AV, Cwirla SE, Pongtornpipat P, Williams BM, Joshi P, Baxter BA, Needels MC, Barrett RW. A mechanistically novel peptide agonist of the IL- 7 receptor that addresses limitations of IL-7 cytokine therapy. PLoS One. 2023 Oct 24;18(1037. Yu X, Ruan M, Wang Y, Nguyen A, Xiao W, Ajena Y, Solano LN, Liu R, Lam KS. Site- Specific Albumin-Selective Ligation to Human Serum Albumin under Physiological Conditions. Bioconjug Chem. 2022 Dec 21;33(12):2332-234038. Parthasarathy R, Subramanian S, Boder ET. Sortase A as a novel molecular "stapler" for sequence-specific protein conjugation. Bioconjug Chem. 2007 Mar-Apr;18(2):469-76.39. Chen I, Dorr BM, Liu DR. A general strategy for the evolution of bond-forming enzymes using yeast display. Proc Natl Acad Sci U S A. 2011 Jul 12; 108(28): 11399-404.40. Barreira et al. (2023) Gene Therapy 30: pp. 122-131).41. Shankar et al. (2017) Cell Gene Therapy Insights 3: pp. 285-300SequencesThe following sequences form part of the disclosure of the present application. A WIPO ST 26 compatible electronic sequence listing is provided with this application, too. For the avoidance of doubt, if discrepancies exist between the sequences in the following table and the electronic sequence listing, the sequences in this table shall be deemed to be the correct ones.The sequences are shown in N^C orientation, or 5’— >3’ orientation, respectively. In some cases, signal peptides may be encompassed in the reproduced sequences. In such case, the sequences shall be deemed disclosed with and without signal peptides. A readily available tool to identify signal peptides in a given protein sequence is SignalP - 6.0 provided by Dansk Technical University under https: / / services.healthtech.dtu.dk / service.php7SignalP. The same applies to His tags, C-Myc tags, FLAG tags, HA tags, Strep tags, TC tags, Poly A tags, or Nuclear Localization Sequences, if existing - all of which are well known to the skilled artisan.Note further that in RNA sequences, “T” is to be read for Uracil. Further, in some cases the respective mRNA or amino acid sequences relates to a selected splice version or isoform of a given gene or protein. In such cases, the skilled person is able to identify the sequences of the other splice versions or isoforms, based on the information provided herein and the respective routine knowledge, plus access to the respective pubic databases. Such splice version or isoforms shall be deemed disclosed herein as well.Note that while SEQ ID NO: 2 is provided as a DNA sequence, it is within the routine of the skilled person to transcribe said DNA into an mRNA encoding for the transposase, e.g. by means of a T7 polymerase.Table 11: Sequence listingUnderlined passages show the linker, VH is N-terminal, VL is C-terminalUnderlined passages show the linker, VL is N-terminal, VH is C-terminal3transcript variant #l / isoform #1, skilled person is able to identify the sequences of the other isoforms based on this information and routine knowledge4transcript variant #3 / isoform #1, skilled person is able to identify the sequences of the other isoforms based on this information and routine knowledge5SEQ ID NOs 11 - 15 define a complete anti CD19 chimeric antigen receptor, when read in N->C direction

Claims

What is claimed is:

1. A lipid nanoparticle (LNP), preferably suitable for transfecting a leukocyte, which nanoparticle a) is decorated with(ii) at least one targeting moiety that binds to and optionally signals via a leukocyte surface antigen selected from, CD3, TCR alpha, beta, gamma or delta chain, CD2, CD4, CD5, CD8, CCR5, CCR7, CD27, CD127, 77CD45RA, CD58, CXCR3, CD122, CD7, CD56, CD161, CD57 (HNK-1, LEU-7 or L2), CD94 (NKG2D), CD 19, CD20, CD 15, CDl lb / CD18 or CD66;(ii) at least one activating moiety that is a gamma chain cytokine, preferably selected from the group consisting of IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21, or IL-12, or a functionally active fragment thereof, and b) further comprises an mRNA encoding for at least one transposase enzyme, and a DNA encoding for at least one antigen receptor.

2. The LNP according to claim 1, which is further decorated with at least one stimulatory moiety comprising means for binding, and / or interacting with, at least one costimulatory molecule expressed on leukocytes.

3. The LNP according to claim 2, wherein a) the costimulatory molecule on the leukocyte to which the stimulatory moiety binds and / or with which it interacts is selected from the group consisting of• CD2, CD28, 4-1BB / CD137, OX-40 / CD134, or CD40L / CD154, ICOS, CD27, DR3, LIGHT / TNFS14, HVEM / TNFRSF14, GITR,• FcyRIII / CD 16, FcyRI I / CD32, FcyRIa / CD64, or• Ig-co-receptors, CD79a (mb-1) or CD79b (B21), or b) the stimulatory moiety which decorates the LNP and interacts with the costimulatory molecule on the leukocyte is the soluble, extracellular domain of OX-40L, CD40, 4-1BBL, CD70, GITR ligand (GITRL), LIGHT / TNFS14, HVEM / TNFRSF14, ICOS Ligand (ICOSL).

4. The LNP according to any one of the aforementioned claims, wherein the targeting moiety according to claim 1 and / or the stimulatory moiety according to claim 3 is selected from the group consisting of c) an antibody, or functional fragment thereof, d) a DARPin or an Affilin.

5. The LNP according to any one of the aforementioned claims, wherein the transposase belongs to a transposase family selected from the group consisting of• Tel -mariner super-family• PiggyBac (PB) transposase family, and / or• hAT super-family.

6. The LNP according to any one of the aforementioned claims, wherein the leukocyte is selected from the group consisting of a T cell, a B lymphocyte, a macrophage or an NK cell.

7. A lipid nanoparticle (LNP), preferably suitable for transfecting a leukocyte, which nanoparticle comprises a) at least one mRNA sequence encoding at least one transposase enzyme, and b) at least one DNA sequence encoding at least one antigen receptor.

8. The LNP according to claim 7, wherein the transposase belongs to a transposase family selected from the group consisting of• Tel -mariner super-family• PiggyBac (PB) transposase family, and / or• hAT super-family.

9. The LNP according to any one of the aforementioned claims, wherein the encoding DNA is provided as a transposable DNA.

10. The LNP according to any one of the aforementioned claims, wherein the DNA encoding at least one antigen receptor and the RNA encoding for at least one transposase are comprised in the LNP in a mass ratio of DNA:RNA of about 1 : up to about 10, preferably 1 : up to about 8, more preferably between about 1 :3 to about 1 :6, more preferably about 1 :4 to about 1 :5.

11. The LNP according to any one of claims 7 - 10, wherein the LNP is decorated with at least one targeting moiety that is suitable to direct the LNP to the leukocyte that is to be transfected.

12. The LNP according to claim 11, wherein the targeting moiety which decorates the LNP binds to at least one surface antigen of a leukocyte, selected from the group consisting of• a subunit of the T cell receptor, such as (i) CD3, (ii) TCR alpha, (iii) TCR beta, (iv) TCR gamma or (v) TCR delta, CD2, CD4, CD5, CD8, CCR5, CCR7, CD27, CD127, CD45RA, CD58, CXCR3, CD 122 or CD7,• CD56, CD161 (NK1.1), CD57 (HNK-1, LEU-7 or L2), or CD94 (NKG2D),• CD19 or CD20, or• CD15, CDl lb / CD18 or CD66b.

13. The LNP according to any one of claims 11 - 12, wherein the targeting moiety which decorates the LNP is at least one of a) an antibody, or functional fragment thereof, or b) an antibody mimetic, including aa DARPin or an Affilin.

14. The LNP according to any one of claims 11 - 13, wherein the surface antigen of the leukocyte exerts an intracellular signal upon binding of the targeting moiety.

15. The LNP according to any one of claims 7 - 14, wherein the LNP is decorated with a) at least one stimulatory moiety that is suitable to stimulate a leukocyte either alone or via an additional costimulatory moiety, and / or b) at least one stimulatory moiety that interacts with at least one costimulatory molecule on the leukocyte.

16. The LNP according to any claim 15, wherein the stimulatory moiety which decorates the LNP interacts with at least one costimulatory molecule on the leukocyte selected from the group consisting of• CD2, CD28, 4-1BB / CD137, OX-40 / CD134, or CD40L / CD154, ICOS, CD27, DR3, LIGHT / TNFS14, HVEM / TNFRSF14, GITR• FcyRIII / CD 16, FcyRI I / CD32, FcyRIa / CD64, orIg-co-receptors, CD79a (mb-1) or CD79b (B21).

17. The LNP according to any one of claims 15 and 16, wherein the stimulatory moiety which decorates the LNP and interacts with at least one costimulatory molecule is at least one selected from c) an antibody, or functional fragment thereof, or d) an antibody mimetic, including a DARPin or an Affilin.

18. The according to any one of claims 15 - 17, wherein the stimulatory moiety which decorates the LNP is the soluble, extracellular domain of at least one of OX-40L, CD40, 4-1BBL, CD70, GITR ligand (GITRL), LIGHT / TNFS14, HVEM / TNFRSF14, ICOS Ligand (ICOSL).

19. The LNP according to any one of claims 7 - 18, wherein the LNP is decorated with at least one activating moiety.

20. The LNP according to claim 19, wherein the activating moiety is a gamma chain cytokine, preferably selected from the group consisting of IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21, or is IL-12.

21. The LNP according to any one of the aforementioned claims, wherein at least one of the targeting moiety, the stimulatory moiety and the activating moiety is coupled to the LNP either by non-covalent or covalent coupling.

22. The LNP according to any one of the aforementioned claims, wherein at least one of the targeting moiety, the activating moiety and / or the stimulatory moiety is bound to the LNP by means of sortase enzyme-mediated covalent conjugation.

23. The LNP according to any one of the aforementioned claims, wherein the DNA encoding for an antigen receptor encodes for a chimeric antigen receptor.

24. The LNP according to any one of the aforementioned claims which comprises, in its lipid sheath, at least three lipids from the group consisting of(i) a cationic / cationizable lipid(ii) a neutral lipid(iii) a steroid or steroid analogue; and(iv) a polymer conjugated lipid, preferably a PEG-lipid.

25. A method for providing a LNP covalently decorated with at least one targeting moiety, activating moiety and / or stimulatory moiety comprising i.) a first step of providing LNPs comprising oligoglycine on the surface, said first step optionally comprising: a. providing a first solution, preferably an organic solution, comprising a glycine-lipid and at least three lipids selected from the group consisting of (i) a cationic / cationizable lipid, (ii) a neutral lipid, (iii) a steroid or steroid analogue; and(iv) a polymer conjugated lipid, preferably a PEG-lipid; b. providing a second solution, preferably an aqueous solution, comprising a polynucleotide, preferably a DNA, encoding an antigen receptor and preferably further comprising an mRNA encoding for a transposase enzyme; c. mixing the first and the second solution to obtain a medium comprising LNPs comprising oligoglycine on its surface; d. optionally subsequently purifying, enriching, or exchanging the medium; and ii.) a second step of conjugating at least one targeting moiety, activating moiety and / or stimulatory moiety, preferably all three moieties, comprising a sortase recognition tag to the LNPs obtained in the first step by a sortase in a suitable medium to obtain LNPs covalently decorated with at least one targeting moiety, activating moiety and / or stimulatory moiety .

26. A LNP covalently decorated with at least one targeting moiety, activating moiety and / or stimulatory moiety obtained, or obtainable, by the method of claim 2527. An in vitro method of transfecting a leukocyte with an LNP according to any one of claims 1 - 24, or 26.

28. A leukocyte comprising an ectopic antigen receptor, which leukocyte has been produced by transfection with an LNP according to any one of claims 1 - 24, or 26., or with the method of claim 27.

29. The LNP according to any one of claims 1 - 24, or 26, or the leukocyte according to claim 28, for (the manufacture of a medicament for) use in the treatment of a human or animal subject• being diagnosed for,• suffering from or• being at risk of developing cancer or an autoimmune disease.

30. A method for treating or preventing a cancer or an autoimmune disease in a human or animal subject, which method comprises administration of the The LNP according to any one of claims 1 - 24, 26 or 29, or the leukocyte according to claim 28, to a patient, in one or more therapeutically sufficient doses.

31. An in vitro method for transfecting leukocytes, the method comprising the steps of: a) obtaining a leukocyte population from a subject, optionally by leukapheresis, and b) transfecting leukocytes of the leukocyte population with at least one antigen receptor, by contacting the leukocytes with at least one LNP according to any one of claims 1 - 24, 26 or 29.

32. A method of treatment of a disease, or the use of transfected leukocytes in such method, wherein the leukocytes obtained by the in vitro transfection method of claim 31 are obtained from a subject in need of treatment, which method comprises a step of administering the transfected leukocytes to said patient.

33. An in vivo method for transfecting leukocytes, the method comprising the steps of contacting at least one leukocyte in a subject to at least one LNP according to any one of claims 1 - 24, 26 or 29.

34. A pharmaceutical composition comprising at least one LNP according to any one of claims 1 - 24, 26 or 29.

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