Synthetic transfer vehicles for delivering RNA to cells

Abstract

The present invention relates to a method of identifying whether an interaction domain capable of nucleating self-assembly of a plurality of fusion proteins comprising said interaction domain and capsid-forming proteins into synthetic transfer vehicles (STVs) provides for RNA transfer efficiency of said STVs from a sender to a receiver cell, comprising: (A) providing a mammalian sender cell; (B) providing a mammalian receiver cell; (C) bringing supernatant from said sender cell comprising the population of STVs into contact with said receiver cell, thereby allowing fusion of said population of STVs with the cell membrane of said reporter cell and delivery of first polynucleotides into the reporter cell; and (D) determining the signal obtained from the reporter cell, said signal being produced by the functional reporter system through protein-fragment complementation of the first and second member of said first split reporter system, said protein-fragment complementation being mediated by protein splicing through a functional intein forming by self-association of the first and second entity of the split intein, said self-association being mediated by the interaction of the protein- protein interaction domains encoded by the first and fourth polynucleotide, respectively; thereby identifying an interaction domain which provides for RNA transfer efficiency of STVs from a sender to a receiver. The present invention further relates to a non-naturally occurring interaction domain obtainable by the method of the present invention.

Description

[0001] PCT patent application

[0002] Applicant: Helmholtz Zentrum Muenchen

[0003] Our ref: HEL18542PCT

[0004] Date: 26 September 2025

[0005] SYNTHETIC TRANSFER VEHICLES FOR DELIVERING RNA TO CELLS

[0006] [1] This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

[0007] TECHNICAL FIELD OF THE INVENTION

[0008] [2] The present invention relates to a method of identifying whether an interaction domain capable of nucleating self-assembly of a plurality of fusion proteins comprising said interaction domain and capsid-forming proteins into synthetic transfer vehicles (STVs) provides for RNA transfer efficiency of said STVs from a sender to a receiver cell, comprising: (A) providing a mammalian sender cell; (B) providing a mammalian receiver cell; (C) bringing supernatant from said sender cell comprising the population of STVs into contact with said receiver cell, thereby allowing fusion of said population of STVs with the cell membrane of said reporter cell and delivery of first polynucleotides into the reporter cell; and (D) determining the signal obtained from the reporter cell, said signal being produced by the functional reporter system through proteinfragment complementation of the first and second member of said first split reporter system, said protein-fragment complementation being mediated by protein splicing through a functional intein forming by self-association of the first and second entity of the split intein, said self-association being mediated by the interaction of the protein-protein interaction domains encoded by the first and fourth polynucleotide, respectively; thereby identifying an interaction domain which provides a readout for RNA transfer efficiency of STVs from a sender to a receiver.

[0009] BACKGROUND OF THE INVENTION

[0010] [3] Evolution guides biological systems to populate ecological niches, with viruses being one of the most successful examples of that principle. Viruses evolved over billions of years for the efficient transfer of nucleic acids. Although highly diverse, most viruses converged toward a remarkable similarity in the size and shape of their capsids. In contrast, generative models for protein design enable the creation of protein architectures that are absent in nature. Here, we investigate whether protein assemblies designed by the methods of the present invention can be functionalized to construct nucleic acid transport vehicles that are independent of evolutionary trajectories. By combining natural protein domains with synthetic protein assemblies of the present invention, we created hundreds of bottom-up RNA transfer vehicles with distinct characteristics. These novel vehicles surprisingly surpass the RNA transfer efficiency of viral architectures and other widely used delivery vehicles by several orders of magnitude. Additionally, their tropism can be programmed by incorporating computationally designed peptide binders and they enable the delivery of various therapeutically relevant cargo RNAs into a wide range of cellular models. We show the in vivo biodistribution of one of these vehicles in a mouse with close to single-cell resolution and use it to deliver a genetic treatment strategy for Duchenne muscular dystrophy into a pig. Our work demonstrates how proteins created by the methods of the present invention comprising generative Al (artificial intelligence) can be applied to overcome limitations of natural protein diversity and be harnessed for the rational engineering of biological systems with desired properties.

[0011] [4] The present invention was made in view of the prior art and a need for improvement thereof as outlined above. The objective of the present invention can therefore inter alia be formulated as to provide improved means and methods for deliverying RNA to cells (e.g., for treatment of genetic disorders, e.g., Duchenne muscular dystrophy). The present invention satisfies this need by the provision of methods, compositions and other means as described herein below, characterized in the claims and illustrated by the appended Examples and Figures.

[0012] SUMMARY OF THE INVENTION

[0013] [5] The technical problem is solved by the subject-matter as defined in the claims.

[0014] [6] In some aspects, the present invention relates to a method of identifying whether an interaction domain capable of nucleating self-assembly of a plurality of fusion proteins comprising said interaction domain and capsid-forming proteins into synthetic transfer vehicles (STVs) provides for RNA transfer efficiency of said STVs from a sender to a receiver cell, comprising: (A) providing a mammalian sender cell; (B) providing a mammalian receiver cell; (C) bringing supernatant from said sender cell comprising the population of STVs into contact with said receiver cell, thereby allowing fusion of said population of STVs with the cell membrane of said reporter cell and delivery of first polynucleotides into the reporter cell; and (D) determining the signal obtained from the reporter cell, said signal being produced by the functional reporter system through protein-fragment complementation of the first and second member of said first split reporter system, said protein-fragment complementation being mediated by protein splicing through a functional intein forming by covalent self-association of the first and second entity of the split intein, said covalent self-association being mediated by the interaction of the proteinprotein interaction domains encoded by the first and fourth polynucleotide, respectively; thereby identifying an interaction domain which provides for RNA transfer efficiency of STVs from a sender to a receiver.

[0015] [7] In some aspects, the present invention further relates to a non-naturally occurring interaction domain obtainable by the method of the present invention. [8] In some aspects, the present invention further relates to a fusion protein comprising a membrane-binding domain, a budding domain, an RNA-binding domain and the interaction domain of the present invention.

[0016] [9] In some aspects, the present invention further relates to a composition, comprising: (i) a polynucleotide encoding a fusion protein comprising: (a) a membrane-binding domain, (b) a budding domain, (c) an RNA-binding domain being capable of binding an RNA packaging signal and (d) the interaction domain of the present invention.

[0017] BRIEF DESCRIPTION OF THE DRAWINGS

[0018]

[0010] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, respectively. The Figures show:

[0019]

[0011] Figure 1 : Design and screening of bottom-up assembled STV RNA carrier, a, Size and shape comparison of viral and Al-designed protein assemblies, b, Principle of mimicking viral release and RNA packaging by expressing synthetic protein assemblies in cells (MBD: Membrane binding domain, L: Late budding domain, RNA BP: RNA binding protein, A: Assembly domain), c, RT-qPCR quantification of RNA release by HE0902-based STV constructs, consisting of different RNA binding proteins (Unpaired Student’s t-test, **P < 0.01 , ***P < 0.001 , mean ± s.d. for n = 6 biological replicates), d, Screening scheme for STV release, uptake, and RNA delivery efficiency based on reconstitution of split luciferases in supernatant and cells, e, Workflow for testing STV release, uptake, and RNA expression over the course of 72 h. f, Luminescence measurement of HiBiT signal in the supernatant of producer cells to quantify the release of STV constructs containing different Al-designed assembly domains by LgBit / HiBiT binding (mean ± s.d. for n = 6 biological replicates), g, NanoLuc measurement in the lysate of LgBit expressing Split-Luc reporter cells to test for uptake of different STV constructs by reconstituting NanoLuc from LgBit and STV containing HiBiT (mean ± s.d. for n = 6 biological replicates), h, Firefly measurement in the lysate of C-split Flue expressing Split-Luc reporter cells to quantify N- split Flue RNA delivery efficiency from different STV constructs (mean ± s.d. for n = 6 biological replicates).

[0020]

[0012] Figure 2: Characterization and programming of the STV-C8 RNA carrier, a, Schematic representation of the optimal STV-C8 construct, consisting of the pleckstrin homology domain from Ursus americanus phospholipase C (UaPHPLC), a synthetic budding domain derived from viral ESCRT recruiting motifs (SynL), a tandem coat protein from Pseudomonas phage PP7 (tdPCP), and an Al-designed assembly domain with C8 symmetry (HE0690). b, RT- qPCR quantification of target RNA release into the supernatant by STV-C8 (mean ± s.d. for n = 3 biological replicates), c, RNA-seq based correlation analysis of producer cell and STV-C8 RNA content (n = 4 biological replicates). Mitochondrial RNAs are colored in red and the EGFP cargo transcript in green, d, Proteomic analysis of STV-C8 protein content by comparing protein content in purified supernatant of budding (+SynL) or non-budding (-SynL) STV-C8 constructs (n = 3 biological replicates). ESCRT-related proteins are colored in purple, e, Cryo-EM based imaging of purified STV-C8 particles in 2D and 3D representation (scale bar: 100 nm). f, Comparison of EGFP mRNA delivery efficiency for STV-C8 and other genetically encoded delivery vehicles into different target cell lines, measured by Flow Cytometry (mean ± s.d. for n = 4 biological replicates). Each mRNA was tagged with its corresponding packaging signal, g, Comparison of the required amount of EGFP mRNA to induce expression of one MFI, quantified by Flow Cytometry, in target cells by LNP or STV-C8 dependent delivery. STV-C8 RNA content was quantified by RT-qPCR by comparing it to an in vitro transcribed reference EGFP RNA (mean ± s.d. for n = 3 biological replicates), h, Induction of GFP expression under the control of the I FN-p promoter in A549 reporter cells upon treatment with STV-C8 or plasmid transfection (scale bar: 50 pm), i, Concept for programming STV-C8 cell-type specificity by incorporating Al-designed minibinders, j, Flow Cytometry-based quantification of EGFP mRNA delivery into HEK293T cells with EGFR or IL7Ra receptor knock-ins by expressing mutant VSV-G together with receptor targeting minibinders (n = 3 biological replicates).

[0021]

[0013] Figure 3: STV-C8 dependent cargo RNA delivery into diverse cellular models, a, Packaging of EGFP mRNA into STV-C8 and delivering into monocyte suspension cells and RPE spheroids, b, Fluorescence imaging of RPE spheroids, 2 days after transduction with STV-C8, containing EGFP mRNA, and co-stained for the RPE marker protein (scale bar: 100 pm), c, Flow Cytometry analysis of human monocytes, untransduced or transduced with STV-C8 containing EGFP mRNA, 24 h after the treatment, d, Packaging of polycistronic mRNA coding for the Ascii transcription factor along with EGFP into STV-C8 and transduction of primary mouse astrocytes, e, Fluorescence imaging of mouse astrocytes, 3 days after transduction with STV-C8 packaging EGFP mRNA (scale bar: 50 pm), f, Quantification of Ascii positive astrocytes, transduced with Ascii containing STV-C8 and stained for Ascii (Unpaired Student’s t-test, ****P < 0.0001 , mean ± s.d. for n = 6 control and n = 4 treated, independent astrocyte cultures), g, Schematic illustration of STV-C8 mediated Cas9 / sgRNA delivery into porcine fibroblasts, resulting in the deletion of dystrophin exon 51 by sgRNAs cleaving in the flanking introns, h, PCR amplification of the dystrophin gene from Cas9 / sgRNA STV-C8 treated porcine fibroblasts 3 days after the treatment, i, STV-C8 mediated delivery of SARS-CoV-2 targeting Cas13d-NCS into virus infected human lung cells, j, Fluorescence imaging of iPSC-derived human lung cells, infected with SARS- CoV-2-GFP (MO110) and treated with STV-C8(Cas13d-NCS / crRNA), 24 h post-infection (scale bar: 100 pm), k, Live imaging of SARS-CoV-2-GFP replication in STV-C8 (Cas13d-NCS / crRNA) treated iPSC-derived human lung cells for 48 h (n = 3 biological replicates). I, Measurement of N- Split-Luc mRNA transfer into Split-Luc reporter cells upon pretreatment of STV-C8 delivery vehicles with human whole blood and serum samples (Unpaired Student’s t-test, mean ± s.d. for n = 6 biological replicates).

[0022]

[0014] Figure 4: STV-C8 biodistribution and delivery of gene editing cargos in mouse and pig models, a, Schematic illustration of in vivo biodistribution analysis of STV-C8 mediated EGFP expression by mouse whole-body clearing and imaging, b, Ventral view of amplified EGFP expression in cleared mouse body, imaged by light-sheet microscopy, 72 h post i.v. injection (scale bar: 5 mm). Depicted rectangles mark magnified regions in panel c. c, Imaging of EGFP signal in cleared lung, liver, spleen, and kidney tissues (scale bar: 500 pm, kidney tissue was imaged from a different plane), d, Schematic illustration of Cas9 / sgRNA delivery into pig muscle by local injection of STV-C8 vehicles, e, PCR analysis of the edited dystrophin gene 72 h post i.m. injection. Upon treatment with STV-C8 (Cas9 / sgRNAs), exon 51 is deleted from the gene, f, Sanger sequencing of the PCR band, corresponding to deleted exon 51 in treated pig muscle cells (depicted is SEQ ID NO: 17,

[0023] CACAGAGTTCCTAAGGTAGAGAGAGGAATNNNGGAATAAAGATAAGAGCTGGCAGAGGTT TA), g, Long-read nanopore sequencing quantification of deletion efficiency in pig muscle (mean ± s.d. for n = 3 technical replicates of the same injection site).

[0024]

[0015] Figure 5: Detailed characterization of fundamental STV domains, a, Structure and size of the initial assembly domain HE0902. b, Design concept of the synthetic budding domain SynL, derived from ESCRT recruiting viral peptides, c, Basic STV design containing different budding domains tested in panel d. d, HiBiT-based quantification of STV release by fusing different budding domains (mean ± s.d. for n = 6 replicates biological replicates), e, Schematic illustration of EGFP mRNA delivery by basic STV construct, f, EGFP mRNA delivery into HEK293T cells, mediated by original STV construct with SynL domain, quantified by Flow Cytometry (mean ± s.d. for n = 4 biological replicates).

[0025]

[0016] Figure 6: Establishment and validation of the dual Split-Luciferase based screening method, a, Analysis of HE0902-based STV release by measuring HiBiT signal in the supernatant of producing cells (mean ± s.d. for n = 6 biological replicates), b, Quantification of HE0902-based STV uptake in C-Split-Luc reporter cells by measuring NanoLuc signal in cell lysates (mean ± s.d. for n = 6 biological replicates), c, Measurement of STV-HE0902 mediated N-Split-Luc mRNA delivery into C-Split-Luc reporter cells by quantifying Firefly Luciferase signal in cell lysates (mean ± s.d. for n = 6 biological replicates), d, Flow Cytometry-based validation of Split-Luc screening results by delivering EGFP mRNA into HEK293T cells (mean ± s.d. for n = 3 biological replicates).

[0026]

[0017] Figure 7: Additional C8 symmetric assemblies. Design of additional protein assemblies by running RFDiffusion with C8 symmetry constraints.

[0018] Figure 8: Screening for improved membrane binding domains, a, Illustration of STV release by PH domain-dependent binding to the plasma membrane of producer cells, b, Structural alignment of pleckstrin homology domains derived from unrestricted or human / metagenomic restricted FoldSeek search, c, Structural similarity of FoldSeek PH domains compared to Rattus norvegicus PHPLC5 domain, d, Sequence similarity of FoldSeek PH domains compared to Rattus norvegicus PHPLC5 domain, e, Schematic workflow for measuring release, uptake, and RNA delivery efficiency of STVs containing different membrane binding domains, f, Quantification of the release of STV variants by measuring HiBiT signal in producer cell supernatant (mean ± s.d. for n = 6 biological replicates), g, Quantification of STV uptake in Split-Luc reporter cells by measuring NanoLuc signal from reconstituted LgBiT / HiBiT in cell lysate (mean ± s.d. for n = 6 biological replicates), h, Quantification of RNA transfer efficiency by measuring Firefly luciferase signal in transduced Split-Luc reporter cell lysate (mean ± s.d. for n = 6 biological replicates), i, Subcellular localization of STVs with different membrane binding domains, determined by immunostaining of STVs in transfected producer cells (scale bar: 10 pm), j, Flow Cytometry analysis of STV’s MBD requirement by EGFP delivery into HEK293T cells (mean ± s.d. for n = 3 biological replicates), k, Flow Cytometry-based validation of screening results by delivering EGFP into HEK293T cells with STVs containing different PH domains (mean ± s.d. for n = 3 biological replicates).

[0027]

[0019] Figure 9: Analysis of purity and particle parameters of STV-C8. a, Characterizing the protein content of purified STV-C8 particles by Silver Stain and Western Blot, b, Analysis of purified STV-C8 particle size by DLS in duplicates (red and blue), c, Summary of STV-C8 particle characteristics in comparison to LNPs.

[0028]

[0020] Figure 10: Categorical analysis of RNA and protein content of STV-C8. a, GO term analysis of at least 3-fold depleted mRNAs in STV-C8 particles, b, GO term analysis of at least 3-fold enriched proteins in STV-C8 particles.

[0029]

[0021] Figure 11 : Programming of STV-C8 cell-type specificity with Al-designed minibinders, a, AlphaFold2 structural prediction of IL7Ra minibinder bound to its target receptor, b, Transduction efficiency of IL7Ra+ / EGFR+ or double-positive HEK293T cells with minibinder- equipped STV-C8 particles, containing EGFP mRNA (Unpaired Student’s t-test, **P < 0.01, ***P < 0.001 , mean ± s.d. for n = 6 biological replicates), c, Flow Cytometry analysis of EGFP expression in IL7Ra+ / EGFR+ or double-positive HEK293T cells, transduced with STV-C8 (EGFP) that were equipped with both IL7Ra and EGFR minibinders simultaneously.

[0030]

[0022] Figure 12: Characterization of STV-C8 packaging capacity, toxicity, and production conditions, a, Flow Cytometry-based analysis of cells transduced with STV-C8, co-packaging EGFP mRNA of 1 kb constant length and mRuby3 of varying 3’UTR lengths (mean ± s.d. for n = 6 biological replicates), b, Analysis of Annexin V positive cells by Flow Cytometry. Cells were transduced with EGFP mRNA-containing vehicles in sufficient concentration to reach 50-60% EGFP+ cells in each condition (mean ± s.d. for n = 3 biological replicates), c, Measurement of N- Split-Luc RNA transfer into Split-Luc reporter cells after 7 d of STV-C8 storage at different temperatures (Unpaired Student’s t-test, *P < 0.05, mean ± s.d. for n = 4 biological replicates), d, Schematic workflow to optimize plasmid amounts for STV-C8 release, uptake, and RNA delivery efficiency, e-g, Comparing STV-C8 release (HiBiT assay in supernatant), STV-C8 uptake (NanoLuc assay in target cell lysate), and RNA transfer (Firefly assay in target cell lysate) upon transfection of different amounts of cargo, packaging and fusogen plasmids (mean ± s.d. for n = 3 biological replicates), h, Summary of tested plasmid amounts and ratios.

[0031]

[0023] Figure 13: Further characterization of RPE spheroid and lung cell models, a, Imaging of STV-C8 (EGFP) treated RPE spheroids (scale bar: 100 pm), b, Confocal microscopy imaging of STV-C8 (EGFP) treated and immunostained spheroid slices, (scale bar: 100 pm), c, Imaging of TLR reporter cells 3 d after treatment with STV-C8 vehicles containing CRISPR gene editing systems (scale bar: 50 pm), d, RT-qPCR characterization of the lung-specific AT2 marker gene and ACE2 in iAT2 differentiated cells (mean ± s.d. for n = 3 biological replicates)

[0032]

[0024] Figure 14: Additional analysis of STV-C8 whole-body tissue distribution, a, Lightsheet imaging of cleared and signal-enhanced mouse body, treated with STV-C8(EGFP), 72 h post i.v. injection, b, Magnified upper and lower body parts of panel a (scale bar: 2 mm), c, Imaging of lung and liver tissues of an independently treated mouse, 24 h post i.v. injection (scale bar: 200 pm).

[0033]

[0025] Figure 15: Evaluation of STV-C8-mediated immunological response and liver damage in vivo, a, Scheme of the experimental setup for testing immunological markers in blood and liver toxicity, b, Analysis of potential liver toxicity (ALT, alanine aminotransferase) and broader toxicological side effects (LDH, lactate dehydrogenase) of animals, treated by systemic STV-C8 injection (mean ± s.d. for n = 5-6 treated animals), c, Testing for immunological markers by RT- qPCR (one-way ANOVA, *P < 0.05, mean ± s.d. for n = 5-6 treated animals).

[0034]

[0026] Figure 16: Continued analysis of intramuscular delivery of mRNAs with STV-C8. a, Quantification of the EGFP mRNA delivery into C2C12 mouse myotubes by Flow Cytometry, b, Long-read nanopore sequencing estimation of deletion efficiency in pig muscle (mean ± s.d. for n = 3 technical replicates of the same injection site), c, Schematic description of STV-C8- mediated intramuscular delivery of mRNAs and subsequent immunological analysis, d and e, Analysis of inflammatory markers of muscle samples by RT-qPCR (one-way ANOVA, ** P < 0.01 ,*** P < 0.001 , ****P < 0.0001 , mean ± s.d. for n = 3 replicates of different injection sites). DETAILED DESCRIPTION OF THE INVENTION

[0035]

[0027] The present invention is described in detail in the following and is also illustrated by the appended examples and figures.

[0036]

[0028] Definitions

[0037]

[0029] As described herein references can be made to UniProtKB Accession Numbers (http: / / www.uniprot.org / , e.g., as available in UniProt release 2024_04 published on July 24, 2024).

[0038]

[0030] As referred herein “EC numbers” (Enzyme Commission numbers) may be used to refer to enzymatic activity according to the Enzyme nomenclature database, Release of February 26, 2020 (e.g., available at https: / / enzyme.expasy.org / ). The EC number refers to Enzyme Nomenclature 1992 from NC-IUBMB, Academic Press, San Diego, Calif., including supplements 1-5 published in Eur. J. Biochem. 1994, 223, 1-5; Eur. J. Biochem. 1995, 232, 1-6; Eur. J. Biochem. 1996, 237, 1-5; Eur. J. Biochem. 1997, 250, 1-6; and Eur. J. Biochem. 1999, 264, 610- 650; respectively.

[0039]

[0031] The term “EC: 3.1.-.-.” as used herein can be interchangeably used with the term ““EC: 3.1.X.Y., wherein X is independently selected from 1 to 31 and Y is independently selected from 1 to 114”. The term “EC: 3.1.-.-.” may refer to endonuclease activity of Cas9.

[0040]

[0032] The term “endonuclease activity” may refer to enzymatic activity that cleave the phosphodiester bond within a polynucleotide chain.

[0041]

[0033] The terms “intein” or “intein activity” may refer to polypeptides (e.g., co-called protein introns) capable of excising themselves out of a polypeptide sequence and joining the remaining flanking regions (e.g., exteins) with a peptide bond.

[0042]

[0034] The term “intein activity” may refer to protein trans-splicing activity.

[0043]

[0035] The term “split-intein” may refer to a sub-group of inteins that are present in two separate complementary entities and catalyze protein splicing in trans upon association of said two complementary entities.

[0044]

[0036] The terms “guide RNA” or “gRNA” may refer to non-coding short RNA sequences which bind to the complementary target DNA sequences and confer target sequence specificity to the CRISPR-Cas9 system.

[0045]

[0037] The term “Cas9” may refer to CRISPR associated protein 9. Cas9 is a dual RNA-guided DNA endonuclease enzyme associated with the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR).

[0046]

[0038] As used herein, term "host cell" may refer to any cell type that is susceptible to transformation, transfection, transduction, or the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication, e.g., recombinant or transgenic host cell. Host cell may be prokaryotic or eukaryotic (e.g., a mammalian non-human cell).

[0047]

[0039] The term “polypeptide" is equally used herein with the term "protein". Proteins (including fragments thereof, preferably biologically active fragments, and peptides, usually having less than 30 amino acids) comprise one or more amino acids coupled to each other via a covalent peptide bond (resulting in a chain of amino acids). The term "polypeptide(s)" as used herein describes a group of molecules, which, for example, consist of more than 30 amino acids. Polypeptides may further form multimers such as dimers, trimers and higher oligomers, i.e. consisting of more than one polypeptide molecule. Polypeptide molecules forming such dimers, trimers etc. may be identical or non-identical. The corresponding higher order structures of such multimers are, consequently, termed homo- or heterodimers, homo- or hetero-trimers etc. An example for a hetero-multimer is an antibody molecule, which, in its naturally occurring form, consists of two identical light polypeptide chains and two identical heavy polypeptide chains. The terms "polypeptide" and "protein" also refer to naturally modified polypeptides / proteins wherein the modification is affected e.g. by post-translational modifications like glycosylation, acetylation, phosphorylation and the like. Such modifications are well known in the art.

[0048]

[0040] Generally, as used herein, the terms ..polynucleotide", ..nucleic acid" or ..nucleic acid molecule" are to be construed synonymously. Generally, nucleic acid molecules may comprise inter alia DNA molecules, RNA molecules, oligonucleotide thiophosphates, substituted ribooligonucleotides or PNA molecules. Furthermore, the term "nucleic acid molecule" may refer to DNA or RNA or hybrids thereof or any modification thereof that is known in the art (see, e.g., US 5525711 , US 471 1955, US 5792608 or EP 302175 for examples of modifications). The polynucleotide sequence may be single- or double- stranded, linear or circular, natural or synthetic, and without any size limitation. For instance, the polynucleotide sequence may be genomic DNA, cDNA, mitochondrial DNA, mRNA, antisense RNA, ribozymal RNA or a DNA encoding such RNAs or chimeroplasts (Gamper et al., Nucleic Acids Res (2000), 28 (21): 4332- 4339). Said polynucleotide sequence may be in the form of a vector, plasmid or of viral DNA or RNA. Also described herein are nucleic acid molecules which are complementary to the nucleic acid molecules described above and nucleic acid molecules which are able to hybridize to nucleic acid molecules described herein. A nucleic acid molecule described herein may also be a fragment of the nucleic acid molecules in context of the present invention. Particularly, such a fragment is a functional fragment. Examples for such functional fragments are nucleic acid molecules which can serve as primers.

[0049]

[0041] Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity”. For purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used may be gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled “longest identity” (obtained using the no-brief option) is used as the percent identity and is calculated as follows: (Identical Residues* 100) / (Length of Alignment-Total Number of Gaps in Alignment). Alternatively, the parameters used may be gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NLIC4.4) substitution matrix. The output of Needle labeled “longest identity” (obtained using the no-brief option) is used as the percent identity and is calculated as follows: (Identical Deoxyribonucleotides*100) / (Length of Alignment-Total Number of Gaps in Alignment).

[0050]

[0042] As used herein, the term “AAV” may refer to Adeno-associated virus. Non-limiting examples of the use of AAVs include their application in delivering therapeutic genes and nucleic acid sequences in gene therapy for the treatment of genetic disorders, such as hemophilia, Duchenne muscular dystrophy, and retinal dystrophies.

[0051]

[0043] As used herein, the term “interaction domain” may refer to a region within a protein that mediates and / or facilitates interaction with other molecules, typically proteins or nucleic acids. Preferably, the interaction domain plays a role in promoting self-assembly.

[0052]

[0044] As used herein, the term “nucleating self-assembly” may refer to the initiation of a process where smaller components begin to organize into a larger, structured complex. In this context, "nucleating self-assembly" may mean that the interaction domain triggers and / or facilitates the autonomous organization of proteins into a structured complex (e.g., synthetic transfer vehicle).

[0053]

[0045] As used herein, the term “plurality” may refer to more than one (e.g., 2, 3, 4, 5, or 6 etc.) fusion proteins, indicating that multiple fusion proteins are involved.

[0054]

[0046] As used herein, the term “fusion protein” may refer to proteins that are engineered or naturally occurring, composed of two or more distinct protein domains or sequences fused together into a single polypeptide. In this case, the fusion protein includes both the interaction domain and capsid-forming proteins.

[0055]

[0047] As used herein, the term “capsid-forming protein” may refer to proteins capable of forming protective shells, i.e. , capsids, e.g., around viral genomes and mediate viral entry into a cell.

[0056]

[0048] As used herein, the term “synthetic transfer vehicles” may refer to artificial or engineered constructs designed to encapsulate and transport genetic material (such as RNA) between cells. Preferably, STVs mimic natural viral particles.

[0057]

[0049] As used herein, the term “RNA transfer efficiency” may refer to the effectiveness or degree to which RNA molecules are successfully transferred from a sender cell to a receiver cell.

[0058]

[0050] As used herein, the term “sender cell” may refer to a cell that produces and / or releases a synthetic transfer vehicle containing the RNA that is to be delivered to another cell. Preferably, a sender cell is a mammalian cell comprising: (i) a first polynucleotide encoding the first member of a first split reporter system providing for a detectable signal, the first entity of a split intein, a protein-protein interaction domain and a non-translated RNA packaging signal being bound by the RNA-binding domain of the fusion protein encoded by the second polynucleotide; (ii) a second polynucleotide encoding a fusion protein comprising (a) a membrane-binding domain, and / or (b) a budding domain, (c) an RNA-binding domain being capable of binding the RNA packaging signal encoded by the first polynucleotide of and (d) an non-naturally occurring interaction domain capable of nucleating self-assembly of a plurality of said fusion protein into STVs secreted from said sender cell in which said fusion proteins are expressed, thereby generating a population of STVs comprising the fusogen encoded by the third polynucleotide and further comprising first polynucleotides bound through their RNA packaging signal by the RNA-binding domain of said fusion protein, and (iii) a third polynucleotide encoding a fusogenic protein.

[0059]

[0051] As used herein, the term “receiver cell” may refer to a cell that takes up the synthetic transfer vehicle and the encapsulated RNA. Preferably, in the context of gene therapy this would typically be the target cell intended to receive therapeutic RNA. As used herein, the term “reporter cell” may refers to a receiver cell as disclosed herein upon contact with a synthetic transfer vehicle (STV) as disclosed herein. Preferably, the “reporter cell” of the present invention is capable of producing a detectable signal indicative of RNA delivery. Particularly, the “reporter cell” of the present invention comprising: (iv) a fourth polynucleotide encoding the second member of the first split reporter system being capable of complementing the first member of the split reporter system to form a functional first reporter system, the second entity of the split intein being capable of selfassociation with the first entity of the split intein to form a functional intein and a protein-protein interaction domain being capable of interaction with the protein-protein interaction domain encoded by the first polynucleotide, that has been brought into contact with the supernatant from the sender cell comprising the population of STVs, thereby allowing fusion of the population of STVs with the cell membrane of said cell and delivery of first polynucleotides into the cell. In nonlimiting examples of the present invention, a reporter cell of the present invention may be genetically engineered to comprise a polynucleotide encoding a component of a split reporter system, such as the second member of a split intein or enzyme system. Upon delivery of the complementary reporter component via STV-mediated RNA transfer this system may enable protein-fragment complementation, resulting in the reconstitution of a functional intein or reporter enzyme and thereby a reporter signal. The reporter cell of the present invention may further comprise a protein-protein interaction domain capable of interacting with a corresponding domain delivered by the STVs. This interaction may facilitate the self-association of the split reporter system, triggering signal generation and thereby signaling RNA cargo uptake. Accordingly, in the context of the present invention, the reporter cell may serve as a functional readout system for RNA transfer efficiency. Therefore, in the context of the present invention the term “reporter cell” may be used interchangeably with the term “receiver cell”, e.g., depending on the functional state of the cell. In the context of the present invention, a receiver cell may become a reporter cell upon contact with synthetic transfer vehicle / s (STVs) as part of the method of the the present invention as disclosed herein, e.g., once the signal-producing molecular interaction has occurred. Accordingly, in the context of the present invention, both terms, i.e., the “receiver cell” and “reporter cell” terms, may refer to the same cell at different functional stages, e.g., as explained herein.

[0060]

[0052] As used herein, the term “split reporter system” may refer to a genetically engineered system in which a reporter protein is divided into two or more separate polypeptide fragments, which individually are non-functional, but can reassemble under certain biological conditions to form a functional reporter. This reassembly typically results in a detectable signal, such as fluorescence, luminescence and / or enzymatic activity that may be used as a reporter of a specific molecular event (e.g., protein-protein interactions, RNA binding, or other cellular processes).

[0061]

[0053] As used herein, the term "first split reporter system" may refer to the first instance of such a system within a method or composition of the present invention. In this context, it may involve: a reporter protein, which may be a molecule used to produce a measurable signal (e.g., green fluorescent protein (GFP), luciferase and / or p-galactosidase); split components, which may be the reporter protein divided into two or more non-functional fragments, each encoded by different polynucleotides or within different sections of a single polynucleotide; and a reconstitution mechanism, which may be when the fragments are designed to reassemble and become functional only when a specific biological or biochemical event occurs, such as the interaction between two proteins or molecules. This reassembly may be to generate a detectable signal.

[0062]

[0054] As used herein, the term “first entity of a split intein” may refer to an intein, which is a protein sequence that can self-excise and facilitate the ligation of surrounding protein sequences (exteins). A split intein may be divided into two separate polypeptides, which, when brought together, undergo splicing to produce a functional protein. The first entity may refer to one of these split intein fragments.

[0063]

[0055] As used herein the term “protein-protein interaction domain” may refer to a functional region of a protein that enables it to specifically interact with another protein / s.

[0064]

[0056] As used herein the term “non-translated RNA packaging signal” may refer to a specific sequence within an RNA molecule that does not get translated into a protein but is important for packaging RNA into viral particles or transfer vehicles. It may serve as a signal for binding proteins or machinery responsible for encapsulating RNA during processes such as gene therapy or viral vector production.

[0065]

[0057] As used herein the term “RNA-binding domain” may refer to a structural domain of a protein that specifically recognizes and binds to an RNA molecule. In this context, the RNA- binding domain of the fusion protein (encoded by the second polynucleotide) may bind to the nontranslated RNA packaging signal.

[0066]

[0058] As used herein the term “membrane-binding domain” may refer to a functional region of the fusion protein that allows it to attach to or associate with cellular membranes. This domain may play a role in positioning the fusion protein near the cell membrane, which may be necessary for the formation of membrane-bound structures such as vesicles or viral particles.

[0067]

[0059] As used herein the term “budding domain” may refer to a specific region of the fusion protein that may facilitate the process of budding, where vesicles or other structures pinch off from the cell membrane. This domain may enable the fusion protein to participate in the formation of synthetic transfer vehicles (STVs), which may bud off from the membrane of a sender cell.

[0068]

[0060] As used herein the term “RNA-binding domain” may refer to a protein domain capable of specifically recognizing and binding to RNA molecules. In this case, the RNA-binding domain may be engineered to bind to an RNA packaging signal.

[0069]

[0061] As used herein the term “RNA packaging signal” may refer to a sequence within an RNA molecule that signals it for packaging into vesicles or viral-like particles. This non-translated sequence may interact with a RNA-binding domain of the fusion protein allowing the RNA to be selectively packaged into the STVs formed by the fusion protein.

[0070]

[0062] As used herein the term “fusogen” may refer to a protein or molecular structure that may facilitate the fusion of a vesicle or particle (such as an STV) with the membrane of a target cell (e.g., recever cell). Accordingly, a fusogen may facilitate membrane fusion allowing the STVs to deliver their contents (e.g., RNA) of a target cell (e.g., receiver cell).

[0071]

[0063] As used herein the term “second member of said first split reporter system” may refer to to the second portion of this split reporter protein. This second member may interact (e.g. complement) with the first member to form a functional reporter allowing for the generation of a detectable signal.

[0072]

[0064] As used herein the term “second entity of said split intein” may refer to a second fragment of the intein that may bind to the first entity of the split intein.

[0073]

[0065] As used herein the term “non-covalent self-association” may refer to ability of the two entities of the split intein (e.g., the first and second parts / entities) to come together and form a functional intein through non-covalent interactions.

[0074]

[0066] As used herein the term “covalent self-association” may refer to the ability of two entities of a split intein (e.g., the first and second parts / entities) to undergo a series of covalent bond rearrangements following non-covalent self-association, leading to the excision of the intein and the ligation of the surrounding protein sequences (exteins).

[0075]

[0067] As used herein the term “self-assembled polypeptides forming naturally-occurring viral architectures” may refer to polypeptides that are capable of spontaneously self-organizing into structural formations that mimic or replicate the protein arrangements observed in viruses through intrinsic biochemical properties of such polypeptides. Preferably, SEQ ID NO: 1 corresponding to EPN24 (WO2023023528) is an example of such “self-assembled polypeptide forming naturally- occurring viral architectures”.

[0076]

[0068] As used herein the term “icosahedral symmetry” may refer to a specific type of geometric arrangement found in some biological structures, particularly viral capsids. An icosahedron is a polyhedron with 20 identical triangular faces, 12 vertices, and 30 edges. In the context of proteins, icosahedral symmetry may describe the organization of protein subunits in a structure that approximates or forms an icosahedron.

[0077]

[0069] As used herein the term “non-icosahedral symmetry” may refer to a structural arrangement that does not follow the specific geometric pattern of an icosahedron, which has 20 equilateral triangular faces, 12 vertices, and 30 edges.

[0078]

[0070] As used herein the term “cyclic symmetry” or “Cn symmetry”, which may be used interchangebly may refer to structures that have a circular arrangement of subunits, typically arranged around a central axis. The "n" in Cn represents the number of repeating subunits that are symmetrically arranged. For example, C6 symmetry would indicate six subunits arranged in a ring-like fashion around a single axis.

[0079]

[0071] As used herein the term “dihedral symmetry” or “Dn symmetry” which may be used interchangebly may refer to a form of symmetry that combines cyclic symmetry with mirror planes. In a system with dihedral symmetry, the structure may include both rotational symmetry (like cyclic symmetry) and reflections across mirror planes. For example, D3 symmetry indicates a structure with threefold rotational symmetry, along with reflection symmetry. This type of symmetry is often seen in protein complexes or biological assemblies with multiple planes of symmetry.

[0080]

[0072] As used herein the term “cubic symmetry” may refer to a type of three-dimensional symmetry in which the structure forms a shape resembling a cube or other regular polyhedrons, e.g., tetrahedrons or octahedrons. In such structures, subunits may be arranged symmetrically along the axes of a cube or other polyhedral shapes.

[0081]

[0073] As used herein the term “helical symmetry” may refer to a type of three-dimensional symmetry in which subunits may be arranged in a helical or spiral fashion, e.g., around a central axis.

[0082]

[0074] As used herein the term “tetrahedral symmetry” may refer to a geometric structure with four triangular faces, forming a tetrahedron. This type of symmetry has four vertices, six edges, and four triangular faces, and is less complex than octahedral symmetry.

[0083]

[0075] As used herein the term “octahedral symmetry” may refer to a geometric structure of an octahedron, which has eight triangular faces, six vertices, and 12 edges.

[0084]

[0076] As used herein the term “planar structure” may refer to a flat two-dimensional geometrical arrangement of components where all the atoms or molecules involved are aligned in a single plane. In this context, the interaction domain may guide the self-assembly of the molecules into a structure that lies flat without significant 3D curvature.

[0085]

[0077] As used herein the term “open structure” may refer to a geometrical arrangement that are not enclosed or tightly packed allowing for more flexible or unconfined spaces. These structures can have voids, pores or less defined boundaries compared to more compact closed assemblies. For example, a scaffold-like structure with gaps or loosely associated protein complexes fall within the meaning of “open structure”.

[0078] As used herein the term “low complexity” may refer to an geometrical arrangement consisting of repetitive or simple components rather than intricate, highly diverse parts. In this context, it suggests that the interaction domain leads to the formation of a structure with minimal variation, often with repetitive motifs or sequences.

[0086]

[0079] As used herein the term “cargo RNA molecule” may refer to a RNA strand designed to deliver genetic information or instructions to a target cell or biological system. In this context, cargo may refer to the RNA's role in carrying specific genetic instructions (coding for a protein). This RNA could be messenger RNA (mRNA), a synthetic RNA molecule, or another RNA type engineered to perform a specific function in gene expression.

[0087]

[0080] As used herein the term “payload protein” may refer to a functional protein that is synthesized based on the genetic instructions carried by the cargo RNA molecule. The term "payload" may refer to the ultimate product or therapeutic protein that is intended to be produced and delivered to achieve a desired biological or therapeutic effect.

[0088]

[0081] A “disease” within the meaning of the present invention may relate to any disease (e.g., a genetic disease or disorder, e.g., a monogenic disorder, i.e., a single gene disorder resulting from a single mutated gene, or multigenic or polygenic disorder, i.e., a condition resulting from the combined effects of multiple genes), particularly to any disease that may be treated by the deletion of a sequence of interest such as an exon. The disease may relate to a disease that is caused by the deletion of an exon of a gene, which induces a frameshift mutation. This frameshift mutation may lead to a non-functional protein or to a protein that is not expressed, e.g. because of a stop codon induced by the frameshift mutation. Non-limiting examples for genes that may be affected by such a mutation are titin and dystrophin. Both of which are important for the function of muscles. Diseases caused by frameshift mutations of titin or dystrophin may include Duchenne muscular dystrophy, hereditary myopathy with early respiratory failure, early-onset myopathy with fatal cardiomyopathy, core myopathy with heart disease, centronuclear myopathy, limb-girdle muscular dystrophy type 2J, familial dilated cardiomyopathy 9, hypertrophic cardiomyopathy and tibial muscular dystrophy. In addition or alternatively, also proteins characterized by repetitive protein domains such as immunoglobulins may be affected. The disease within the meaning of the invention may further relate to diseases that are caused by the presence of an exon, which is not present in a healthy subject, e.g., a duplication, triplication etc. of an exon. Such a duplication, triplication etc. may be present in the dystrophin gene and / or may also lead to Duchenne muscular dystrophy. In a preferred embodiment, the disease is Duchenne muscular dystrophy (DMD). DMD is a severe type of muscular dystrophy characterized by muscle weakness usually beginning around the age of four in boys and worsens quickly. Typically muscle loss occurs first in the thighs and pelvis followed by those of the arms. Most patients are unable to walk by the age of 12. The disorder is X-linked recessive. About two thirds of cases are inherited from a person's mother, while one third of cases are due to a new mutation. It is caused by a mutation in the dystrophin gene at locus Xp21 , located on the short arm of the X chromosome and is inherited in an X-linked recessive pattern. Dystrophin is responsible for connecting the cytoskeleton of each muscle fiber to the underlying basal lamina (extracellular matrix), through a protein complex containing many subunits. The absence of dystrophin permits excess calcium to penetrate the sarcolemma (the cell membrane). Alterations in calcium and signaling pathways cause water to enter into the mitochondria, which then burst.“Sequence of interest” as used herein may relate to a nucleotide sequence, preferably of the genome of a target cell that is to be excised, i.e. removed, from a gene to restore the reading frame or to repair any other type of mutation that renders the gene non-functional. One example of a gene that is affected by a frameshift leading to a truncated and non-functional protein is dystrophin. Dystrophin is a very large protein comprising many exons. Deletion of one exon may lead to a frameshift mutation. As outlined herein, a prominent example is the deletion of exon 52 of dystrophin, which could be treated by deletion of exon 51 of dystrophin. Accordingly, the sequence of interest may be exon 51 of the dystrophin gene. Preferebly, a deletion of exon 51 of the dystrophin gene may restore the reading frame of the dystrophin gene and thereby enables the translation of a truncated but functional dystrophin.

[0089] ***

[0090]

[0082] Selective pressure is the driver of biological systems towards a local minimum on the evolutionary landscape, enabling them to occupy an ecological niche1-3. Conceptualizing evolutionary trajectories as a sum vector, defined by the combination of various selective pressures, raises the possibility of studying how these trajectories change if certain elements are altered or removed. Such a holistic view could open new perspectives for the repurposing of biological systems in bioengineering. Within this evolutionary framework, similar selection pressure on otherwise unrelated species can lead to homoplasy, the convergence of biological features4,5. Viruses, for instance, are highly optimized vehicles for gene transfer, but despite their diversity, they converged on similar features. Most viruses rely on large supramolecular protein capsids, composed of thousands of subunits, which self-assemble into icosahedral symmetry to enclose and protect their genome6-9. Viral capsids are presumably selected for their resilience in harsh environmental conditions. However, when repurposed as vectors for genetic engineering, they are handled in controlled environments. This raises the question of whether features evolution selected for might become unnecessary or even disadvantageous when placing a biological system in a new context outside of its original ecological niche. Recently developed Al models for protein design could be harnessed to explore this question. These models create protein structures that are physically feasible but do not occur naturally10-14, enabling the manipulation of the evolutionary vector with non-natural protein architectures that are free of the constraints of natural proteins.

[0091]

[0083] Here, we exemplify this idea by constructing bottom-up RNA transfer vehicles consisting of natural protein domains, combined with Al-designed synthetic protein assemblies. These Synthetic Transfer Vehicles (STVs) are distinct from any known natural RNA transfer vehicle by exhibiting unique characteristics, such as non-icosahedral symmetry, open structures, and low complexity of the assembled protein. We develop a multi-dimensional screening system that enables the testing of hundreds of designs and identify an exemplary STV-C8 construct, which is built from an unusual planar symmetry, as the most efficient structure for RNA delivery. We characterize the shape, content, and packaging capacity of exemplary STV-C8 and program its tropism by combining it with computationally designed peptide binders. Regardless of its distinct structure, STV-C8 is several orders of magnitude more efficient in RNA transfer compared to natural counterparts and clinically used lipid nanoparticles. We demonstrate the versatility of STV- C8 by delivering various cargo RNAs, such as reporter RNAs, gene editors, programmable antivirals, and transcription factors, into a wide variety of cellular models from multiple species. We perform a comprehensive in vivo biodistribution analysis of STV-C8 at near single-cell resolution in a mouse model. Additionally, we deliver the CRISPR / Cas9 system into the muscle of a pig to delete exon 51 of the dystrophin gene as a treatment approach for Duchenne muscular dystrophy (DMD), emphasizing the clinical potential of STVs (e.g., STV-C8, SEQ ID NO: 102) of the present invention.

[0092]

[0084] In some aspects, the present invention relates to a method of identifying whether an interaction domain capable of nucleating self-assembly of a plurality of fusion proteins comprising said interaction domain and capsid-forming proteins into synthetic transfer vehicles (STVs) provides for RNA transfer efficiency of said STVs from a sender to a receiver cell, comprising: (A) providing a mammalian sender cell; (B) providing a mammalian receiver cell; (C) bringing supernatant from said sender cell comprising the population of STVs into contact with said receiver cell, thereby allowing fusion of said population of STVs with the cell membrane of said reporter cell and delivery of first polynucleotides into the reporter cell; and (D) determining the signal obtained from the reporter cell, said signal being produced by the functional reporter system through protein-fragment complementation of the first and second member of said first split reporter system, said protein-fragment complementation being mediated by protein splicing through a functional intein forming by non-covalent self-association of the first and second entity of the split intein, said non-covalent self-association being mediated by the interaction of the protein-protein interaction domains encoded by the first and fourth polynucleotide, respectively; thereby identifying an interaction domain which provides for RNA transfer efficiency of STVs from a sender to a receiver.

[0093]

[0085] In some further aspects, the present invention further relates to a method of identifying whether an interaction domain capable of nucleating self-assembly of a plurality of fusion proteins comprising said interaction domain and capsid-forming proteins into synthetic transfer vehicles (STVs) provides for RNA transfer efficiency of said STVs from a sender to a receiver cell, comprising:

[0086] (A) providing a mammalian sender cell comprising:

[0094]

[0087] (i) a first polynucleotide encoding the first member of a first split reporter system providing for a detectable signal, the first entity of a split intein, a protein-protein interaction domain and a non-translated RNA packaging signal being bound by the RNA-binding domain of the fusion protein encoded by the second polynucleotide,

[0095]

[0088] (ii) a second polynucleotide encoding a fusion protein comprising (a) a membranebinding domain, and / or (b) a budding domain, (c) an RNA-binding domain being capable of binding the RNA packaging signal encoded by the first polynucleotide of and (d) an interaction domain capable of nucleating self-assembly of a plurality of said fusion protein into STVs secreted from said sender cell in which said fusion proteins are expressed, thereby generating a population of STVs comprising the fusogen encoded by the third polynucleotide and further comprising first polynucleotides bound through their RNA packaging signal by the RNA-binding domain of said fusion protein, and

[0096]

[0089] (iii) a third polynucleotide encoding a fusogenic protein,

[0097]

[0090] (B) providing a mammalian receiver cell comprising:

[0098]

[0091] (iv) a fourth polynucleotide encoding the second member of said first split reporter system being capable of complementing the first member of said split reporter system to form a functional first reporter system, the second entity of said split intein being capable of selfassociation with the first entity of said split intein to form a functional intein and a protein-protein interaction domain being capable of interaction with the protein-protein interaction domain encoded by the first polynucleotide;

[0099]

[0092] (C) bringing supernatant from said sender cell comprising the population of STVs into contact with said receiver cell, thereby allowing fusion of said population of STVs with the cell membrane of said reporter cell and delivery of first polynucleotides into the reporter cell; and

[0100]

[0093] (D) determining the signal obtained from the reporter cell, said signal being produced by the functional reporter system through protein-fragment complementation of the first and second member of said first split reporter system, said protein-fragment complementation being mediated by protein splicing through a functional intein forming by self-association of the first and second entity of the split intein, said self-association being mediated by the interaction of the protein-protein interaction domains encoded by the first and fourth polynucleotide, respectively,

[0101]

[0094] thereby identifying an interaction domain which provides (e.g., a readout) for RNA transfer efficiency of STVs from a sender to a receiver.

[0102]

[0095] In some further aspects, the present invention further relates to the method of the present invention, further comprising: (E) comparing said signal obtained in (D) with the signal obtained for a STV comprising self-assembled polypeptides forming naturally-occurring viral architectures, preferably an icosahedral symmetry, preferably a STV as defined in claim 1 (A)(ii), wherein the interaction domain comprising the amino acid sequence shown in SEQ ID NO: 1 , wherein, if the signal obtained in (D) is higher than the signal obtained for a STV comprising self-assembled polypeptides forming naturally-occurring viral architectures, preferably an icosahedral symmetry, preferably having the amino acid sequence shown in SEQ ID NO: 1 , such signal is indicative of a STV with high RNA delivery to receiver cells.

[0103]

[0096] In some further aspects, the present invention further relates to the method of the present invention, wherein said membrane-binding domain has an amino acid sequence which is at least 70% identical to the amino acid sequence shown in SEQ ID NO: 2.

[0104]

[0097] In some further aspects, the present invention further relates to the method of the present invention, wherein said RNA-binding domain has an amino acid sequence which is at least 70% identical to the amino acid sequences shown in SEQ ID NO: 4.

[0105]

[0098] In some further aspects, the present invention further relates to the method of the present invention, wherein said interaction domain an amino acid sequence which is at least 70% identical to any one of the amino acid sequences shown in: SEQ ID NO: 5 (HE0690); SEQ ID NO: 6 (HE0490); SEQ ID NO: 7 (HE0499); and / or SEQ ID NO: 8 (HE0505).

[0106]

[0099] In some further aspects, the present invention further relates to a polynucleotide comprising a nucleotide sequence encoding the interaction domain of the present invention.

[0107]

[0100] In some further aspects, the present invention further relates to a fusion protein comprising a membrane-binding domain, a budding domain, an RNA-binding domain and the interaction domain of the present invention.

[0108]

[0101] In some further aspects, the present invention further relates to a fusion protein of the present invention, wherein said membrane-binding domain has an amino acid sequence which is at least 70% identical to any one of the amino acid sequence shown in the amino acid sequence shown in SEQ ID NO: 2.

[0109]

[0102] In some further aspects, the present invention further relates to a fusion protein of the present invention, wherein said budding domain has an amino acid sequence which is at least 70% identical to any one of the amino acid sequence shown in the amino acid sequence shown in SEQ ID NO: 3.

[0110]

[0103] In some further aspects, the present invention further relates to a fusion protein of the present invention, wherein said RNA-binding domain has an amino acid sequence which is at least 70% identical to any one of the amino acid sequence shown in the amino acid sequence shown in SEQ ID NO: 4.

[0111]

[0104] In some further aspects, the present invention further relates to a fusion protein of the present invention, wherein said interaction domain an amino acid sequence which is at least 70% identical to any one of the amino acid sequences shown in: SEQ ID NO: 5 (HE0690); SEQ ID NO: 6 (HE0490); SEQ ID NO: 7 (HE0499); and / or SEQ ID NO: 8 (HE0505).

[0112]

[0105] In some further aspects, the present invention further relates to a fusion protein of the present invention, wherein said fusion protein comprising a membrane-binding domain having the amino acid sequence shown in SEQ ID NO: 2, a budding domain having the amino acid sequence shown in SEQ ID NO: 3, an RNA-binding domain having the amino acid sequence shown in SEQ ID NO: 4 and an interaction domain having the amino acid sequence shown in SEQ ID NO: 5 (HE0690); SEQ ID NO: 6 (HE0490); SEQ ID NO: 7 (HE0499); and / or SEQ ID NO: 8 (HE0505).

[0113]

[0106] In some further aspects, the present invention further relates to a polynucleotide comprising a nucleotide sequence encoding a fusion protein of the present invention.

[0114]

[0107] In some further aspects, the present invention further relates to a non-naturally occurring interaction domain obtainable by the method of the present invention.

[0115]

[0108] In some further aspects, the present invention further relates to a fusion protein comprising a membrane-binding domain, a budding domain, an RNA-binding domain and the interaction domain of the present invention.

[0116]

[0109] In some further aspects, the present invention further relates to a composition, comprising: (i) a polynucleotide encoding a fusion protein comprising: (a) a membrane-binding domain, (b) a budding domain, (c) an RNA-binding domain being capable of binding an RNA packaging signal and (d) the interaction domain of the present invention.

[0117]

[0110] In some aspects, the present invention (e.g., STVs of the present invention) relates to delivering / using CRISPR / Cas and gRNA, preferably for exon skipping of defective exons, further preferably in therapy of the Duchenne muscular dystrophy (DMD) (e.g., as described in WO 2021 / 064162 A1 , which is incorporated herein in its entirety), most preferably the STVs of the present invention are used as carriers for / of the split-CRISPR / Cas system as described in WO 2021 / 064162 A1.

[0118]

[0111] In some further aspects, the present invention makes use of STVs of the present invention for delivering genome-targeting nucleic acids that can direct the activities of an associated endonuclease to a specific target sequence within a target nucleic acid. The genome-targeting nucleic acid can be RNA. A genome-targeting RNA is referred to as a "guide RNA" or "gRNA" herein. A guide RNA can comprise at least a spacer sequence that hybridizes to a target nucleic acid sequence of interest, and a CRISPR repeat sequence. In Type II systems, the gRNA also comprises a second RNA called the tracrRNA sequence. In the Type II guide RNA (gRNA), the CRISPR repeat sequence and tracrRNA sequence hybridize to each other to form a duplex. In the Type V guide RNA (gRNA), the crRNA forms a duplex. In both systems, the duplex can bind a site-directed polypeptide, such that the guide RNA and site-direct polypeptide form a complex. The genome-targeting nucleic acid can provide target specificity to the complex by virtue of its association with the site-directed polypeptide. The genome-targeting nucleic acid thus can direct the activity of the site-directed polypeptide. The functionality of a genome-targeting nucleic acid can be tested by analyzing the DNA that should have been modified. If the desired modification is present, the genome-targeting nucleic acid(s) target the endonuclease to the correct position and excises the correct sequence of interest from the genome. Suitable methods include, but are not limited to, Mismatch cleavage assay, Sequence trace decomposition analysis, Indel Detection by Amplicon Analysis (IDAA), Digital PCR, Immunofluorescence analysis or Clonal analysis. Deletion of exon 51 of the dystrophin gene may restore the reading frame of the dystrophin gene and thereby enables the translation of a truncated but functional dystrophin.

[0119]

[0112] The present invention is summarized by the following items, which are the preferred embodiments of the invention:

[0120] 1 . A method of identifying whether an interaction domain capable of nucleating self-assembly of a plurality of fusion proteins comprising said interaction domain and capsid-forming proteins into synthetic transfer vehicles (STVs) provides for RNA transfer efficiency of said STVs from a sender to a receiver cell, comprising:

[0121] (A) providing a mammalian sender cell comprising:

[0122] (i) a first polynucleotide encoding the first member of a first split reporter system providing for a detectable signal, the first entity of a split intein, a protein-protein interaction domain and a non-translated RNA packaging signal being bound by the RNA-binding domain of the fusion protein encoded by the second polynucleotide,

[0123] (ii) a second polynucleotide encoding a fusion protein comprising (a) a membrane-binding domain, and / or (b) a budding domain, (c) an RNA- binding domain being capable of binding the RNA packaging signal encoded by the first polynucleotide of and (d) an interaction domain capable of nucleating self-assembly of a plurality of said fusion protein into STVs secreted from said sender cell in which said fusion proteins are expressed, thereby generating a population of STVs comprising the fusogen encoded by the third polynucleotide and further comprising first polynucleotides bound through their RNA packaging signal by the RNA-binding domain of said fusion protein, and

[0124] (iii) a third polynucleotide encoding a fusogenic protein,

[0125] (B) providing a mammalian receiver cell comprising:

[0126] (iv) a fourth polynucleotide encoding the second member of said first split reporter system being capable of complementing the first member of said split reporter system to form a functional first reporter system (e.g., which can optionally comprise one or more further entities), the second entity of said split intein being capable of (e.g., a non-covalent or covalent) selfassociation with the first entity of said split intein to form a functional intein and a protein-protein interaction domain being capable of interaction with the protein-protein interaction domain encoded by the first polynucleotide; (C) bringing supernatant from said sender cell comprising the population of STVs into contact with said receiver cell, thereby allowing fusion of said population of STVs with the cell membrane of said reporter cell (e.g., that can also be referred to as receiver cell) and delivery of first polynucleotides into the reporter cell; and

[0127] (D) determining the signal obtained from the reporter cell, said signal being produced by the functional reporter system through protein-fragment complementation of the first and second member of said first split reporter system, said protein-fragment complementation being mediated by protein splicing through a functional intein forming by (e.g., a non-covalent or covalent) self-association of the first and second entity of the split intein, said (e.g., a non-covalent or covalent) self-association being mediated by the interaction of the protein-protein interaction domains encoded by the first and fourth polynucleotide, respectively, thereby identifying an interaction domain which provides for RNA transfer efficiency of STVs from a sender to a receiver, optionally selecting said interaction domain which provides for RNA transfer efficiency of STVs from a sender to a receiver. The method of any one of the preceding items, further comprising:

[0128] (E) comparing said signal obtained in (D) with the signal obtained for a STV comprising self-assembled polypeptides forming naturally-occurring viral architectures, preferably an icosahedral symmetry, preferably a STV as defined in item 1 (A)(ii), wherein the interaction domain comprises the amino acid sequence shown in SEQ ID NO: 1 (I 301 / EPN24, e.g., which does not comprise a RNA-binding protein)

[0129] (MHGLQDDPDLQALLKGSQLLKVKSSSWRRERFYKLQEDCKTIWQESRKVMRS PESQLFSIEDIQEVRMGHRTEGLEKFARDIPEDRCFSIVFKDQRNTLDLIAPSPA DAQHWVQGLRKIIHHSGSMDQRQKLQSRPEPTAPPEESFRSGVETTTPPQKQ EPIDKELYPLTSLRSLFGNDPSSQKIEELFKKHKIVAVLRANSVEEAKKKALAVFL GGVHLIEITFTVPDADTVIKELSFLKEMGAIIGAGTVTSVEQCRKAVESGAEFIVS PHLDEEISQFCKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKA MKGPFPNVKFVPTGGVNLDNVCEWFKAGVLAVGVGSALVKGTPVEVAEKAKA FVEKIRGCTEQKLISEEDL), wherein, if the signal obtained in (D) is higher than the signal obtained for a STV comprising self-assembled polypeptides forming naturally-occurring viral architectures, preferably an icosahedral symmetry, preferably having the amino acid sequence shown in SEQ ID NO: 1 (I 301 / EPN24), such signal is indicative of a STV with high RNA delivery to receiver cells. The method of item 2, wherein the interaction domain having the amino acid sequence shown in SEQ ID NO: 1 (I 301 / EPN24), further comprises a RNA-binding protein, e.g., comprising SEQ ID NO: 20

[0130] (ASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQKRK YTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNP IPSAIAANSGIY), e.g., as shown in Horns et al. 2023 (Ref. 24 herein). The method of any one of the preceding items, wherein said fusion protein encoded by said second polynucleotide further encodes a first member of a second split reporter system providing for a detectable signal which is different to the signal provided by first split reporter system. The method of any one of the preceding items, wherein said fourth polynucleotide further encodes a second member of the second split reporter system being capable of complementing the first member of the second split reporter system to form a functional second reporter system. The method of any one of the preceding items, wherein said fusogenic protein is derived from a SNARE protein (Soluble N-ethylmaleimide-sensitive factor Attachment protein Receptor), dynamin, an FF protein, a FAST protein (Fusion-Associated Small Transmembrane proteins), or a viral fusogenic glycoprotein. The method of any one of the preceding items, wherein said viral fusogenic glycoprotein is a glycoprotein from retroviridae, herpesviridae, poxviridae, hepadnaviridae, flaviviridae, togavoridae, coronaviridae, hepatitis D virus, orthomyxoviridae, paramyxoviridae, filoviridae, rhabdoviridae, bunyaviridae, or orthopoxivridae. The method of any one of the preceding items, wherein said viral fusogenic glycoprotein is HBsAg of HBV, E1 or E2 protein of HCV, hemagglutinin (HA) or neuraminidase (NA) of Influenza, glycoprotein G of VSV, glycoprotein GP of Ebola or Marburg virus, glycoproteins Gp120 or Gp41 of lentiviruses, envelop protein (DENV E) or pre-membrane protein (prM DENV) of Dengue virus, envelope glycoproteins of Hantaan virus, glycoprotein E2 of Chikungunya virus, glycoproteins HN and F of Newcastle virus, gp85 and gp37 of Rous sarcoma virus, protein E aor prM of Murray Valley encephalitis virus, or HERV protein or reactivated HERV-protein from human endogenous retroviruses. The method of any one of the preceding items, wherein said fusogenic protein is engineered to co-display a targeting molecule; [e.g., as in Lei et al., J. Biol. Eng. 3, 8 (2009), doi.org / 10.1186 / 1754-1611-3-8], The method of any one of the preceding items, wherein said targeting molecule is an antibody. The method of any one of the preceding items, wherein the first split reporter system after protein-fragment complementation of said first and second member provides for determining the efficiency of RNA delivery to receiver cells. The method of any one of the preceding items, wherein the first split reporter system after protein-fragment complementation of said first and second member provides for a fluorescent signal or bioluminescent signal. The method of any one of the preceding items, wherein the first split reporter system is a split-beta-galactosidase system, split-beta-lactamase system, split-fluorescent protein system, or split-luciferase system. The method of any one of the preceding items, wherein the first member of said second split reporter system alone provides for determining the efficiency of STV release from sender cells. The method of any one of the preceding items, wherein the second split reporter system after protein-fragment complementation of said first and second member provides for determining the efficiency of STV uptake by receiver cells. The method of any one of the preceding items, wherein the second split reporter system after protein-fragment complementation of said first and second member provides for a fluorescent signal or bioluminescent signal. The method of any one of the preceding items, wherein the second split reporter system is a split-beta-galactosidase system, split-beta-lactamase system, split-fluorescent protein system, or split-luciferase system. The method of any one of the preceding items, wherein said split-intein system encoded by the first and fourth polynucleotide is gp41-1 (N2 / C2), gp41-8 (N2 / C2), NrdJ-1 (N2 / C2), IMPDH-1 (N2 / C2), SspGyrB (N2 / C2), TvoVMA (N2 / C2), PhoRadA (N1 / C1), PhoRadA (N2 / C2), PhoRadA (N3 / C3), Cth-Ter (N2 / C2), MP-M-DnaB (N2 / C2), NrdA (n2 / C2); e.g., as in [Pinto et al. Nat Commun 11 , 1529 (2020), doi.org / 10.1038 / s41467-020-15272-2], The method of any one of the preceding items, wherein said protein-protein interaction domain encoded by the first and fourth polynucleotide is a peptide of a pair of coiled-coil dimer-forming peptides or selected from the group consisting of: scFab, ScFv, nanobodies, DARPINs or Anticaline. The method of any one of the preceding items, wherein said pair of coiled-coil dimerforming peptide is the N5-N6 pair, or N7-N8 pair; e.g., as in [Plaper et al. Sci Rep 11 , 9136 (2021 ) , doi . org / 10.1038 / s41598-021 -88315-3] . The method of any one of the preceding items, wherein said RNA packaging signal encoded by the first polynucleotide comprises a non-translated hairpin structure.

[0131] Z4 The method of any one of the preceding items, wherein said hairpin structure is the hairpin structure bound by the RNA-binding domain of the bacteriophage PP7 coat protein. The method of any one of the preceding items, wherein said membrane-binding domain of the fusion protein encoded by the second polynucleotide is capable of fusing with the inner part of the cell membrane of a mammalian cell. The method of any one of the preceding items, wherein said membrane-binding domain has or comprises an amino acid sequence which is at least 70% (e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, preferably at least 80%, 85%, 90%, 95% or 99%, preferably at least 80%) identical to the amino acid sequence shown in SEQ ID NO: 2

[0132] (GLQDDEDLQVLLKGSQLLKVKSNSWRRERFYKLQEDCKTIWQESRKVMRTPESHLFS IEDIQEVRKGHRTEGMEKFARDVPEDRCFSIVFKDQRNTLDLISPSPAEAQHWVRGLHK IIH). The method of any one of the preceding items, wherein said budding domain of the fusion protein encoded by the second polynucleotide is capable of recruiting proteins of the endosomal sorting complexes required for transport (ESCRT) pathway. The method of any one of the preceding items, wherein said budding domain has or comprises an amino acid sequence which is at least 70% (e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, preferably at least 80%, 85%, 90%, 95% or 99%, preferably at least 80%) identical to the amino acid sequences shown in SEQ ID NO: 3

[0133] (PTAPPGSGSPTAPPEYSGSGPSAPPMEEKLLDLGGSGPYKEGSGSPPPPYVGSGLYP SLSGGFPIVSGGPLPPVGSGSYLDL). The method of any one of the preceding items, wherein said RNA-binding domain of the fusion protein encoded by the second polynucleotide is capable of binding a RNA packaging signal. The method of any one of the preceding items, wherein said RNA-binding domain has or comprises an amino acid sequence which is at least 70% (e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, preferably at least 80%, 85%, 90%, 95% or 99%, preferably at least 80%) identical to the amino acid sequences shown in SEQ ID NO: 4

[0134] (SKTIVLSVGEATRTLTEIQSTADRQIFEEKVGPLVGRLRLTASLRQNGAKTAYRVNLKLD QADVVDSGLPKVRYTQVWSHDVTIVANSTEASRKSLYDLTKSLVATSQVEDLVVNLVPL GR). The method of any one of the preceding items, wherein said interaction domain of the fusion protein encoded by the second polynucleotide is capable of nucleating selfassembly. The method of any one of the preceding items, wherein said interaction domain has or comprises an amino acid sequence which is at least 70% (e.g., at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, preferably at least 80%, 85%, 90%, 95% or 99%, preferably at least 80%) identical to any one of the amino acid sequences shown in:

[0135] SEQ ID NO: 5 (HE0690)

[0136] (LKVKIKVKDNPAVARGVLRLADKLKKAGVDVEIEIDLYGDEEQALATLAAMEAEVEELA);

[0137] SEQ ID NO: 6 (HE0490)

[0138] (ELKKRWRYVATRLFTEILSLEPLIGRETALELLLETARILYKASGELELILEVAREEMRRA GVPEEDIEALLAELRAWA);

[0139] SEQ ID NO: 7 (HE0499)

[0140] (KLLEVAVLKAIAELLGLAILDPRAIPLAREALEKLRKIHPSEIIKEMCDMGERILELIEE); and / or

[0141] SEQ ID NO: 8 (HE0505)

[0142] (DLLKEAEELVKKILETDPEANPAALNLYTILKTYVDIGAEKQAKKILELLKWAEHLEKK). An non-naturally occurring interaction domain obtainable (e.g., obtained) by the method of any one of the preceding items. The interaction domain of any one of the preceding items, wherein said interaction domain self-assembles into an assembly having non-icosahedral symmetry; e.g., as in [https: / / neurosnap.ai / blog / post / 65b4310a66fbcdc5d780129e], The interaction domain of any one of the preceding items, wherein said interaction domain self-assembles into an assembly having cyclic symmetry, dihedral symmetry, cubic symmetry, helical symmetry. The interaction domain of any one of the preceding items, wherein cubic symmetry is tetrahedral symmetry or octahedral symmetry. The interaction domain of any one of the preceding items, wherein the order of cyclic symmetry is Cn, with n being > 2 (e.g., C2, C3 or C4). The interaction domain of any one of the preceding items, wherein the order of dihedral symmetry is Dn, with n being > 2 (e.g., D2 or D3). The interaction domain of any one of the preceding items, wherein said interaction domain self-assembles into a planar structure and / or an open structures, and / or has low complexity. The interaction domain of any one of the preceding items, wherein said interaction domain has or comprises an amino acid sequence which is at least 70% (e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, preferably at least 80%, 85%, 90%, 95% or 99%, preferably at least 80%) identical to any one of the amino acid sequence shown in the amino acid sequence shown in:

[0143] SEQ ID NO: 5 (HE0690)

[0144] (LKVKIKVKDNPAVARGVLRLADKLKKAGVDVEIEIDLYGDEEQALATLAAMEAEVEELA);

[0145] SEQ ID NO: 6 (HE0490)

[0146] (ELKKRWRYVATRLFTEILSLEPLIGRETALELLLETARILYKASGELELILEVAREEMRRA GVPEEDIEALLAELRAWA);

[0147] SEQ ID NO: 7 (HE0499)

[0148] (KLLEVAVLKAIAELLGLAILDPRAIPLAREALEKLRKIHPSEIIKEMCDMGERILELIEE); and / or

[0149] SEQ ID NO: 8 (HE0505)

[0150] (DLLKEAEELVKKILETDPEANPAALNLYTILKTYVDIGAEKQAKKILELLKVVAEHLEKK). A polynucleotide comprising a nucleotide sequence encoding the interaction domain of any one of the preceding items. A fusion protein comprising a membrane-binding domain, a budding domain, an RNA- binding domain and the interaction domain of any one of the preceding items. The fusion protein of any one of the preceding items, wherein said membrane-binding domain has or comprises an amino acid sequence which is at least 70% (e.g., at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, preferably at least 80%, 85%, 90%, 95% or 99%, preferably at least 80%) identical to the amino acid sequence shown in SEQ ID NO: 2

[0151] (GLQDDEDLQVLLKGSQLLKVKSNSWRRERFYKLQEDCKTIWQESRKVMRTPESHLFS IEDIQEVRKGHRTEGMEKFARDVPEDRCFSIVFKDQRNTLDLISPSPAEAQHWVRGLHK IIH). The fusion protein of any one of the preceding items, wherein said budding domain has or comprises an amino acid sequence which is at least 70% (e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, preferably at least 80%, 85%, 90%, 95% or 99%, preferably at least 80%) identical to any one of the amino acid sequence shown in the amino acid sequence shown in SEQ ID NO: 3

[0152] (PTAPPGSGSPTAPPEYSGSGPSAPPMEEKLLDLGGSGPYKEGSGSPPPPYVGSGLYP SLSGGFPIVSGGPLPPVGSGSYLDL). The fusion protein of any one of the preceding items, wherein said RNA-binding domain has or comprises an amino acid sequence which is at least 70% (e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, preferably at least 80%, 85%, 90%, 95% or 99%, preferably at least 80%) identical to the amino acid sequence shown in SEQ ID NO: 4

[0153] (SKTIVLSVGEATRTLTEIQSTADRQIFEEKVGPLVGRLRLTASLRQNGAKTAYRVNLKLD QADVVDSGLPKVRYTQVWSHDVTIVANSTEASRKSLYDLTKSLVATSQVEDLVVNLVPL GR). The fusion protein of any one of the preceding items, wherein said interaction domain has or comprises an amino acid sequence which is at least 70% (e.g., at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, preferably at least 80%, 85%, 90%, 95% or 99%, preferably at least 80%) identical to any one of the amino acid sequence shown in:

[0154] SEQ ID NO: 5 (HE0690) (LKVKIKVKDNPAVARGVLRLADKLKKAGVDVEIEIDLYGDEEQALATLAAMEAEVEELA);

[0155] SEQ ID NO: 6 (HE0490)

[0156] (ELKKRWRYVATRLFTEILSLEPLIGRETALELLLETARILYKASGELELILEVAREEMRRA GVPEEDIEALLAELRAWA);

[0157] SEQ ID NO: 7 (HE0499)

[0158] (KLLEVAVLKAIAELLGLAILDPRAIPLAREALEKLRKIHPSEIIKEMCDMGERILELIEE); and / or

[0159] SEQ ID NO: 8 (HE0505)

[0160] (DLLKEAEELVKKILETDPEANPAALNLYTILKTYVDIGAEKQAKKILELLKVVAEHLEKK). The fusion protein, preferably of any one of the preceding items, comprising a membranebinding domain having the amino acid sequence shown in SEQ ID NO: 2, a budding domain having the amino acid sequence shown in SEQ ID NO: 3, an RNA-binding domain having the amino acid sequence shown in SEQ ID NO: 4 and an interaction domain having the amino acid sequence shown in SEQ ID NO: 5 (HE0690)

[0161] (LKVKIKVKDNPAVARGVLRLADKLKKAGVDVEIEIDLYGDEEQALATLAAMEAEVEELA);

[0162] SEQ ID NO: 6 (HE0490)

[0163] (ELKKRVVRYVATRLFTEILSLEPLIGRETALELLLETARILYKASGELELILEVAREEMRRA GVPEEDIEALLAELRAWA);

[0164] SEQ ID NO: 7 (HE0499)

[0165] (KLLEVAVLKAIAELLGLAILDPRAIPLAREALEKLRKIHPSEIIKEMCDMGERILELIEE); and / or

[0166] SEQ ID NO: 8 (HE0505)

[0167] (DLLKEAEELVKKILETDPEANPAALNLYTILKTYVDIGAEKQAKKILELLKVVAEHLEKK). A polynucleotide comprising a nucleotide sequence encoding the fusion protein of any one of the preceding items. A composition, comprising:

[0168] (i) a polynucleotide encoding a fusion protein, preferably of any one of the preceding items, comprising: (a) a membrane-binding domain, preferably of any one of the preceding items, (b) a budding domain, preferably of any one of the preceding items, (c) an RNA-binding domain being capable of binding an RNA packaging signal, preferably of any one of the preceding items; and (d) the interaction domain of any one of the preceding items, preferably of item 28 or 29. The composition of any one of the preceding items, comprising:

[0169] (i) a polynucleotide encoding a fusion protein of any one of the preceding items, comprising: (a) a membrane-binding domain of any one of the preceding items, (b) a budding domain of any one of the preceding items, (c) an RNA-binding domain being capable of binding an RNA packaging signal of any one of the preceding items; and (d) the interaction domain of any one of the preceding items, preferably of item 28 or 29. The composition of any one of the preceding items, further comprising:

[0170] (ii) a polynucleotide encoding a cargo RNA molecule comprising a packaging signal. The composition of any one of the preceding items, further comprising:

[0171] (iii) a third polynucleotide encoding a fusogenic protein. The composition of any one of the preceding items, comprising or further comprising: one or more CRISPR / Cas and one or more gRNAs, preferably for gene therapy (e.g., exon skipping of defective exon / s), e.g., in the Duchenne muscular dystrophy (DMD) gene (e.g., as described in WO 2021 / 064162 A1 , which is incorporated herein in its entirety), preferably said composition (e.g., STV-C8, SEQ ID NO: 102) is a carrier for / of a split- CRISPR / Cas system components / elements. The composition of any one of the preceding items, comprising or further comprising:

[0172] (a’) a first vector comprising a nucleic acid sequence encoding:

[0173] (i’) a first fragment of an endonuclease,

[0174] (ii’) a first fragment of an intein, and

[0175] (iii’) a first guide RNA (gRNA); and

[0176] (b) a second vector comprising a nucleic acid sequence encoding:

[0177] (i”) a second fragment of the endonuclease,

[0178] (ii”) a second fragment of the intein, and

[0179] (iii”) a second guide RNA (gRNA); wherein the first gRNA binds to a region, which is located 5’ to a sequence of interest comprised in a nucleic acid sequence in the genome, preferably DNA, of a target cell, wherein the second gRNA binds to a region located 3’ to the sequence of interest comprised in the nucleic acid sequence in the genome, preferably DNA, of a target cell; wherein the first fragment and the second fragment of the intein are capable of associating into a functional intein, wherein the functional intein is capable of ligating the first and the second fragment of the endonuclease to form a functional endonuclease; wherein the functional endonuclease is capable of excising the sequence of interest. The composition of any one of the preceding items, wherein the endonuclease is Cas9, preferably Streptococcus pyogenes Cas9 (SpCas9), The composition of any one of the preceding items, further comprising a population of sender cells. The composition of any one of the preceding items, wherein said cargo RNA molecule encodes a payload protein. The composition of any one of the preceding items, wherein said payload protein is capable of modulating the expression, concentration, localization, stability, and / or activity of the one or more endogenous proteins of a receiver cell. The composition of any one of the preceding items, wherein the payload protein is a therapeutic protein, preferably a therapeutic protein configured to prevent or treat a disease of a subject (e.g., Duchenne muscular dystrophy), preferably the disease is selected from the group consisting of Duchenne muscular dystrophy, hereditary myopathy with early respiratory failure, early-onset myopathy with fatal cardiomyopathy, core myopathy with heart disease, centronuclear myopathy, limb-girdle muscular dystrophy type 2J, familial dilated cardiomyopathy 9, hypertrophic cardiomyopathy and tibial muscular dystrophy, preferably Duchenne muscular dystrophy, further preferably the disease is a muscular disease or Duchenne muscular dystrophy, optionally characterized by a deletion of exon 52 of the dystrophin gene. The composition of any one of the preceding items, which is a pharmaceutical composition and / or diagnostic composition (e.g., for treatment and / or prevention of a genetic disorder, e.g., Duchenne muscular dystrophy), preferably for treatment and / or prevention of Duchenne muscular dystrophy. The composition of any one of the preceding items, comprising STV-C8 (SEQ ID NO: 102) as defined herein (e.g., as in Example 1 and / or Example 2 herein). The composition of any one of the preceding items for use as a medicament and / or for use in a therapy (preferably human therapy) and / or for use in the manufacture of a medicament and / or pharmaceutical composition (e.g., for treatment of Duchenne muscular dystrophy), preferably for treatment of Duchenne muscular dystrophy. The method, interaction domain, fusion protein, polynucleotide and / or composition of any one of the preceding items, wherein said SEQ ID NOs: 2-8 are used as one or more tandem / s repeats and / or one or more douplicates. The method, interaction domain, fusion protein, polynucleotide and / or composition of any one of the preceding items, wherein said self-assembly domains are selected from the group consisting of: HE0490 (e.g., having D3 symmetry), HE0499 (e.g., having D3 symmetry), HE0505 (e.g., having D3 symmetry), and HE0690 (e.g., having C8 symmetry). The method of any one of the preceding items, wherein said method is an in vitro or ex vivo method. The method of any one of the preceding items, wherein said cell (e.g., sender and / or receiver cell) is an isolated and / or recombinant cell. The method, interaction domain, fusion protein, polynucleotide and / or composition of any one of the preceding items, wherein said intein / s (e.g., with extein / s) enable protein splicing similar to RNA splicing (intron / exon), preferably in therapy of Duchenne muscular dystrophy (DMD). The method, interaction domain, fusion protein, polynucleotide and / or composition of any one of the preceding items, carried out as shown in Example 1 (e.g., comprising or consisting of STV-C8 (SEQ ID NO: 102)) and / or Example 2 herein (e.g., as shown in one or more of Figure 1-14, 15-16 and / or in Table 1 herein). The method, interaction domain, fusion protein, polynucleotide and / or composition of any one of the preceding items, comprising (or consisting of) and / or encoding for one or more sequences selected from Table 1 herein, preferably comprising one or more sequences selected from the group consisting of: SEQ ID NOs: 29-67. The method, interaction domain, fusion protein, polynucleotide and / or composition of any one of the preceding items, comprising (or consisting of) and / or encoding for one or more sequences selected from the group consisting of: SEQ ID NOs: 102-103, preferably SEQ ID NO: 102 (STV-C8). The method, interaction domain, fusion protein, polynucleotide and / or composition of any one of the preceding items, wherein said split-reporter system means or methods are replaced by a qPCR and / or sequencing means or methods of the supernatant from the producer cell. Use of the interaction domain, fusion protein, polynucleotide and / or composition of any one of the preceding items fordeliverying RNA to / into a cell / s (e.g., in therapy of Duchenne muscular dystrophy (DMD)), preferably said use is in vitro, ex vivo or in vivo use, further preferably in vitro or ex vivo use.

[0113] It is noted that as used herein, the singular forms “a”, “an”, and “the”, include plural references unless the context clearly indicates otherwise. Thus, for example, reference to “a reagent” includes one or more of such different reagents and reference to “the method” includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.

[0180]

[0114] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0181]

[0115] The term "and / or" wherever used herein includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term".

[0182]

[0116] The term “less than” or in turn “more than” does not include the concrete number.

[0183]

[0117] For example, less than 20 means less than the number indicated. Similarly, more than or greater than means more than or greater than the indicated number, e.g., more than 80 % means more than or greater than the indicated number of 80 %.

[0184]

[0118] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. When used herein the term “comprising” can be substituted with the term “containing” or “including” or sometimes when used herein with the term “having”. When used herein “consisting of' excludes any element, step, or ingredient not specified.

[0185]

[0119] The term “including” means “including but not limited to”. “Including” and “including but not limited to” are used interchangeably.

[0186]

[0120] It should be understood that this invention is not limited to the particular methodology, protocols, material, reagents, and substances, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0187]

[0121] All publications cited throughout the text of this specification (including all patents, patent application, scientific publications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.

[0188]

[0122] The content of all documents and patent documents cited herein is incorporated by reference in their entirety.

[0189] EXAMPLES OF THE INVENTION

[0190]

[0123] An even better understanding of the present invention and of its advantages will be evident from the following examples, offered for illustrative purposes only. The examples are not intended to limit the scope of the present invention in any way.

[0191]

[0124] Example 1 : Creating bottom-up RNA transfer vehicles from synthetic protein assemblies

[0192]

[0125] Methods

[0193]

[0126] Molecular cloning

[0194]

[0127] All clonings were performed using standard molecular techniques. Fragments for cloning were generated by PCR using Platinum SuperFi II Master Mix (Thermo Fisher) and appropriate oligonucleotides (IDT DNA), by plasmid digests using standard restriction enzymes (NEB), or synthesized as Gene Fragments (TWIST Biosciences or IDT DNA). Fragment assemblies were performed using the NEBuilder HiFi DNA Assembly Mix (NEB) or Instant Sticky-end Ligase Master Mix (NEB). Assembled fragments were transformed into self-made chemically competent E. coli DH5a cells. Correct clones were identified by plasmid preparation (Monarch Plasmid Miniprep Kit, NEB) and Sanger Sequencing (Azenta) or RCA directly on cells (Microsynth). Subsequently, plasmids were isolated using the Plasmid Maxiprep Kit (QIAGEN) and used for transfection. All cloned sequences of this study are listed in Table 1.

[0195]

[0128] Table 1. Exemplary sequences of the present invention used in Example 1

[0196]

[0197]

[0129] Plasmid transfection

[0198]

[0130] One day prior transfection, cells were seeded at 3.0x104cells per well for 96 well plates, 2.2x105for 24 well plates, 7.5x105for 6 well plates, and 4.0x106for 10 cm dishes. Cells were transfected with JetOptimus DNA transfection reagent (Polyplus transfection). For 96 well 75 ng DNA per well were transfected, for 24 well plates 300 ng DNA, for 6 well plates 1 pg, and for 10 cm dishes 5 pg DNA.

[0199]

[0131] Cell culture and cell lines

[0200]

[0132] HEK293T cells were cultivated at 37°C, 5% CO2 in an H2O-saturated atmosphere, and maintained in DMEM (Gibco) supplemented with 10% FBS (Gibco) and 1% penicillinstreptomycin (Gibco). HEK293T Split-Luciferase reporter cell line was generated by Cas9 cleavage at the AAVS1 locus and homology-directed integration of a donor construct containing LgBiT, the C-terminal fragment of Firefly Luciferase, separated by a P2A sequence, and the puromycin resistance gene. Three days after transfection, the cells were selected for two weeks with 2 pg / ml puromycin (Thermo Fisher). HEK293T cells stably expressing EGFR or IL7Ra were generated by means of amplifying the EGFR sequence from AddGene plasmid #23935 (a gift from William Hahn & David Root), whereas IL7Ra was synthesized (TWIST Biosciences). Both coding sequences were cloned into the AAVS1 knock-in donor plasmid, transfected with AAVS1 targeting Cas9, and selected with 2 pg / ml puromycin. Dual-positive EGFR and IL7Ra receptor cells were generated by cloning IL7Ra into an AAVS1 donor plasmid containing a blasticidin resistance gene, and cells were transfected and selected in 10 pg / ml blasticidin medium (Thermo Fisher).

[0201]

[0133] Quantification of STV-mediated target RNA release into the cell culture supernatant

[0202]

[0134] Supernatants from the STV-releasing cells were collected and filtered after 48 h. RNA was extracted with Monarch Total RNA Miniprep Kit (NEB), and isolated RNA was used as template for RT-qPCR with Luna Universal One-Step RT-qPCR Kit (NEB), along with a primer / FAM-probe set (custom design, Metabion), specific for EGFP mRNA. The reaction was analyzed on a QuantStudio 7 Flex device (Thermo Fisher).

[0203]

[0135] Integration of diffusion-designed symmetric oligomers into STV design

[0204]

[0136] Previously designed RFdiffusion symmetric oligomers were filtered for successfully assembled oligomers based on size exclusion data (Ref. 10). Additionally, all D2 symmetric oligomers were excluded. The resulting 39 sequences having SEQ ID NO: 29-67 (see Table 1 herein) were synthesized (eBlocks, IDT DNA) and cloned as a C-terminal fusion to the additional STV components (PHPLC, SynL, and tdPCP).

[0205]

[0137] Integrating structure-mined membrane binding domains into STV design

[0206]

[0138] The pleckstrin homology domain-containing PDB: 1MAI was used as input structure for structure-based homology search with FoldSeek (Refs. 22, 37). 10 sequences from three categories (other species, human, metagenome) were selected based on the highest homology to the input structure. Each sequence was synthesized (IDT DNA) and fused to the N-terminus of the previously identified ideal STV construct, containing SynL, tdPCP, and HE0690.

[0207]

[0139] Sequence and structural alignments of the structure-mined membrane binding domains

[0208]

[0140] The amino acid sequences of the structure-mined MBDs were aligned to that of Rattus norvegicus PHPLCb, using the residues visible in the X-ray structure (PDB: 1MAI). The alignment was performed with the MAFFT version 7 --add tool (Ref. 38), using default settings (strategy: auto, scoring matrix: BLOSUM62, gap opening penalty = 1.53, offset value = 0.0). For the structural alignment, the structure of the MBD region was extracted from the AlphaFold2 (Ref. 39) (human and other species MBDs) or ESMFold (Ref. 23) (metagenomic MBDs) prediction of the respective structure-mined proteins containing these MBDs. These structures were aligned, and the RMSD to PDB: 1 MAI was calculated using the PyMOL super alignment tool.

[0209]

[0141] Screening of symmetric oligomer and membrane binding proteins for RNA release and uptake

[0210]

[0142] Cells were seeded in 96 well format and transfected with each of the oligomer or membrane STV constructs along with plasmids coding for VSV-G and N-SplitLuc-PP7 (in a 2:1 :7 ratio). 24 h post-transfection, 5 pl of supernatant was collected from the transfected cells, mixed with 45 pl PBS, and measured using Nano-Gio HiBiT Lytic Detection System (Promega) at a Centro LB960 device (Berthold Laboratories), using 0.5 sec integration time. 48 h posttransfection, 120 pl of supernatant was collected and filtered through a 0.45 pm PVDF 96 well filter plate (Sigma Aldrich) by centrifugation (1 ,500 g, 4°C, 20 min). Cleared supernatant was added to a seeded 96 well plate of C-split luciferase reporter cells. 24 h later Nano-Gio DualLuciferase Reporter Assay (Promega) was performed on the cells after complete removal of the supernatant. STV uptake was quantified by light emission from the NanoLuc substrate. N- splitLuc-PP7 mRNA uptake and expression were measured by the light emission from Firefly Luciferase substrate.

[0211]

[0143] Validation of STV-mediated transfer of EGFP mRNA by Flow Cytometry

[0212]

[0144] HEK293T producer cells were transfected in 24 well format with plasmids coding for STV constructs, VSV-G, and EGFP-PP7 (2:1 :7 ratio). STV-containing supernatant was collected for two consecutive days, filtered through a 0.45 pm PVDF membrane filter, and concentrated 5-10 fold with Lenti-X Concentrator (Takara Bio) in fresh DM EM. 10-20 pl of resuspended STVs were added to a 96 well plate of HEK293T cells. After 24 h, the treated cells were detached using StemPro Accutase (Thermo Fisher), mixed with FACS buffer (EDTA / BSA), and filtered through cell strainer-containing tubes. Subsequently, samples were gated for living, single cells and EGFP mRNA uptake and expression were analyzed by Flow Cytometry (BD FACSaria III, BD Biosciences). Data analysis was performed using the FlowJo software (BD Biosciences).

[0213]

[0145] Design of additional oligomers with C8 symmetry

[0214]

[0146] Additional oligomers featuring C8 symmetry were generated using the open-source version of RFdiffusion, along with the script provided for symmetric oligomers (Ref. 10). These computations were performed on a single A100 GPU.

[0215]

[0147] Determination of subcellular STV localization

[0148] HEK293T cells were transfected with STV constructs containing different membranebinding domains. 24 h later, cells were fixed with 10% Formalin (Sigma Aldrich) and permeabilized in 1 % BSA / 0.5% Triton X-100 containing PBS. Permeabilized cells were incubated with primary anti-HA antibody (Sigma Aldrich, cat. H3663), overnight at 4°C. Subsequently, the cells were washed and stained with an Alexa 488 coupled, secondary donkey anti-mouse antibody (Thermo Fisher, cat. A21202), overnight at 4°C. Stained cells were mounted with Prolong Diamond reagent (Thermo Fisher) and imaged at an Axio Imager M2 fluorescence microscope (Carl Zeiss).

[0216]

[0149] Characterization of packaging capacity by Flow Cytometry

[0217]

[0150] EGFP-PP7-STVs were produced in 24 well format as previously described. Additionally, producer cells were transfected with mRuby3-PP7 constructs containing UTR sequences of variable lengths. Concentrated STVs were added to HEK293T target cells. After 24 h, EGFP and mRuby3 expression was quantified using Flow Cytometry, as described previously.

[0218]

[0151] Concentration of STVs by ultracentrifugation for analytical and experimental purposes

[0219]

[0152] Producer cells, seeded in Poly-L-Lysin (Sigma Aldrich) coated 10 cm dishes, were transfected with plasmids coding for STV-C8 components required for the respective experiment. If not specified otherwise, supernatants were collected for three consecutive days and stored until day 3 at 4°C. The collected supernatant was centrifuged for 5 min at 1 ,000 g and passed through a 0.45 pm PVDF membrane filter. Filtered supernatant was added to a cushion of 20% (w / v) sucrose (Sigma Aldrich) in PBS. Subsequent ultracentrifugation was performed at 26,000 rpm for 2 h and 4°C using a SW28 rotor in an Optima L-60 ultracentrifuge (Beckman Coulter). After the centrifugation, the supernatant and the sucrose solution were removed, and the pellet was resuspended in 50 pl ice-cold 1x PBS (Thermo Fisher) on an orbital shaker at 150 rpm for 45 min at 4°C. T o remove debris, the resuspended pellet was centrifuged at 1 ,000 g for 5 min at 4°C and stored at -80°C. Following this process, samples were concentrated approximately 300-fold.

[0220]

[0153] Determination of STV purity for downstream analysis

[0221]

[0154] STV-C8 samples were concentrated via ultracentrifugation, and the sample purity was determined by silver staining. Samples were prepared in 2x Laemmli buffer (Sigma Aldrich) for 10 min at 98°C. The SDS-PAGE was run on a 4-15% gradient TGX gel (BioRad) using a 1x Tris / Glycine / SDS running buffer (BioRad) for 60 min at 130 V. Subsequently, the gel was silver stained according to the manufacturer’s description (Serva). A gel was run in parallel with the same samples and blotted onto a nitrocellulose membrane for 60 min, 100 V at 4°C in transfer buffer (Tris / Gylcine-buffer, BioRad). The STV-C8 protein position on the membrane was determined by imaging with Nano-Gio HibiT Blotting system (Promega) in a Fusion SL Vilber machine (Peqlab). The HibiT signal on the membrane was used as a reference to identify STV proteins on the corresponding silver-stained gel.

[0222]

[0155] Characterization of STV vesicles by Cryo-Electron Tomography

[0223]

[0156] In Poly-L-Lysin (Sigma Aldrich) coated 10 cm dishes, seeded producer cells were transfected with plasmids coding for STV-C8 and EGFP-PP7. 24 h after transfection, the cells were washed with PBS, and serum-free DMEM was added to the cells. After another 24 h, the supernatant was collected and concentrated via ultracentrifugation as described previously. For subsequent cryo-EM analysis, the purified STV-C8 particles were diluted to 109particles / pl in PBS. The samples were applied to holey R 3.5 / 1 carbon 200 mesh copper grids (Quantifoil), covered with a homemade 3 nm thick continuous carbon film by flotation. The grids were treated by glow discharge (at 4 mA for 10 s), then blotted and cryo-cooled into liquid ethane using a Vitrobot IV (Thermo Fisher) with the chamber operating at 95% humidity and at 10°C. For each tomogram, movies were automatically acquired using Tomo5 software on the Titan Krios G4 equipped with a cold-FEG operated at 300 kV and equipped with a Falcon IVi camera and a Selectris X energy filter (Thermo Fisher). A magnification of 81 ,000x was applied at a pixel size of 1.63 A. Each movie was acquired in the EER format and fractionated into 270 frames for a total dose of 2 elk2. Each tomogram was composed of 61 movies, acquired according to the dose symmetric scheme with angles between 60° and -60° and a 2° increment between movies. Processing was performed using Relion 5 beta3 software (Ref. 40). MotionCor2 was used to apply the gain reference and to align the EER fractionation by groups of 45 frames (Ref. 41). CTF estimation was performed using CTFFIND 4.1 , tilt-series alignment was performed using AreTomo2 (Refs. 42, 43). The tomogram was reconstructed using Relion 5 beta3, and denoising was performed using CryoCARE. Data segmentation was performed with MemBrain-seg for membranes and manually in Amira for particles (Ref. 44).

[0224]

[0157] Characterization of STV-C8 RNA content

[0225]

[0158] STV-C8 particles were produced and purified as described in the previous section. RNA was isolated from the particles, as well as from corresponding producer cells, using the Monarch Total RNA Miniprep Kit (NEB). Subsequently, Illumina RNA-Seq Library Prep and sequencing with 20 million paired-end reads per sample were performed on a NovaSeq device. Sequencing reads were mapped to the human reference transcriptome using the STAR aligner and differential expression analysis was performed using DESeq2. Library preparation, sequencing, and data analysis were performed by Azenta (Leipzig).

[0226]

[0159] Characterization of STV-C8 protein content

[0160] STV-C8 were produced and purified as described in the previous section. Total protein was extracted by lysing the sample with lysis buffer (Preomics) supplemented with complete Protease Inhibitor (Roche). The released protein was quantified using a BCA assay (Thermo Fisher). 10 pg of protein per sample was further processed by Filter Aided Sample Preparation (FASP) and subsequently measured on a QExactive HFx device (Thermo Fisher). Data analysis was performed in MaxQuant 2.4.9.0 using a merged database of SwissProt human protein database and the sequences of exogenously expressed proteins. Statistical analysis was performed in Perseus.

[0227]

[0161] RNA and protein Gene Set Enrichment Analysis

[0228]

[0162] Significantly enriched or depleted genes (p adjusted < 0.005, log2FC +3 / -3) or proteins (- log q < 0.05, log2FC +3 / -3) were selected, and a gene set enrichment analysis was performed using gProfiler2 with default options (e111_eg58_p18_30541362).

[0229]

[0163] Benchmarking of EGFP mRNA delivery efficiency of STVs compared to SEND, EPN, and VLP

[0230]

[0164] SEND / MmPeg10 was ordered from Addgene (#174858, a gift from Feng Zhang), and EPN-MCP, VLP-MCP, EGFP-MS2, SEND-EGFP constructs were ordered as synthesis (Twist Bioscience) and cloned into a CAG promoter expression backbone. For each system, the corresponding capsid scaffold and cargo RNA plasmids were co-transfected with VSV-G plasmid in 24 well format. Supernatants were produced for 48 h and concentrated as previously described. Concentrated vehicles were added to a 96 well plate of HEK293T, Vero E6, N2a, and HepG2 cells. 24 h later EGFP expression was analyzed by Flow Cytometry as described earlier.

[0231]

[0165] Benchmarking of STV against LNP characteristics

[0232]

[0166] A plasmid encoding EGFP under the control of the T7 promoter was cloned. The plasmid was linearized via digestion downstream of the stop codon. The reaction was purified (Monarch DNA Cleanup Kit, NEB) and used as template for in vitro transcription (HiScribe T7 Quick High Yield RNA Synthesis Kit, NEB). Subsequently, the RNA was purified (Monarch RNA Cleanup Kit, NEB) and capped with the Vaccinia Capping System (NEB). The reaction was purified again and polyadenylated with E. coli Poly(A) Polymerase (NEB). After a final purification step, the EGFP coding mRNA was diluted to 150 ng / pl in 20 mM citrate buffer (pH 4.0). LNPs were composed of ALC-0315 (Cayman Chemical, cat. 34337), DOPE (Avanti Polar Lipids, cat. 850725), Cholesterol (ChemCruz, cat. sc-202539), and DMG-PEG 2000 (Avanti Polar Lipids, 880151) in the respective ratio (50:10:38.5:1.5). The lipid and RNA solutions were quickly mixed at a 1 :3 volume ratio, resulting in a final weight ratio of 40:1 . 1 pl of the prepared sample was diluted in 3 ml of PBS in a cuvette (Sarstedt) and analyzed by DLS using a Zetasizer Pro (Malvern Panalytical). EGFP mRNA-containing STV-C8 particles were prepared via ultracentrifugation, as described previously. The absolute STV-C8 protein content was determined by extrapolation from a HiBiT Control Protein (Promega) standard curve. EGFP-mRNA content in STV-C8 was determined by absolute RT-qPCR quantification (Luna Universal One-Step RT-qPCR Kit, NEB) with an in vitro transcribed EGFP mRNA standard and STV-C8 particle number was determined by DLS. Different concentrations of STV-C8(EGFP) and EGFP mRNA containing LNPs were added to HEK293T cells and EGFP expression was analyzed by Flow Cytometry. For the concentration of each vehicle, in which approx. 50% of cells turned EGFP positive, the required amount of EGFP mRNA for both vehicles was calculated and compared.

[0233]

[0167] Analysis of STV-C8-induced interferon signalling

[0234]

[0168] A549-IFN-GFP cells (a gift from Ralf Bartenschlager) that report interferon signaling by GFP expression were transfected with luciferase plasmid DNA as a positive control for interferon stimulation and treated with STV-C8 particles, containing a luciferase mRNA. GFP expression upon treatment was monitored after 24 h at an EVOS imaging device (Thermo Fisher).

[0235]

[0169] Comparison of LNP and STV-C8-induced cytotoxicity

[0236]

[0170] HEK293T cells were seeded in 96 well plate format and transfected with 50 ng EGFP mRNA containing LNPs and transduced with purified STV-C8 (EGFP) particles. Both particles were used at a concentration that induced EGFP expression in approx. 50% of cells. After 24 h, cells were detached with Trypsin 0.05% (Thermo Fisher), resuspended in Annexin V binding assay buffer (10 mM HEPES, 140 mM NaCI, and 2.5 mM CaCI2, pH 7.4), and labeled 1 :100 with Annexin V-iFluor 680 (Abeam). Subsequently Annexin V staining intensity was quantified using Flow Cytometry.

[0237]

[0171] Establishing cell-type specific STV-C8 by peptide binder engineering

[0238]

[0172] Previously designed EGFRn, EGFRc, and IL-7Ra minibinders (Ref. 29) were exposed on the STV-C8 surface by expressing them as a fusion construct, consisting of a signal peptide, minibinder sequences, and a transmembrane domain, along with STV-C8 components and an LDLR-binding deficient mutant of VSV-G (K63Q, R370Q (Ref. 28)). Transfections were performed in 6 well plates with EGFP mRNA cargo, the supernatant was collected for 48 h and concentrated with LentiX concentrator (TakaraBio). 30 pl of concentrated supernatant was transferred to either WT-HEK293T cells or HEK293T cells stably expressing the EGFR or IL-7Ra receptors. 24 h later, the EGFP expression was analyzed by Flow Cytometry as described earlier.

[0239]

[0173] STV-C8 mediated EGFP mRNA delivery into RPE spheroids

[0174] Human retinal organoids were differentiated from the hiPSCs-F49B7 cell line, derived from healthy donors, and tested for pluripotency markers as well as germ layer differentiation potential. hiPSC were seeded on 6 well plates coated with Matrigel (Corning) and cultured in mTeSR plus medium (STEMCELL Technologies). The medium was changed every two days. At 70% confluency, iPSCs were passaged in small clumps using 0.5 mM EDTA (0.5 M, pH 8.6, Thermo Fisher). On day 0, hiPSCs were dissociated as small aggregates using 0.5 mM EDTA. The aggregates were suspended in cold Matrigel (GFR, Corning) and incubated at 37°C for 20 min to allow gelling. hiPSCs / Matrigel aggregates were gently dispersed in the Neural Induction Medium (DMEM / F12+GlutaMax, 1% B27 with Vit A supplement, 0.5% N2 supplement, 0.1 mM 2- Mercaptoethanol, 2 mM GlutaMAX, and 1% penicillin / streptomycin, all from Thermo Fisher). The aggregates were cultivated in Ultra-Low Adherent 6 well plate Culture (Costar, Corning). On day 5, the floating cysts were seeded on Matrigel-coated 6 well plates. On Day 15, the cysts were detached by adding Dispase (0.5 mg / mL in DMEM / F12, STEMCELL Technologies) for 3-4 mins at 37°C, followed by washing with DMEM / F12 medium and growing in the Retinal Differentiation Medium (DMEM / F12 +GlutaMax, 2% B27 without vitamin A, 1 % NEAA, and 1 % penicillin / streptomycin, all from Thermo Fisher). On Day 25, the immature retinal organoids were transferred to Retinal Maturation Medium (DMEM / F12 +GlutaMax, 8% FBS, 2% B27 without vitamin A, 1% NEAA, 1% A / A, all from Thermo Fisher, and 1 % 100 mM taurine from Sigma- Aldrich). Half of the medium was changed every 2-3 days, and all organoids were cultured in a humidified incubator at 37°C and 5% CO2 until the end of the experiment. On day 200, RPE spheroids were dissected from human retinal organoids. RPE spheroids were sorted into 96-well U Bottom Ultra-Low Attachment Nucleon Sphera (Thermo Scientific). Each well consisted of 3-4 RPE spheroids. RPE spheroids were transduced with 10 pl of STV-C8(EGFP) / VSV-G or STV- C8(EGFP). The RPE spheroids were fixed two days after the treatment and then gradually dehydrated in 10% sucrose at RT, 30% at RT, and 50% overnight at 4°C. The spheroids were embedded in O.C.T (Tissue-Tek O.C.T. compound, Sakura) and immediately frozen at-80°C until solidification. RPE spheroids were sectioned into 10 pm thickness using a cryostat (Leica CM3050 S, Leica Biosystems). Cryosections were rehydrated and incubated in a 5% chemoblocker solution (Merck) for 30 min, followed by 30 min incubation in 0.3% triton-X. Anti-RPE65 (Proteintech, cat: 17939-1 -AP) and anti-GFP (Santa Cruz, cat. sc-101536) primary antibodies were diluted in 5% chemo-blocking solution and incubated overnight at 4°C. The cells were washed three times in PBS. Goat anti-rat Alexa Fluor 488 (1 :300, Thermo Fisher) and donkey anti-rabbit Alexa Fluor 555 (Thermo Fisher) secondary antibodies were diluted in 5% chemoblocking solution and incubated for 1 h at room temperature. Finally, the sections were washed with PBS and mounted using Fluoroshield with DAPI (Sigma Aldrich). Immunolabeled RPE spheroids were imaged using a Leica TCS SP8 spectral confocal laser scanning microscope (Leica Microsystems).

[0175] EGFP delivery into human monocytes

[0240]

[0176] Primary human monocytes (ATCC, cat. CRL-3622) were seeded in 96 well format. 5 l of concentrated EGFP mRNA containing STVs were added to the cells, and EGFP expression was analyzed by Flow Cytometry 24 h later.

[0241]

[0177] Isolation of primary astroglia from mouse postnatal cortex and Ascii mRNA delivery

[0242]

[0178] Primary astrocytes were isolated from the cerebral cortex of postnatal day 5 C57BL / 6N mice. The cortex was isolated, cut into small pieces, and mechanically dissociated by vigorous pipetting. Subsequently, the cell suspension was centrifuged for 7 min at 1 ,300 rpm, the cell pellet was plated in a T25 flask and cultivated for 7-13 days in DMEM / F-12 GlutaMAX, supplemented with 10% FBS, 10% penicillin / streptomycin, 5% horse serum, 4.5% D-(+)-glucose, 2% B27, 10 ng / ml bFGF, 10 ng / ml EGF (all from Thermo Fisher). Upon reaching 90% confluency, the cells were passaged using 0.2% Trypsin / EDTA (Thermo Fisher) and approx. 75.000 cells were seeded onto Poly-D-Lysin (Sigma Aldrich) coated glass coverslips. 24 h later, 15 pl of concentrated EGFP or Asci 1-P2A- EGFP containing STV-C8 was added to the cells. After 48 h, cells were fixed in 10% formalin (Sigma Aldrich) and incubated with anti-GFP (Abeam, cat. ab13970) or anti-Mash1 (Abeam, ab211327) primary antibody in PBS containing 1% BSA (Sigma Aldrich) and 0.3% Triton X-100 (Sigma Aldrich) overnight at 4°C. After washing, the cells were stained with Alexa488 coupled donkey anti-chicken (Dianova, cat. 703-546-155) or Alexa594 coupled donkey antirabbit (Thermo Fisher, cat. A21207) secondary antibody for 1-2 h in darkness at room temperature. Subsequently, cells were DAPI stained, coverslips were mounted using Aqua Poly / Mount (Polyscience), and samples were imaged using an Axio Imager M2 fluorescence microscope (Carl Zeiss).

[0243]

[0179] Exon 51 deletion of dystrophin gene in primary porcine fibroblasts

[0244]

[0180] Two Cas9 sgRNA plasmids containing a PP7 motif in the stem-loop of the sgRNA, along with porcine dystrophin targeting spacers, were cloned. The sgRNAs target intron 50 (SEQ ID NO: 9, AGAGTTCCTAAGGTAGAGAG) and intron 51 (SEQ ID NO: 10, ATAAAGATAAGAGCTGGCAG) to delete exon 51 (Ref. 30). Additionally, a plasmid coding for NLS and NES fused Cas9, along with a 3’UTR PP7 motif, was cloned. HEK293T producer cells were seeded in poly-Lysin coated 10 cm dishes and co-transfected with Cas9 mRNA and the two sgRNA plasmids (1 :1 :1 ratio), along with STV-C8 and VSV-G coding plasmids. STV-C8 particles were collected and concentrated via ultracentrifugation, as described before. Primary fibroblasts from USH1Cp R3pigs were seeded in a collagen-coated 48 well plate in DMEM medium, supplemented with 1% NEAA 10 mM HEPES, 15% FBS (all from Thermo Fisher) and 2- Mercaptoethanol (Merck). Seeded cells were treated with 20 pl STVs for 72 h. Subsequently, genomic DNA was extracted (Monarch Genomic DNA Purification Kit, NEB), and a 2 kb fragment covering the deleted region was amplified (Primer: SEQ ID NO: 11 , CCCATGACATTTACCCTATTATTATCCC and SEQ ID NO: 12,

[0245] GCTAATGTTCATTTTAAAAAGGAATCTGTC) using Platinum SuperFi II Master Mix (Thermo Fisher). The PCR product was run on a 1.5% agarose gel and imaged.

[0246]

[0181] Treatment of SARS-CoV-2 infected iPSC-derived human lung cells with STV- delivered Cas13d-NCS

[0247]

[0182] For lung cell differentiation, hiPSCs (ISFi001-A - RRID: CVCL_YT30) were cultured in StemMACS medium (Miltenyi Biotec) on plates coated with Geltrex Reduced Growth Factor (Thermo Fisher). 70% confluent iPSC colonies were isolated as single cell suspension with Accutase (Thermo Fisher), 5 min at 37°C, neutralized with StemMACS medium, centrifuged for 3 min at 200 g, room temperature, and 1.0-1.2* 106cells were seeded onto non-adherent 6-well plates (Corning, 3471) in StemMACS medium supplemented with 10 pM Y2763 (Enzo Life Sciences). Differentiation basal medium (DBM) was prepared with DMEM / F12 1 :1 GlutaMAX (Thermo Fisher) supplemented with 1x NEAA (Thermo Fisher), 0.1 % Albumax (Thermo Fisher), 1 x B27 (Thermo Fisher). Formation of embryonic bodies (EB) was induced by changing the medium to 50% StemMACS medium / 50% DBM with 20 ng / ml Activin A (Bio-Techne). Medium was replaced entirely to DBM with 20 ng / ml Activin for 48 h. Definitive endoderm (DE; Days 0 to 5) was induced by plating EBs onto Geltrex Reduced Growth Factor-coated plates at 7 EBs per cm2of culture surface in DBM supplemented with 150 ng / ml Activin A and 25 ng / ml bone morphogenic protein 4 (BMP4) (Thermo Fisher) for 5 days with daily medium changes. Anteriorization of DE (Days 6 to 10) was elicited by changing DBM supplements to 50 ng / ml EGF (Invitrogen) and 50 ng / ml bFGF (Thermo Fisher), 3 pM SB431542 (Miltenyi Biotec) and 10 ng / ml Noggin (Sigma Aldrich) for 5 days with medium changes every day. Lung progenitors giving rise to alveolar epithelial cells type II (Days 10 to 17) were generated by changing the medium to DBM containing 50 ng / ml BMP2 (Thermo Fisher), 50 ng / ml FGF10 (Peprotech), 50 ng / ml BMP4, 50 ng / ml bFGF, and 50 ng / ml WNT3A (Bio-Techne) for 7 days. Successful differentiation into alveolar epithelial cells was confirmed by expression analysis of ACE2 and SLC34A2 by RT- qPCR (Luna Universal One-Step RT-qPCR, NEB). Additionally, NLS and NES containing Cas13d-NCS33was cloned into a PP7 motif containing backbone in the 3’UTR, and a PP7 motif was attached 3’ to a crRNA, targeting the SARS-CoV-2 3’UTR region (SEQ ID NO: 13, GUCAUCCAAUUUGAUGGCACCUG). Subsequently, lung progenitor cells were seeded into Geltrex-coated 96-well plates at a density of 2x104cells / well (Merck) and differentiated for 7 days in differentiation medium. Differentiated lung cells were transduced with 40 pl concentrated STV- C8, containing Cas13d-NCS / SARS-CoV-2 or non-target crRNA. 24 h later, the cells were infected with SARS-CoV-2-GFP (MQI10), and viral replication was monitored for 72 h in an Incucyte S3 live imaging system (Sartorius).

[0183] Analysis of STV-C8 inactivation in human blood samples

[0248]

[0184] Peripheral blood mononuclear cells (PBMCs) were isolated by diluting blood 2-4 times the volume of PBS. 35 ml of the diluted blood suspension was carefully layered onto 15 ml of Ficoll (density = 1 .077 g / mL) in a Falcon tube and centrifuged without brake at 400 g for 30 min at 20°C. After centrifugation, the upper layer was aspirated, leaving PBMCs at the interphase. The PBMC layer was transferred to a fresh Falcon tube, filled with PBS, and centrifuged again at 300 g for 10 min at 20°C. The resulting cell pellet was resuspended in PBS, and cell counting was performed using Trypan blue staining. For long-term storage, PBMCs were frozen at the density of 1x107cells / ml in FBS supplemented with 20% DMSO. Blood samples were collected in EDTA- free tubes for the isolation of blood serum. The tubes were gently inverted several times to mix the blood and then allowed to clot at 4°C for 3-4 h. After clotting, the samples were centrifuged at 2500 g for 10 min at room temperature (RT). Using a sterile pipette, the top clear layer (serum) was carefully transferred to new sterile microcentrifuge tubes or storage vials. For long-term storage, aliquoted serum was stored at -80°C. STV-C8(N-Split-Luc) were produced in 24 well plates and collected for 48 h. The collected supernatant was concentrated, using LentiX (TakaraBio) and concentrated as described. 30 pl of concentrated STV-C8 particles were mixed with 30 pl of 1 :10 diluted serum, 30 pl of resuspended PBMCs (approximately 3.0x105cells) or PBS and incubated at 37°C for 60 min. After the incubation period, 50 pl of the STV-C8 with PBMCs or serum mixes were transferred to a 96 well plate of Split-luc reporter cells. The next day, N-Split-Luc RNA expression was analyzed using ONE-GloEX Luciferase (Promega) assay.

[0249]

[0185] Testing of STV-C8 storage conditions

[0250]

[0186] STV-C8(N-Split-Luc) were produced in 6 well format for 2 days, concentrated, using LentiX (TakaraBio), and stored for 7 days at 4°C or -80°C. Subsequently, 50 pl of stored samples were added to Split-Luc reporter cells. The next day, N-Split-Luc RNA expression was analyzed using ONE-GloEX Luciferase (Promega) assay.

[0251]

[0187] Delivering of OpenCRISPR-1 with STV-C8

[0252]

[0188] The coding sequence of OpenCRISPR-1 was ordered (Twist Bioscience) and cloned into a CAG promoter containing expression plasmid. The coding sequence was fused to two NLS and one NES signal, and the PP7 aptamer was added to the 3’UTR. Additionally, a sgRNA containing the PP7 aptamer in the stem-loop region and a spacer targeting the stop codon in eTLR cells (SEQ ID NO: 14, GCUCCCACAACGAAGACUGAC) was cloned45. STV-C8 particles, containing OpenCRISPR-1 or Cas9 and the sgRNA, were produced in 6 well format for 3 days, concentrated, using LentiX (TakaraBio), and 20 pl concentrated particles were added to a 96 well plate of eTLR cells. 3 days later, the cells were imaged at an EVOS imaging device (Thermo Fisher).

[0189] Analysis of mouse whole-body biodistribution of STV-C8 mediated EGFP expression

[0253]

[0190] STV-C8 (EGFP) particles were produced in coated 10 cm dishes for 3 days and concentrated by ultracentrifugation, as described before. 50 l of concentrated STV-C8 (EGFP) samples were injected intravenously into four-week-old female Balb / c WT mice. The mice were sacrificed 24 or 72 h after injection and intracardially perfused with heparinized PBS (10 ll / rnl heparin) and 4% paraformaldehyde (PFA). The skin was removed, and the bodies were fixed in 4% PFA overnight at 4°C. As previously described (Ref. 34), for vDISCO whole-body staining and clearing the following steps were performed: In brief decolorization (% CUBIC reagent #1 in PBS), decalcification (10% (wt / vol) EDTA in PBS), dehydration (with tetrahydrofuran), delipidation (with dichloromethane), refractive-index matching with a mixture of benzyl alcohol and benzyl benzoate (BAAB), and signal-enhancement with anti-GFP nanobodies (Chromotek, anti-GFP-AF647). A Blaze light sheet system (LaVision BioTec) with an axial resolution of 4 pm was used for light sheet imaging. Full-scale mouse body imaging was performed using a 4x magnification objective (Olympus XFLUOR 4x corrected / 0.28 NA [WD=10 mm]). High-magnification tile scans were obtained with 22% overlap, and the light-sheet width was reduced to 80%. For the z-step, the size was set to 6 pm, with a time exposure of 40 ms in the background channel (488 nm) and 60 ms in the signal channel (640 nm, 647-boosted GFP signal). A Fiji plugin was used to stitch the raw TIFF files to a full plane. The individual planes were merged into a 3D file format with Imaris converter and visualized by Imaris viewer (both Oxford Instruments) (Ref. 34).

[0254]

[0191] In vivo treatment of porcine muscle cells to delete exon 51 from the dystrophin gene

[0255]

[0192] STV-C8 (Cas9 / sgRNAs) were produced in coated 10 cm dishes and concentrated by ultracentrifugation as described before. The pig was sedated by intramuscular injection of ketamine and azaperone. Subsequently, the injection site was shaved and disinfected, and 1 ml of concentrated STV-C8 sample was injected at 1 .75 cm depth, using a 22G safety needle, into the right hind leg (M. biceps femoris). The animal was clinically monitored post-injection. After 3 days, the animal was sedated and euthanized by i.v. injection of pentobarbital. Several muscle samples around the injection site, as well as samples from the uninjected back (latissimus dorsi), were prepared, and genomic DNA (Monarch Genomic DNA Purification Kit, NEB) was extracted. A PCR, using Platinum SuperFi II Master Mix (Thermo Fisher), was performed (Primer: SEQ ID NO: 15, CCCATGACATTTACCCTATTATTATCCC and SEQ ID NO: 16, GCTAATGTTCATTTTAAAAAGGAATCTGTC) to assess the deletion efficiency on an agarose gel. The resulting bands at 2 kb (wild type) and 1 kb (genomic deletion) were extracted from the gel and Sanger sequenced (Microsynth). Additionally, the PCR product was sequenced using Oxford Nanopore sequencing (Eurofins Genomics), and the deletion frequency was analyzed using Geneious Prime (Dotmatics).

[0193] Statistical Analysis

[0256]

[0194] Statistical tests and graphical representations of the numerical data were performed using GraphPad Prism.

[0257]

[0195] RESULTS

[0258]

[0196] Viral capsid-forming proteins consist of multiple domains that orchestrate the packaging of genetic material as well as the assembly and release of the capsid at the plasma membrane of infected cells. The curved surface of assembled viral capsids induces the first step of particle release by membrane bending, and it has been proposed that not only the fully assembled capsid multimer induces membrane bending but also partially assembled protomers15’16. This observation raises the question of whether the mechanism can be harnessed for creating RNA transfer vehicles from scratch by using simple, low-dimensional protein assemblies. To explore this possibility, we leverage Al-designed symmetric protein assemblies with diverse symmetries for building hundreds of vehicles in a bottom-up approach.

[0259]

[0197] Screening of synthetic protein assemblies

[0260]

[0198] Generative models for protein design, such as RFdiffusion (Ref. 10), can generate virtually infinite numbers of protein assemblies with various shapes, including icosahedral, dihedral, and cyclic symmetries that largely differ in size and architecture compared to natural capsids (Fig. 1a). To implement such synthetic protein assemblies in the context of a bottom-up designed RNA transfer vehicle, we fused them to three structural domains that are typically part of capsid-forming proteins: a membrane-binding domain, a late budding domain, and an RNA-binding domain (Fig. 1 b) (Ref. 16). We refer to this design as a Synthetic Transfer Vehicle, STV. As the initial scaffold for STVs, we employed the well-characterized protein assembly HE0902 (Fig. 5a) (Ref. 10), and fused it to a membrane-binding domain derived from the pleckstrin homology domain of Rattus norvegicus phospholipase C delta (PHPLC5). In addition, we created a synthetic budding domain composed of budding motifs from multiple viruses. This synthetic late-budding domain (SynL) exceeds the budding efficiency of the natural HIV p6 L-domain (Fig. 5b-d). To enable RNA packaging, we added high-affinity RNA binding proteins to the construct (Refs. 17, 18). We transfected HEK293T cells with these initial STV constructs, along with RNAs containing the corresponding packaging signal, and quantified the RNA release. All constructs successfully transferred their RNA cargo into the supernatant, with the STV construct built on tandem PCP (tdPCP) being the most efficient (Fig. 1c). Furthermore, we demonstrated that co-expression of VSV-G as fusogenic protein enabled these synthetic particles to deliver EGFP cargo RNA into target cells (Fig. 5e-f).

[0199] HE0902 displays icosahedral symmetry as most viral capsids. Encouraged by the initial proof that STV-HE0902 enables efficient RNA release and transfer, we explored embedding nonnatural symmetries into the STV scaffold. To comprehensively characterize these vehicles, we developed a screening method that allowed monitoring of three relevant dimensions: STV release (1), STV uptake (2), and RNA transfer efficiency (3). We created a reporter cell line expressing the N-split part of NanoLuciferase (NIuc), LgBiT (Ref. 19), as well as the C-split part of Firefly Luciferase (Flue), fused to an N6 coil and the GP41-1 C-intein (Refs. 20, 21). The STV constructs contained a Hi BiT tag that enabled quantification of STV release into the supernatant of producer cells (1). STV uptake into reporter cells was measured by reconstitution of NIuc from LgBiT, expressed in the reporter cell line, and the STV-delivered HiBiT tag (2). In producer cells, we coexpressed a PP7-tagged mRNA coding for the N-split part of Flue, fused to the GP41-N-intein and an N5 coil. Reconstitution of wild-type Flue reported the transfer efficiency of this mRNA from coiled-coil enhanced intein splicing in target reporter cells (Fig. 1d). To validate the screening method, we co-transfected producer cells with HiBiT-tagged STV-HE0902, PP7-tagged N-split Flue cargo RNA along with VSV-G and successfully measured STV-HE0902 release into the supernatant, STV-HE0902 uptake into reporter cells as well as N-split Flue mRNA transfer and expression (Fig. 6a-c). Building on the established method, we screened 39 STV constructs consisting of synthetic assembly domains with icosahedral, dihedral, or cyclic symmetries (Fig. 1e). Many of these STV constructs were efficiently released from producer cells and delivered their cargo RNA into target cells. Strikingly, we observed that STVs built on non-natural dihedral and cyclic symmetries largely outperformed their icosahedral counterparts (Fig. 1f-h). We confirmed the high delivery efficiency for another cargo RNA, EGFP mRNA, with the four bestperforming designs, HE0490, HE0499, HE0505 (all D3 symmetry), and HE0690 (08 symmetry) into wild-type target cells (Fig. 6d). These results suggest that Al-designed protein assemblies with non-natural symmetries can be harnessed for creating virtually infinite numbers of synthetic RNA delivery vehicles. To demonstrate this large potential for scaling, we designed an additional 30 assemblies with C8 symmetry, the symmetry of the best-performing structure HE0690 (Fig. 7). Although being built on the same 08 symmetry, these newly created structures are highly diverse, allowing them to be screened for customized characteristics such as altered packaging density or low immunogenicity.

[0261]

[0200] To further scale the design space of STVs, we combined the identified HE0690 assembly domain with a diverse panel of membrane-binding domains. We selected these membranebinding domains by performing a structure-based search using FoldSeek (Ref. 22), with the PHPLC5 domain from Rattus norvegicus as the template structure. We selected 29 domains from an unrestricted search across all species, a restricted search for human proteins, and a search for metagenomic proteins within the ESMAtlas (Fig. 8a-d) (Ref. 23). We created a library of membrane binding domains fused to SynL-tdPCP-HE0690 and tested them using the established screening scheme for STV release, STV uptake, and RNA transfer efficiency (Fig. 8e). This analysis revealed the membrane binding domain from Ursus americanus (Ila) PHPLC, a previously uncharacterized PH domain, as the most efficient domain for RNA packaging and transfer (Fig. 8f-h). Additionally, we confirmed the robust membrane localization of UaPHPLC- STVs in producer cells and validated their ability to efficiently transfer EGFP mRNA into target cells (Fig. 8i-k). The STV construct, consisting of UaPHPLC and SynL-tdPCP-HE0690, is subsequently named STV-C8 (Fig. 2a).

[0262]

[0201] Characterizing and programming of STV-C8

[0263]

[0202] After having optimized the domain composition, we characterized the properties of STV- C8 as a novel bottom-up assembled RNA transfer vehicle. STV-C8 particles enriched their cargo RNA more than 10,000-fold in the supernatant (Fig. 2b). To further analyze the characteristics of STV-C8, we established a purification method by ultracentrifugation (Fig. 9a), and analyzed the size distribution, particle number, and absolute protein / RNA content per particle (Fig. 9b, c). Furthermore, we characterized the RNA content of purified STV-C8 particles and found strong enrichment of the EGFP cargo RNA (Fig. 2c). In line with previous reports (Ref. 24), mitochondrial RNAs were depleted from STV-C8 particles, presumably because they are not accessible for packaging (Fig. 10a). In addition to RNA, we characterized the protein content of STV-C8 particles and found a strong enrichment of ESCRT related proteins that are involved in budding (Fig. 2d and Fig. 10b). Next, we analyzed the size and shape of STV-C8 particles by Cryo-EM. The analysis revealed that multiple STV-C8 assemblies were released in membrane-enclosed vesicles. Although STV-C8 assemblies are much smaller than viral capsids, they induce the release of vesicles of similar size to enveloped viruses (Fig. 2e) (Ref. 7). This finding points to a biophysical optimum of vesicle sizes and supports the initial hypothesis that non-natural synthetic protein assemblies act similarly as natural capsid protomers by initiating budding through membrane bending. Furthermore, these observations suggest that STV-C8 packages the cargo RNA on the surface of its oligomers while the surrounding membrane provides protection of the cargo RNA.

[0264]

[0203] Given its fundamentally different architecture, we wondered how efficiently STV-C8 delivers cargo RNAs into cells compared to its natural counterparts. Therefore, we benchmarked the efficiency of EGFP mRNA transfer into target cells against commonly used genetically encoded systems, Virus-like particles (VLP) (Ref. 24), Enveloped Protein Nanocages (EPN24) (Refs. 16, 24) and Selective Endogenous Encapsidation For Cellular Delivery (SEND) (Ref. 25). For each of these vehicles, we added their corresponding packaging signal to an EGFP mRNA and compared their delivery efficiency in four cell lines from three species. Across all cell lines tested, we found the efficiency of STV-C8 to be at least an order of magnitude higher (Fig. 2f). Additionally, we benchmarked STV-C8 against clinically used Lipid Nanoparticles (LNP) and found a more than 100,000-fold lower RNA dose requirement for STV-C8 to induce the same expression level (Fig. 2g). Unmodified mRNA is a potent trigger for the innate immune response. Therefore, we tested whether STV-C8-delivered RNA induces interferon signaling. Unlike plasmid DNA, which is a known trigger of the innate immune response, treatment with RNA-containing STV-C8 did not cause a detectable interferon response in A549-IFN reporter cells, suggesting that the biological production process of the cargo RNA alleviates the response (Fig. 2h).

[0265]

[0204] In addition to efficiency, a critical aspect of delivery vehicles is their cell-type specificity. Recently, mutant versions of VSV-G have been created that retain endosomal escape activity but abolish binding to the target receptor LDLR. When combined with antibodies, such a blinded VSV- G can guide VLPs to preferentially target alternative receptors (Refs. 16-28). To overcome the limited availability of antibodies, we wondered whether the concept of using artificially designed proteins could be extended to the programming of cell-type specificity. We added computationally designed peptide binders against EGFR or IL7Ra to STV-C8 particles (Ref. 29), by fusing the binders to a signal peptide along with a transmembrane domain and expressed these constructs together with blinded VSV-G (K63Q / R370Q) in STV-C8(EGFP) producing cells. Upon budding of STV-C8 (EGFP) from the plasma membrane, these synthetic binding modules were co-packaged and mediated cell-type specificity (Fig. 2i and Fig. 11a). We added programmed STV-C8(EGFP) to EGFR or IL7Ra target cells and measured their uptake into receptor-expressing cells using Flow Cytometry. We found a strong preference of programmed STV-C8 for their respective target cell line (Fig. 2j) and confirmed that cells expressing both receptors were permissive for STV-C8 (EGFP) containing either EGFR or IL7Ra binders (Fig. 11b). Additionally, we recognized that the uptake of STV-C8 (EGFP) containing both binders on their surface was further increased in target cells expressing both receptors, suggesting an additive effect that might be further explored for precise cell type-specific targeting (Fig. 11c).

[0266]

[0205] Viruses, such as AAVs and Lentiviruses, are widely used for delivering genetic material into cells, but they have limited packaging capacity. Therefore, we characterized the cargo size that can efficiently be packaged by STV-C8. We co-packaged an EGFP mRNA of constant length along with mRuby3 mRNAs of varying lengths. Across the mRNA sizes tested (1-10 kb), we detected similar expression levels of EGFP and mRuby3 in target cells, suggesting that STV-C8 does not have a packaging limit within the relevant length range (Fig. 12a). We hypothesized that RNA packaging on the surface of HE0690 oligomers instead of inside a capsid shell relaxes the size constraints of STV-C8 compared to viruses. Additionally, we could not detect signs of STV- C8-induced apoptosis (Fig. 12b), confirmed that STV-C8 are stable for at least one week under 4°C storage conditions, facilitating their practical use (Fig. 12c), and characterized the optimal cargo RNA / STV-C8 / fusogen ratio (Fig. 12d-h).

[0267]

[0206] Delivering RNAs into diverse cellular models

[0207] We showed that STV-C8 is a transport vehicle with unique characteristics, enabling highly efficient transfer of reporter RNAs into various cell lines. Building on these results, we delivered a panel of relevant cargo RNAs into complex cellular models. First, we tested the ability of STV- C8 to deliver the EGFP cargo RNA into a multilayer cellular model. We transduced iPSC-derived retinal pigment epithelium (RPE) spheroids with STV-C8 (EGFP) and detected EGFP expression in the inner layers of the spheroids, confirming their ability to penetrate deeper tissue (Fig. 3a, b and Fig. 13a,b). We also demonstrated high delivery rates in primary human monocytes as a model for suspension cells (Fig. 3c). Next, we isolated primary cortical astrocytes from mouse brains and confirmed a high transduction rate of STV-C8(EGFP) also in this model (Fig. 3d,e). Additionally, we delivered mRNA encoding the pro-neuronal transcription factor Ascii into cortical astrocytes and induced Ascii expression in more than 30% of cells (Fig. 3f). One of the most relevant application areas of novel delivery vehicles is the efficient transfer of gene editors. We added the PP7 packaging signal to the 3’IITR of the Cas9 mRNA and cloned two sgRNAs containing the PP7 signal in the tetraloop of the sgRNA along with spacers targeting the intronic region up- and downstream of exon 51 of the dystrophin gene. Deletion of exon 51 of the dystrophin gene has been shown to be a viable strategy for rescuing a high proportion of DMD- related phenotypes (Refs. 30, 31). We produced STV-C8(Cas9 / sgRNAs) containing particles and treated primary porcine fibroblasts with them (Fig. 3g). Amplification of the dystrophin locus from treated cells revealed a deletion frequency of approximately 40% (Fig. 3h). Recently, sequences of novel CRISPR proteins were created in silico by a protein language model. We demonstrated that one of these proteins, OpenCRISPR-1 (Ref. 32), can efficiently be delivered with STV-C8 and induced similar editing rates as wild-type Cas9 in Traffic Light Reporter (TLR) cells (Fig. 13c). These data illustrate that end-to-end programmable therapeutics could be within reach by combining bottom-up created delivery vehicles, equipped with synthetic receptor binders, and Al- designed cargo RNAs. To further demonstrate the flexibility of STV-C8, we packaged and delivered the programmable antiviral Cas13d-NCS (Ref. 33). We added the PP7 signal to the 3’IITR of Cas13d-NCS mRNA and to the 3’part of a crRNA targeting the SARS-CoV-2 genome. We produced STV-C8 (Cas13d-NCS / crRNA) particles, delivered them into iPSC-derived human lung cells, and infected these cells with SARS-CoV-2-GFP (Fig. 3i and Fig. 13d). The viral replication was monitored in a live imaging setup, showing that STV-C8 delivered Cas13d-NCS almost entirely blocked the virus (Fig. 3j,k). To further validate the therapeutic potential of STV- 08, we tested their stability in human blood samples and found that they were unaffected by the blood treatment (Fig. 3I).

[0268]

[0208] Delivering cargo RNAs into animal models

[0269]

[0209] We demonstrated that STV-C8 effectively delivers a wide variety of cargo RNAs into diverse cellular models. Based on these results, we next tested the system in animal models. Notably, to our knowledge, no Al-designed proteins have been studied in vivo so far. We injected mice intravenously with STV-C8 (EGFP) and applied an advanced whole-body clearing and imaging technique to comprehensively analyze the biodistribution of STV-C8 mediated EGFP expression at near single-cell resolution (Fig. 4a) (Ref. 34). Surprisingly, we detected strong and highly specific expression in the lung but no expression in the liver, the common target tissue for most lipid-based vehicles (Fig. 4b, c and Fig. 14a-c). The high resolution of the imaging method enabled us to detect a punctuated expression pattern in the lung, suggesting an additional layer of cell-type specificity within the tissue. To extend the in vivo proof-of-concept beyond a reporter RNA, we packaged the CRISPR / Cas9 system, deleting exon 51 from the dystrophin gene into STV-C8 and injected them into the muscle of a pig (Fig. 4d). We amplified the dystrophin locus and confirmed the successful deletion of exon 51 in the STV-C8(Cas9 / sgRNAs) treated muscle (Fig. 4e,f).SEQ ID NO: 18 depicts a full-length DNA sequence of the dystrophin gene (without deletion), whereas SEQ ID NO: 19 depicts a full-length DNA sequence of the dystrophin gene (with deletion). To further characterize the deletion efficiency, we employed long-read nanopore sequencing and found a deletion frequency of up to 50% (Fig. 4g). Dystrophin exon 51 is a hotspot for mutations, and its deletion was shown to be a viable treatment strategy to rescue DMD- induced phenotypes (Refs. 30, 31). Therefore, these data in a large animal model emphasize the clinical potential of STV-C8.

[0270]

[0210] Discussion

[0271]

[0211] Viruses are highly diverse, yet most converged towards packaging their genomes in large, multimeric protein shells with icosahedral symmetry (Refs. 5-9). Here, we raised the question of whether these characteristics are essential for efficient RNA transport or whether it is possible to discover new avenue by creating transport vehicles with synthetic characteristics. We demonstrated that protein assemblies designed by RFdiffusion (Ref. 10), can be harnessed to build synthetic RNA transfer vehicles from scratch. By establishing a multi-dimensional screening platform, we tested hundreds of designs with diverse structures, we found a novel vehicle architecture that outperformed the RNA transfer efficiency of nature-derived vehicles and applied the identified vehicle to deliver a wide variety of cargo RNAs into various cellular and animal models. Unlike previous studies that mimicked natural capsids (Refs. 16, 24), we leveraged Al- based protein design to create unusual symmetric protein assemblies with high precision, flexibility and functionality. Our findings suggest that viral capsids have been shaped by multiple streams of selective pressure, not only for packaging and transferring their genomes but also for features such as environmental stability or co-packaging of accessory proteins. These aspects could be less important when repurposing such vehicles for gene delivery, potentially explaining the high efficiency of STVs despite their non-natural structure.

[0272]

[0212] The vehicles we created here are an ideal use case for the abilities of current protein design methods, which are very effective in creating static, symmetric structures but limited in generating dynamic proteins or enzymatic activities (Refs. 10, 11 , 35). Future advancements, such as incorporating molecular dynamics data or developing multi-modal models that integrate structure, sequence, and functional annotations, could address these limitations, leading to more granular and flexible designs (Refs. 14, 36). Applying such tools to overcome the limitations of natural protein diversity could unlock new directions in various areas of biological research and enable the development of programmable therapeutics.

[0273]

[0213] Example 2: Further analysis of immunogenicity and toxicological side effects of the STVs of the present invention in vivo

[0274]

[0214] The immunogenicity of the STVs of the present invention prepared as described in Example 1 herein (see above, incorporated herein by reference) has been further analysed (see Figures 15 and 16).

[0275]

[0215] Methods:

[0276]

[0216] Probing potential immunological response and liver toxicity upon systemic STV-C8 injection in mice

[0277]

[0217] PBS, as an untreated control, or 50 pL of STV-C8 was injected intravenously into female C57BL / 6J mice (11 weeks old). 1 and 3 days post-injection, mice were euthanized using carbon dioxide (CO2) inhalation in accordance with institutional animal care guidelines. Blood samples were collected via cardiac puncture, and liver tissues were immediately excised for subsequent analysis. Serum alanine aminotransferase (ALT) levels were measured using the ALAT (GPT) FS (IFCC mod.) assay on the respons 910 random-access clinical chemistry analyzer (DiaSys Diagnostic Systems). Liver tissue was homogenized, and total RNA was extracted from the aqueous phase using the NucleoSpin RNA Mini Kit (Macherey-Nagel). 400 ng of RNA was reverse transcribed into cDNA using the PrimeScript RT Reagent Kit (TaKaRa). Subsequently, RT-qPCR on RNA samples was performed using PowerUp SYBR Green Master Mix (Applied Biosystems) on a QuantStudio 3 Real-Time PCR System (Applied Biosystems).

[0278]

[0218] Analysis of immunological response to local STV-C8 injection in pig muscle

[0279]

[0219] RNA was extracted from the different muscle tissues by phenol-chloroform extraction. The induction of inflammation-related genes upon STV-C8 treatment was evaluated by RT-qPCR in four experimental groups, using TBP as an endogenous control gene. The target group consisted of muscle samples from STV-C8-injected regions. Non-injected muscles from the same animal and muscle samples from untreated animals were used as negative controls, while muscle samples from rejected tissue were used as a positive indicator of strong inflammatory response.

[0280]

[0220] Conclusions:

[0221] We could not detect immunological or toxicological side effects of systemic STV-C8 injection in the mouse (Figure 15), nor any immunological response for the local application of STV-C8-treated muscle in the pig (Figure 16). Therefore, Figures 15 and 16 specifically demostrate the lack of immunogenicity and toxicological side effects of the synthetic transfer vehicles (STVs) of the present invention. These results further demonstrate that administration of the STVs of the present invention does not elicit an immune response, thereby supporting their suitability for repeated and / or therapeutic use as described herein, e.g., without risk of adverse immunological reactions.

[0281] ***

[0282]

[0222] One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. Further, it will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The compositions, methods, procedures, treatments, molecules and specific compounds described herein are presently representative of certain embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention are defined by the scope of the claims. The listing or discussion of a previously published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0283]

[0223] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including,” containing”, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by exemplary embodiments and optional features, modification and variation of the inventions embodied herein may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0284]

[0224] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. All documents, including patent applications and scientific publications, referred to herein are incorporated herein by reference for all purposes.

[0285]

[0225] Other embodiments are within the following claims. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0286]

[0226] REFERENCES (Refs.)

[0287]

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Claims

CLAIMS1 . A method of identifying whether an interaction domain capable of nucleating self-assembly of a plurality of fusion proteins comprising said interaction domain and capsid-forming proteins into synthetic transfer vehicles (STVs) provides for RNA transfer efficiency of said STVs from a sender to a receiver cell, comprising:(A) providing a mammalian sender cell comprising:(i) a first polynucleotide encoding the first member of a first split reporter system providing for a detectable signal, the first entity of a split intein, a proteinprotein interaction domain and a non-translated RNA packaging signal being bound by the RNA-binding domain of the fusion protein encoded by the second polynucleotide,(ii) a second polynucleotide encoding a fusion protein comprising (a) a membrane-binding domain, and / or (b) a budding domain, (c) an RNA-binding domain being capable of binding the RNA packaging signal encoded by the first polynucleotide of and (d) an interaction domain capable of nucleating selfassembly of a plurality of said fusion protein into STVs secreted from said sender cell in which said fusion proteins are expressed, thereby generating a population of STVs comprising the fusogen encoded by the third polynucleotide and further comprising first polynucleotides bound through their RNA packaging signal by the RNA-binding domain of said fusion protein, and(iii) a third polynucleotide encoding a fusogenic protein,(B) providing a mammalian receiver cell comprising:(iv) a fourth polynucleotide encoding the second member of said first split reporter system being capable of complementing the first member of said split reporter system to form a functional first reporter system, the second entity of said split intein being capable of self-association with the first entity of said split intein to form a functional intein and a protein-protein interaction domain being capable of interaction with the protein-protein interaction domain encoded by the first polynucleotide;(C) bringing supernatant from said sender cell comprising the population of STVs into contact with said receiver cell, thereby allowing fusion of said population of STVs with the cell membrane of said reporter cell and delivery of first polynucleotides into the reporter cell; and(D) determining the signal obtained from the reporter cell, said signal being produced by the functional reporter system through protein-fragment complementation of the first and second member of said first split reporter system, said protein-fragment complementation being mediated by protein splicing through a functional intein forming by self-association of the first and second entity of the split intein, said selfassociation being mediated by the interaction of the protein-protein interaction domains encoded by the first and fourth polynucleotide, respectively, thereby identifying an interaction domain which provides (e.g., a readout) for RNA transfer efficiency of STVs from a sender to a receiver.

2. The method of claim 1 , further comprising:(E) comparing said signal obtained in (D) with the signal obtained for a STV comprising self-assembled polypeptides forming naturally-occurring viral architectures, preferably an icosahedral symmetry, preferably a STV as defined in claim 1(A)(ii), wherein the interaction domain comprising the amino acid sequence shown in SEQ ID NO: 1(MHGLQDDPDLQALLKGSQLLKVKSSSWRRERFYKLQEDCKTIWQESRKVMRSPE SQLFSIEDIQEVRMGHRTEGLEKFARDIPEDRCFSIVFKDQRNTLDLIAPSPADAQH WVQGLRKIIHHSGSMDQRQKLQSRPEPTAPPEESFRSGVETTTPPQKQEPIDKEL YPLTSLRSLFGNDPSSQKIEELFKKHKIVAVLRANSVEEAKKKALAVFLGGVHLIEIT FTVPDADTVIKELSFLKEMGAIIGAGTVTSVEQCRKAVESGAEFIVSPHLDEEISQFC KEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVP TGGVNLDNVCEWFKAGVLAVGVGSALVKGTPVEVAEKAKAFVEKIRGCTEQKLISE EDL), wherein, if the signal obtained in (D) is higher than the signal obtained for a STV comprising self-assembled polypeptides forming naturally-occurring viral architectures, preferably an icosahedral symmetry, preferably having the amino acid sequence shown in SEQ ID NO: 1 , such signal is indicative of a STV with high RNA delivery to receiver cells.

3. The method of any one of the preceding claims, wherein said membrane-binding domain has an amino acid sequence which is at least 70% identical to the amino acid sequence shown in SEQ ID NO: 2(GLQDDEDLQVLLKGSQLLKVKSNSWRRERFYKLQEDCKTIWQESRKVMRTPESHLFSIEDIQEVRKGHRTEGMEKFARDVPEDRCFSIVFKDQRNTLDLISPSPAEAQHWVRGLHKIIH).

674. The method of any one of the preceding claims, wherein said budding domain has an amino acid sequence which is at least 70% identical to the amino acid sequence shown in SEQ ID NO: 3(PTAPPGSGSPTAPPEYSGSGPSAPPMEEKLLDLGGSGPYKEGSGSPPPPYVGSGLYPSL SGGFPIVSGGPLPPVGSGSYLDL).

5. The method of any one of the preceding claims, wherein said RNA-binding domain has an amino acid sequence which is at least 70% identical to the amino acid sequences shown in SEQ ID NO: 4(SKTIVLSVGEATRTLTEIQSTADRQIFEEKVGPLVGRLRLTASLRQNGAKTAYRVNLKLDQ ADWDSGLPKVRYTQVWSHDVTIVANSTEASRKSLYDLTKSLVATSQVEDLWNLVPLGR).

6. The method of any one of the preceding claims, wherein said interaction domain an amino acid sequence which is at least 70% identical to any one of the amino acid sequences shown in:SEQ ID NO: 5 (HE0690)(LKVKIKVKDNPAVARGVLRLADKLKKAGVDVEIEIDLYGDEEQALATLAAMEAEVEELA);SEQ ID NO: 6 (HE0490)(ELKKRWRYVATRLFTEILSLEPLIGRETALELLLETARILYKASGELELILEVAREEMRRAG VPEEDI EALLAELRAWA);SEQ ID NO: 7 (HE0499)(KLLEVAVLKAIAELLGLAILDPRAIPLAREALEKLRKIHPSEIIKEMCDMGERILELIEE); and / orSEQ ID NO: 8 (HE0505)(DLLKEAEELVKKILETDPEANPAALNLYTILKTYVDIGAEKQAKKILELLKVVAEHLEKK).

7. An non-naturally occurring interaction domain obtainable by the method of any one of the preceding claims.

8. The interaction domain of any one of the preceding claims, wherein said interaction domain has an amino acid sequence which is at least 70% identical to any one of the amino acid sequence shown in:SEQ ID NO: 5 (HE0690)(LKVKIKVKDNPAVARGVLRLADKLKKAGVDVEIEIDLYGDEEQALATLAAMEAEVEELA);SEQ ID NO: 6 (HE0490)68(ELKKRWRYVATRLFTEILSLEPLIGRETALELLLETARILYKASGELELILEVAREEMRRAG VPEEDIEALLAELRAWA);SEQ ID NO: 7 (HE0499)(KLLEVAVLKAIAELLGLAILDPRAIPLAREALEKLRKIHPSEIIKEMCDMGERILELIEE); and / orSEQ ID NO: 8 (HE0505)(DLLKEAEELVKKILETDPEANPAALNLYTILKTYVDIGAEKQAKKILELLKVVAEHLEKK).

9. A polynucleotide comprising a nucleotide sequence encoding the interaction domain of any one of the preceding claims.

10. A fusion protein comprising a membrane-binding domain, a budding domain, an RNA-binding domain and the interaction domain of any one of the preceding claims.

11. The fusion protein of any one of the preceding claims, wherein said membrane-binding domain has an amino acid sequence which is at least 70% identical to any one of the amino acid sequence shown in the amino acid sequence shown in SEQ ID NO: 2(GLQDDEDLQVLLKGSQLLKVKSNSWRRERFYKLQEDCKTIWQESRKVMRTPESHLFSIE DIQEVRKGHRTEGMEKFARDVPEDRCFSIVFKDQRNTLDLISPSPAEAQHWVRGLHKIIH).

12. The fusion protein of any one of the preceding claims, wherein said budding domain has an amino acid sequence which is at least 70% identical to any one of the amino acid sequence shown in the amino acid sequence shown in SEQ ID NO: 3(PTAPPGSGSPTAPPEYSGSGPSAPPMEEKLLDLGGSGPYKEGSGSPPPPYVGSGLYPSL SGGFPIVSGGPLPPVGSGSYLDL).

13. The fusion protein of any one of the preceding claims, wherein said RNA-binding domain has an amino acid sequence which is at least 70% identical to any one of the amino acid sequence shown in the amino acid sequence shown in SEQ ID NO: 4 (SKTIVLSVGEATRTLTEIQSTADRQIFEEKVGPLVGRLRLTASLRQNGAKTAYRVNLKLDQ ADVVDSGLPKVRYTQVWSHDVTIVANSTEASRKSLYDLTKSLVATSQVEDLVVNLVPLGR).

14. The fusion protein of any one of the preceding claims, wherein said interaction domain has an amino acid sequence which is at least 70% identical to any one of the amino acid sequence shown in:SEQ ID NO: 5 (HE0690)(LKVKIKVKDNPAVARGVLRLADKLKKAGVDVEIEIDLYGDEEQALATLAAMEAEVEELA);SEQ ID NO: 6 (HE0490)(ELKKRWRYVATRLFTEILSLEPLIGRETALELLLETARILYKASGELELILEVAREEMRRAG VPEEDIEALLAELRAWA);SEQ ID NO: 7 (HE0499)(KLLEVAVLKAIAELLGLAILDPRAIPLAREALEKLRKIHPSEIIKEMCDMGERILELIEE); and / orSEQ ID NO: 8 (HE0505)(DLLKEAEELVKKILETDPEANPAALNLYTILKTYVDIGAEKQAKKILELLKVVAEHLEKK).

15. The fusion protein of any one of the preceding claims, comprising a membrane-binding domain having the amino acid sequence shown in SEQ ID NO: 2, a budding domain having the amino acid sequence shown in SEQ ID NO: 3, an RNA-binding domain having the amino acid sequence shown in SEQ ID NO: 4 and an interaction domain having the amino acid sequence shown in SEQ ID NO: 5 (HE0690)(LKVKIKVKDNPAVARGVLRLADKLKKAGVDVEIEIDLYGDEEQALATLAAMEAEVEELA);SEQ ID NO: 6 (HE0490)(ELKKRVVRYVATRLFTEILSLEPLIGRETALELLLETARILYKASGELELILEVAREEMRRAG VPEEDIEALLAELRAWA);SEQ ID NO: 7 (HE0499)(KLLEVAVLKAIAELLGLAILDPRAIPLAREALEKLRKIHPSEIIKEMCDMGERILELIEE); and / orSEQ ID NO: 8 (HE0505)(DLLKEAEELVKKILETDPEANPAALNLYTILKTYVDIGAEKQAKKILELLKVVAEHLEKK).

16. A polynucleotide comprising a nucleotide sequence encoding the fusion protein of any one of the preceding claims.

17. A composition, comprising:(i) a polynucleotide encoding a fusion protein comprising (a) a membrane-binding domain, (b) a budding domain, (c) an RNA-binding domain being capable of binding an RNA packaging signal and (d) the interaction domain of any one of the preceding claims.

18. The composition of any one of the preceding claims, further comprising:(ii) a polynucleotide encoding a cargo RNA molecule comprising a packaging signal.

19. The composition of any one of the preceding claims, further comprising:70(iii) a third polynucleotide encoding a fusogenic protein.

20. The composition of any one of the preceding claims, further comprising a population of sender cells.

21. The composition of any one of the preceding claims, wherein said cargo RNA molecule encodes a payload protein.

22. The composition of any one of the preceding claims, wherein said payload RNA encodes an effector protein which is capable of modulating the expression, concentration, localization, stability, and / or activity of the one or more endogenous proteins of a receiver cell.

23. The composition of any one of the preceding claims, wherein the payload RNA is preferably a therapeutic RNA, configured to prevent or treat a disease of a subject, preferably said disease is Duchenne muscular dystrophy.

24. The composition of any one of the preceding claims, which is a pharmaceutical composition.

25. The composition of any one of the preceding claims for use in a therapy, preferably Duchenne muscular dystrophy therapy.

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