WO2025219570A1 - Composition for inhibiting protein translocation and methods using the same - Google Patents

Abstract

The present invention relates to a composition for inhibiting protein translocation in cells harbouring a target nucleic acid (NA) molecule such as a ribonucleic acid (RNA) molecule, the composition comprising a peptide construct, comprising a protein secretion signal amino-acid sequence ("SP sequence"), and a nucleic acid mimic (NAM) sequence such as a peptide nucleic acid (PNA) sequence comprising 5 to 30 consecutive bases complementary to a sequence of the target NA molecule; and a delivery vehicle for delivering the peptide construct to cells. The present invention further relates to a method for inhibiting protein translocation in cells harbouring a target NA molecule, comprising the step of delivering to the cells a peptide construct, comprising an SP sequence, and a NAM sequence such as a PNA sequence comprising 5 to 30 consecutive bases complementary to a sequence of the target NA molecule. The present invention further relates to uses of such compositions and methods for therapy of the human or animal body, plant treatment and pest control, among other uses.

Description

COMPOSITION FOR INHIBITING PROTEIN TRANSLOCATION AND METHODS USING THE SAME

TECHNICAL FIELD

The field of the present invention relates to compositions for inhibiting protein translocation in cells harbouring a target nucleic acid (NA) molecule like ribonucleic acid (RNA) or deoxyribonucleic acid ( DNA) and methods for inhibiting protein translocation in cells harbouring a target NA molecule , as well as uses of such compositions and methods for therapy of the human or animal body, plant treatment and pest control , among other uses .

BACKGROUND

Protein translation is the process by which the genetic information encoded in messenger RNA (mRNA) is used to synthesi ze proteins . This process involves two main steps : transcription and translation . Transcription is the process of creating an mRNA molecule from a DNA template , while translation is the process of using this mRNA molecule to produce a protein .

In bacteria, translation occurs on ribosomes that are located throughout the cytoplasm . The mRNA molecule is bound to the ribosome , which reads the genetic code and uses it to synthesi ze the corresponding protein . The Sec translocon is a protein-conducting channel that allows proteins to be transported across or inserted into the bacterial cell membrane . The Sec translocon is composed of several subunits , including SecY, SecE , and SecG, which form a stable complex ( termed "SecYEG" ) in the bacterial membrane . Periplasmic, outer membrane and secretory proteins are fully translated before translocation across the inner membrane proceeds . This process is facilitated by SecA, a cytoplasmic ATPase that provides the energy needed for protein translocation .

In eukaryotes , translation of a si zeable portion of all proteins ( typically secretory, membrane or certain organelle- targeted proteins ) occurs on ribosomes that are bound to the endoplasmic reticulum (ER) . The ribosome assembled on the mRNA is brought to the ER at the beginning of translation by a complex of proteins called the signal recognition particle (SRP) , which recognizes a specific N-terminal sequence of amino acids (the signal sequence) of the nascent polypeptide chain. Once the SRP-mRNA complex reaches the ER, the mRNA- ribosome complex is transferred to the Sec61 translocon, which is similar in structure and function to the bacterial SecYEG translocon. The Sec61 translocon allows newly synthesized proteins to be translocated across or inserted into the ER membrane, where they reside or can be sorted for transport to their final destination.

Inhibiting protein translocation usually has catastrophic effects on cells, as many proteins are not able to reach their proper destination. One consequence of inhibiting protein translocation is the accumulation of misfolded or unfolded proteins in the cytoplasm. These proteins can be toxic to cells, as they can form aggregates that interfere with normal cellular processes and activate stress response pathways. In addition, the cell may begin lacking essential functional membrane proteins. Over time, this can lead to cell death and tissue damage.

Accordingly, compounds that generally inhibit protein translocation can be classified as toxins.

One of these toxins is Eeyarestatin I, which is a synthetic compound found in a screen of a compound library (Fiebiger et al, 2004) . This toxin was later found to bind to Sec61 and render it translocation-incompetent (Cross et al, 2009) . In cultured cells, the toxin had a wide-ranging effect on protein export, thereby perturbing the secretory pathway. Eeyarestatin I was shown to be highly cytotoxic to cultured cells .

Another such toxin is mycolactone produced by Mycobacterium ulcerans , which is the causative agent of a necrotizing skin disease known as Buruli ulcer (Demangel & High, 2018) . The key target of mycolactone is the core unit of Sec61. The binding of the toxin blocks the engagement and insertion stages of Sec61-dependent translocation. Overall, this interaction significantly hampers the Sec61 translocon' s ability to facilitate the entry and passage of new membranebound and secreted proteins through the ER membrane. Given the critical role of the ER as the gateway to the mammalian secretory pathway, controlling the initial transfer to the whole endomembrane system, cells affected by mycolactone display a diminished capacity for generating various proteins, including secretory cytokines and plasma membrane receptors. Continuous exposure to mycolactone typically results in cell toxicity, as it activates stress responses that turn on the transcription factor ATF4, leading to cell death through apoptosis .

Mycolactone is discussed as a potential therapeutic agent in Domenger et al, 2023, and in Ricci & Demangel, 2024.

The problem with these toxins is that their mode of action (inhibition of protein translocation in eukaryotic cells) is non-specific. In other words, these toxins generally do not discriminate between healthy cells and diseased cells (e.g., cells infected by a virus, cells harboring an undesired genomic mutation, or tumor cells) . While cells with an increased metabolism such as tumor cells may be particularly affected by these toxins (meaning that they could be used for chemotherapy) , healthy eukaryotic cells will generally be severely affected as well (which may lead to off-target toxicity and intolerable side effects) . Another downside of many known toxins targeting Sec61-dependent protein translocation is that they generally do not exert their toxic effect in prokaryotic cells (or, at most, to a limited extent) . Put differently, these toxins may be difficult to use as antibiotics.

An object of the present invention is therefore to provide compositions and methods to specifically and efficiently inhibit (Sec61- or SecYEG-dependent ) protein translocation in certain populations of cells to compromise cell viability while reducing off-target toxicity as much as possible. SUMMARY OF THE INVENTION

The present invention provides a composition for inhibiting protein translocation in cells harbouring a target NA molecule (in the cytoplasm) , preferably an RNA molecule such as an mRNA molecule. The composition comprises a peptide construct and preferably a delivery vehicle for delivering the peptide construct to cells. The peptide construct comprises a protein secretion signal amino-acid sequence ("SP sequence") , and a nucleic acid mimic (NAM) sequence comprising 5 to 30 consecutive bases, preferably complementary to a sequence of the target NA molecule.

The NAM is preferably selected from the group consisting of peptide nucleic acids (PNAs) , phosphorodiamidate morpholino oligomers (PMOs) , locked nucleic acids (LNAs) , and threose nucleic acids (TNAs) . In a particular preference, the NAM is a PNA.

The peptide construct is also called "SP-NAM" herein. In the particularly preferred case of the NAM being a PNA, the peptide construct is also called "SP-PNA" herein.

The present invention further provides an (in vivo or in vitro) method for inhibiting protein translocation in cells harbouring a target NA molecule (in the cytoplasm) , preferably an RNA molecule such as an mRNA molecule. The method comprises at least the step of: delivering to the cells a peptide construct, comprising an SP sequence, and a NAM sequence (preferably PNA, PMO, LNA or TNA; in particular a PNA sequence) comprising 5 to 30 consecutive bases preferably complementary to a sequence of the target NA molecule.

Typically, the SP sequence is covalently linked to the NAM sequence (such as the PNA sequence) , preferably by a linker peptide or bioconjugation tools, for instance, click-chemistry tools. Suitable biocon ugation strategies are, for instance, disclosed in Stephanopoulos & Francis, 2011 (incorporated herein by reference in its entirety) .

In an aspect, the inventive composition is provided for use in therapy, in particular for use in the inventive method. In particular, the inventive composition is for use in prevention or treatment of an infection, for use in prevention or treatment of a neoplasm, preferably a malignant neoplasm, especially a solid tumor or a haematological malignancy, for use in prevention or treatment of a (pathological ) somatic mutation, for use in prevention or treatment of an autoimmune disease or an inflammatory disease or for use in treatment of cell senescence .

In a further aspect , the following uses are provided : Use of the inventive composition or method for treating a plant or fungus , use of the inventive composition or method for pest control and use of the inventive composition or method for non-human animal population control or control of an invasive species .

In another aspect , a method for producing a peptide construct for inhibiting protein translocation in cells harbouring a target NA molecule ( in the cytoplasm) , preferably an RNA molecule such as an mRNA molecule , is provided . The method comprises the steps of providing a peptide comprising an SP sequence , providing a NAM (preferably PNA, PMO, LNA or TNA; in particular a PNA) comprising at least 5 consecutive bases preferably complementary to a sequence of the target NA molecule , and linking the peptide to the NAM to obtain the peptide construct .

The method may further comprise the step of binding the peptide construct to a delivery vehicle for delivering the peptide construct to cells or encapsulating the peptide construct in a delivery vehicle for delivering the peptide construct to cells , to obtain the composition of the invention .

The present invention represents the first generali zable approach for speci fically inhibiting protein translocation in subpopulations of cells . Protein translocation is a crucial process in both prokaryotic and eukaryotic cells , and its inhibition has ( in the present case , desired) detrimental ef fects on cell viability . The inventive approach utili zes a bi- functional peptide construct , comprising an SP sequence ("SP module" ) and a NAM sequence complementary to a target NA ("NAM module" , in particular "PNA module" in case the NAM is PNA which is preferred) . This SP-NAM ( in particular SP-PNA) may be delivered to a cell via a delivery vehicle . I f the target NA is not present in the cytoplasm or only at a very low level , inhibition of protein translocation will not occur ( or, in the worst case , be transient ) . Importantly, it was found in the course of the present invention that in particular SP-PNA is not toxic in the absence of target NA. By contrast , in the presence of a certain level of complementary target NA molecules in the cytoplasm, the SP-NAM ( in particular SP-PNA) forms a larger complex with the target NA and binds to the Sec translocon via the SP module . As this larger "SP-NAM-NA" complex ( in particular "SP-PNA-NA" complex ) cannot be translocated, this essentially leads to "clogging" of the translocon and makes it unavailable for the translocation of physiological polypeptides . The inventive concept is also exempli fied in Figs . 4 and 5 . The obstruction of translocon function over a certain threshold triggers cell death, as observed in the course of the present invention . This speci fic inhibition of protein translocation allows for targeted treatment of various conditions , such as viral or bacterial infections , cancer, and genetic disorders , while leaving non-targeted cells relatively unaf fected . Importantly, the inventive concept is also applicable to cells without a nucleus such as bacterial cells , thereby opening a new avenue for highly targeted antibiotics . The inventive concept can be used in various in vi tro and in vi vo applications , including therapy, plant or fungus treatment , pest control , non-human animal population control , and invasive species control . DETAILED DESCRIPTION OF THE INVENTION

The detailed description given below relates to all of the above aspects of the invention unless explicitly excluded.

The heterotrimer SecYEG translocates or inserts many proteins through or into the plasma membrane of bacteria and archaea. Sec61apy is a close analog of SecYEG and performs similar functions in the ER membrane of eukaryotes. These essential protein complexes function in two modes (see also Fig. 1) : (i) in the co-translational mode, where the substrate protein is threaded through the translocon while translation is in progress; (ii) in the post-translational mode, where the substrate protein is fully translated prior to insertion into the translocon. In this latter mode, a signal peptide (SP) is generally sufficient for binding the substrate protein to the translocon (see Simon & Blobel, 1992 and Vorhees & Hedge, 2016) .

Although various other proteins exist that facilitate membrane protein insertion or translocation across the membrane, the redundancy is only partial. In consequence, SecYEG or Sec61apy inhibition or misfunction generally leads to cell death (see e.g., Stirling et al, 1992 and Deshaies & Schekmann, 1987) .

SPs are a generally N-terminal extension of proteins utilizing the Sec translocation pathway. Their function is to target the substrate proteins to the translocon. For the Sec pathway, SPs are usually about 20 amino acids long, and comprise a short (1-5 residues-long) positively charged N- terminus, followed by the hydrophobic span (7-15 residues- long) , and ends with a C-terminal region (3-7 residues-long) with an SP I cleavage site, the "AXA" motif (see e.g., Choo et al, 2005) .

An attempt to utilize the high affinity of SPs to Sec translocons is disclosed in WO 2009/041830 A2. Disclosed are essentially constructs with a toxic cargo attached to an SP. Similar to the toxins described further above, this approach cannot be used to target a subpopulation of cells. In consequence, it is therefore not applicable e.g., to the therapy of cancer or to fight viral infections.

"Jamming" or "clogging" the translocon has only been discussed in the literature in the context of using research tools to elucidate details about the translocation mechanism itself and potential rescue of translocon clogging by proteases (see e.g., Ast et al, 2016 and van Stelten et al, 2009) . However, industrial application was never envisioned, let alone was the inventive solution to enable specificity to certain cell populations by linking an SP to a NAM (let alone a PNA) complementary to a target NA molecule ever suggested.

In all living cells, DNA serves as the primary carrier of genetic information. In order to generate proteins, the cell needs to copy (transcribe) a relevant section (gene) into mRNA. Subsequently, the mRNA is used to synthesize (translate) the respective protein based on the specific sequence.

Both DNA and RNA are biopolymers made from a set of four different nucleobases: adenine (A) , thymine (T) or uracil (U) in RNA, cytosine (C) , and guanine (G) . G and C as well as A and T (or U in RNAs) form pairs, respectively, causing DNA strands to establish characteristic double strands with complementary DNA, as their bases pair up. The longer the matching base-pair sequence, the higher the melting temperature necessary to separate the tightly binding double strands. In contrast, RNA-RNA interactions are much weaker and thus RNA usually does not form stable double strands. RNA molecules rather fold on themselves by pairing shorter complementary stretches, forming 3-dimensional structures.

The inherent ability of nucleobases within DNA and RNA to direct sequence-specific binding through complementary base pairing has been leveraged in artificial molecules engineered to mimic this behavior (see e.g., Duffy et al, 2020) . These molecules, commonly referred to in the art as NAMs (also called NA analogs or xeno NAs) , represent a class of synthetic oligomers specifically designed to recognize and bind to target sequences within natural DNA or RNA molecules. In NAMs, nucleobases are positioned along an artificial backbone structure. This enables NAMs to hybridize with a complementary target NA strand through the formation of specific hydrogen bonds - usually according to canonical Watson-Crick base pairing rules, wherein adenine pairs with thymine or uracil, and guanine pairs with cytosine. Typically, NAMs incorporate one or more of the standard (or canonical) nucleobases: adenine, guanine, cytosine, thymine and uracil.

While having the discussed base-pairing capability for sequence recognition, NAMs diverge from natural nucleic acids in the chemical composition of their backbone structures. Unlike the deoxyribose-phosphate backbone of DNA or the ribose-phosphate backbone of RNA, NAMs may feature modified sugars, alternative linkages between units, or entirely synthetic backbone scaffolds for their backbone. These structural modifications are frequently introduced to confer properties advantageous over natural NAs, such as significantly enhanced stability against enzymatic degradation by cellular nucleases, improved binding affinity towards the target sequence, altered pharmacokinetic profiles, or reduced immunogenicity. Consequently, NAMs are powerful tools for applications requiring specific targeting of NA sequences, such as the present invention.

Any NAMs may be used in the present invention, as long as they fulfil the following criteria: (i) high resistance to nucleases and/or proteases, (ii) high specificity to the target NA sequence and (iii) good deliverability into target cells. Suitable NAMs are reviewed e.g., in Duffy et al, 2020, and Bege & Borbas, 2022.

Particularly suitable for these purposes are PMOs, LNAs, TNAs and PNAs . Thus, in the entire context of the present invention (i.e., each of the inventive compositions and methods) , the NAM is preferably selected from the group consisting of PMOs, LNAs, TNAs and PNAs.

In PMOs, the conventional ribose or deoxyribose sugar moieties are replaced by morpholine rings, and the standard anionic phosphodiester linkages are substituted with uncharged phosphorodiamidate linkages. This unique backbone composition renders PMOs electrically neutral and confers exceptional stability within biological systems, demonstrating high resistance to degradation by both cellular nucleases and proteases. PMOs function primarily by binding to target NA sequences via canonical Watson-Crick base pairing, based on the specific hydrogen bonding patterns of their incorporated nucleobases. Due to their stability and neutral charge, PMOs are particularly well-suited for therapeutic applications and they exhibit favorable pharmacokinetic properties for in vivo use. The structure and properties of PMOs are discussed in detail in Maksudov et al, 2023. PMOs have been approved by the US Food and Drug Administration (FDA) for the treatment of Duchenne muscular dystrophy (DMD) since 2016, e.g. eteplirsen.

LNAs are NAMs wherein the conformational flexibility of the sugar ring, characteristic of natural NAs, is constrained (see e.g, Hagedorn et al, 2018, for a detailed review) . While LNAs typically retain the standard phosphodiester linkages found in natural NAs, and thus maintain an overall negative charge, the locked sugar conformation significantly enhances their binding affinity (reflected in an increased melting temperature) towards complementary NA sequences compared to unmodified oligonucleotides of the same sequence. Furthermore, the structural modification provides increased resistance to degradation by nucleases.

TNAs constitute another category of NAMs distinguished by the replacement of the natural pentose sugar with an unnatural tetrose sugar, specifically threose (see e.g., Lee et al, 2023) . Similar to LNAs, TNAs typically utilize the standard phosphodiester linkages to connect the threose units, resulting in a negatively charged backbone analogous to that of natural NAs. Despite the significant alteration in the sugar component, TNAs are capable of forming stable duplexes with complementary DNA and RNA strands through Watson-Crick base pairing. The threose sugar renders TNA oligomers highly resistant to degradation by a wide range of cellular nucleases, contributing to their stability in biological environments. The ability of TNA to mediate sequence-specific hybridization while resisting enzymatic breakdown makes it suitable for therapeutic applications. The synthesis and design of SP-NAM constructs for diverse applications may involve tailored approaches to accommodate different NAM chemistries. For PMOs, solid-phase phosphoramidate chemistry may be employed on morpholine rings, with terminal reactive groups such as amines or azides introduced for conjugation to the SP module. TNAs may also be synthesized using solid-phase protocols. LNAs may incorporate LNA monomers via standard phosphoramidite chemistry. A linker between the SP module (typically N-terminus) and NAM module (typically C-terminus) may be provided to ensure proper translocon engagement. Flexible linkers composed of glycineserine repeats (e.g., GGGS or GGSGS) may be used, with adjustments in length depending on the NAM type - PMOs, for instance, may require slightly longer linkers to accommodate their bulkier structure. Bioconjugation strategies may vary by NAM chemistry: By way of example, PMOs may be linked via click chemistry or NHS ester coupling to lysine residues in the SP; LNAs may employ terminal alkyne groups conjugated to azidomethyl-modif led cysteines; and TNAs may utilize EDC/NHS- mediated amide bond formation with C-terminal carboxylic acids on the SP. Sequence selection may involve selecting NAM sequences complementary to target NA molecules (e.g., 10-30 bases for PNAs, 10-20 bases for PMOs/LNAs) using tools like BLAST .

Another class of NAMs are PNAs. They were first described by Egholm et al., 1993. While in DNA and RNA phosphate bonds interconnect individual desoxyribonucleic base subunits by linking the ribose sugars of each building block, in PNAs, the base subunits are linked via amide bonds, similar to the backbone of peptides or proteins (see Fig. 2) . The change in backbone scaffold leads to certain key features of PNAs, which render them extremely interesting research tools, as well as drug candidates.

Due to negative charges in the phosphate ester skeleton of DNA, repulsion between the double strands in DNA-DNA double helices occurs, which negatively influences the effective melting temperature of paired sequences. In the case of PNA, the altered backbone shows no charges, hence there is no electrostatic repulsion. This results in the formation of very stable double strands with complementary DNA or RNA, with significantly increased melting temperatures even in short stretches of e.g., ten base pairs (see Fig. 2) .

In addition, PNA can be synthesized with relative ease using regular peptide synthesis methods with base building blocks (see e.g., Nandhini et al, 2023) . If desired, amino acids or functional groups can be added to either side of the molecule utilizing the same chemistry, as to provide additional functionalities. Suitable bioconjugation strategies are for instance disclosed in Stephanopoulos & Francis, 2011 (incorporated herein by reference in its entirety) . Other ways of synthesizing the PNAs used herein include click chemistry or manufacturing via Sortase A (see Example 1) .

A lot of research has also been done on modifying the PNAs, in order to further increase their affinity to nucleic acids and to increase the probability to enter the cytoplasm of target cells (see e.g., Suparpprom et al, 2022) .

One area of modification focuses on the backbone of PNAs. Although the neutral nature of the peptide backbone provides certain advantages, including improved binding affinity and specificity for complementary DNA or RNA, modifications to this backbone can further optimize these interactions. For instance, incorporating flexibility through the insertion of linkers or spacers can enhance hybridization properties by reducing steric hindrance, thereby allowing for more efficient binding to target sequences.

Another promising modification involves the nucleobases of PNAs. By altering the structure of the nucleobases, it is possible to fine-tune the hybridization properties of PNAs, such as their melting temperatures and sequence specificity.

Taken together, the PNA used in the present invention may thus be unmodified or modified, e.g., by attaching cationic lipid groups to one or more of the nucleobases. Suitable PNA modifications are also disclosed e.g., in WO 2009/113828 A2 and WO 2018/122610 Al, both of which are incorporated herein by reference. PNAs have been the focus of several studies and are currently used as probes as shown in Fig. 3. The use of PNAs is further discussed in the prior art, e.g., in Muangkaew & Vilaivan, 2020, Pradeep et al, 2023, Das & Pradhan, 2021, Singh et al, 2020, Saarbach et al, 2019, and Montazersaheb et al, 2018.

PNA-based antisense antibiotics were also proposed as a novel class of agents against antibiotic-resistant bacteria, where PNA agents are used to specifically silence crucial target genes or to block functionally important regions of the ribosomal RNA (rRNA) of these pathogens (see Montazersaheb et al, 2018, Chen et al, 2021, Tsylents et al, 2023, and Lee et al, 2019) .

MacLelland et al, 2024, provides an overview on therapeutic and diagnostic applications of antisense PNAs.

US 2004/0220095 Al relates to PNA conjugates for the treatment of diseases associated with human immunodeficiency virus (HIV) . US 6,734,161 Bl relates to PNAs having antibacterial activity.

Unrelated to PNAs, CA 3 026 340 Al discloses a fusion protein containing: a functional domain that improves the protein expression from mRNA; and a pentatricopeptide repeat (PPR) protein capable of binding RNA bases or binding an RNA base sequence, with respect to a target mRNA.

Several modified PNAs are presently under clinical development by Olipass Corporation, a South Korea based company specialized in the development of PNA therapeutics. CLP-1002, which is a PNA with modified nucleobases, is presently in a phase 2a clinical trial. Further PNA therapeutics under development are disclosed e.g., in WO 2018/051175 Al, WO 2018/069764 Al, WO 2019/022434 Al and WO 2018/029517 Al, all of which are incorporated herein by reference .

The peptide construct preferably comprises at least the following two modules: an SP typically at the N-terminus, and a (preferably C-terminal) PNA. The PNA should be long enough to specifically complement a target nucleic acid at physiological temperatures (cf . Fig. 4) . The SP module may be about 20 amino acids long and specifically interacts with the translocon of interest, i.e., the SecYEG translocon and/or the Sec61 translocon. The PNA module provides the specificity to bind the target NA. Binding of the target NA results in a stable globular structure that cannot be threaded through the translocon. Thus, the translocon is blocked, and further translocation processes are abolished.

The two modules are preferably connected by a flexible peptide linker. The linker may be a short peptide segment not forming stable secondary structures. It should be long enough to give the required degree of freedom for the SP module to interact sufficiently with the translocon and short enough to avoid the cleavage of the peptide construct by endogenous proteases .

Upon delivery to the cytoplasm of a target cell (e.g., a diseased cell such as a cancer cell) harboring the target NA in the cytoplasm, the peptide construct ("SP-NAM", in particular "SP-PNA") typically forms a complex with the target NA via its NAM module (in particular PNA module) and therefore obstructs the Sec translocon when bound thereto via the SP module (see Fig. 5) . This blockage of the translocon may lead to cell stress and death, which triggers the immune system to attack and in addition, exposes other diseased (or pathogenic) cells. Conversely, generally no blockage of the translocon occurs in a non-target cell (e.g., a healthy cell such as a non-cancerous cell) due to the absence (or reduced cytoplasmic concentration) of the target NA. Accordingly, the SP-NAM (in particular SP-PNA) is released from the translocon, and the non-target cell stays generally unaffected.

The SP sequence (or SP module) typically targets the peptide construct to a translocon, preferably a Sec61 translocon or a SecYEG translocon. This translocon preferably is a bacterial, archaeal, fungal, animal or plant translocon, preferably wherein the translocon is a vertebrate translocon, especially a mammalian translocon such as a human translocon.

According to a particularly preferred embodiment, the SP sequence has a length of 10 to 40 amino acids, preferably 11 to 35 amino acids , more preferably 12 to 30 amino acids , even more preferably 13 to 27 amino acids , yet even more preferably 14 to 26 amino acids , especially 15 to 25 amino acids or even 16 to 24 amino acids .

It is further preferred that the SP sequence enters the translocon while the NAM sequence ( in particular PNA sequence ) stays accessible for its target NA, preferably wherein the SP starts at an N-terminus of the peptide construct .

According to a further preferred embodiment , the SP sequence comprises a positively charged N-terminal segment , a hydrophobic segment and optionally a C-terminal cleavage site . It is however strongly preferred that the SP sequence does not comprise such a cleavage site ( see also below) . The N-terminal segment of the SP sequence is typically rich in positively charged amino acids such as lysine (K) and arginine (R) . This positive charge allows for electrostatic interaction with negatively charged phospholipid headgroups on the cytoplasmic face of the ER membrane . The hydrophobic segment is a stretch of typically 7- 15 amino acids that are hydrophobic in nature and allows for interaction with the lipid bilayer e . g . of the bacterial membrane or of the ER membrane , thereby promoting insertion of the SP sequence into the translocon . The C- terminal segment of the SP sequence may contain a speci fic recognition site for signal peptidase or other proteases involved in the processing and release of mature proteins from the translocon . It is however strongly preferred that such a recognition site is not present .

Generally, signal peptidases are enzymes that cleave signal sequences from nascent polypeptide chains during protein translocation across membranes in prokaryotic and eukaryotic cells . Signal peptidase I is a speci fic type of signal peptidase which recogni zes a conserved sequence moti f at the j unction between the hydrophobic region of an SP sequence and the mature protein domain, and cleaves the polypeptide chain at this site .

According to a preference , the SP sequence ( and, preferably the entire peptide construct ) does not comprise a cleavage site for signal peptidase I , preferably for any signal peptidase. This increases the likelihood that the translocon remains clogged in cells harbouring the target NA in the cytoplasm.

Wildtype signal peptides do not have a fixed sequence - the natural SPs diverge in length and charge of the N-terminal charged region, and in the length and hydrophobicity of the hydrophobic span.

To give an estimation of translocon recognition probability of a given sequence (i.e., to give a numeric indicator of the suitability of a given sequence as a SP sequence) , the prediction tool Signal? 5.0 may be used (see Almagro Armenteros, et al. 2019) . This tool is also available under https : // services . healthtech . dtu . dk/ services/ SignalP- 5.0/. This tool allows the prediction of the probability, P, of a respective SP for interacting with both, eukaryotic and prokaryotic translocons. Alternatively, its successor, Signal? 6.0, may be used (see Teufel, et al., 2022) , which is available under https : // services . healthtech . dtu . dk/ services/ Signal?- 6.0/. Signal? 6.0 is based on a transformer protein language model with a conditional random field for structured prediction. To minimize the artifacts due to border effects, not only the SP itself but the longer SP-linker sequence should be used for calculating the SP-score. For instance, Gasl may be chosen as an adequately potent SP for both eukaryotes and prokaryotes (see Figs. 6 and 7) . The translocon binding probability may be significantly increased by single amino-acid substitutions. Gasl is a SP from 1 , 3-beta-glucanosyltransf erase from Saccharomyces cerevisiae (see Figs. 8 and 9) . The SPs of prion protein and insulin are further suitable examples of potent SPs .

Further suitable examples of SP sequences are disclosed in WO 2015/127094 Al (incorporated herein by reference) , in particular in Table 1 thereof.

According to a particularly preferred embodiment, at least 10, preferably at least 11 or even at least 12, more preferably at least 13 or even at least 14, even more preferably at least 15 or even at least 16, yet even more preferably at least 17 or even at least 18, especially at least 19 or even at least 20 consecutive amino acids of the SP sequence have a predicted translocon recognition probability of at least 0.4, preferably at least 0.6, more preferably at least 0.7, yet even more preferably at least 0.8, especially at least 0.9, preferably as calculated by SignalP 5.0 or 6.0.

According to another particularly preferred embodiment, at most 3, preferably at most 2, more preferably at most 1, especially no amino acids of the SP sequence have a predicted cleavage probability by a signal peptidase of at least 0.3, preferably at least 0.4, more preferably at least 0.5, yet even more preferably at least 0.6, especially at least 0.7; preferably as calculated by SignalP5.0 or 6.0.

A mechanism of destruction of clogged translocon involves promiscuous membrane proteases - FtsH for prokaryotes (for instance UniProt P0AAI3 in E. coll; see for instance van Stelten et al, 2009) , the ER protein Ste24 in yeasts (e.g. UniProt P47154 in S. cerevisiae) , and ZMPSTE24 (FACE-1) in mammals, e.g., UniProt 075844 in humans; cf. Ast et al., 2016. It was shown that high level of expression of rapidly folding constructs which are targeted to Sec translocons but cannot be translocated is lethal to the cell due to the destruction of vital Sec translocons by FtsH (Cosma et al, 1995) .

Accordingly, in a preferred embodiment, the (targeted) cells have FtsH activity (e.g., if they are bacterial) , or ZMPSTE24 or Ste24 (if they are eukaryotic, e.g. mammalian) activity. The expression "having activity" means that the respective protease is expressed in the cell and is preferably enzymatically active. This may be tested, for example, by employing specific enzymatic activity assays that quantify the cleavage of substrate molecules known to be processed by the protease in question. Such assays typically involve the use of fluorogenic, chromogenic, or luminescent substrates that yield a detectable signal upon proteolytic cleavage. The intensity of the signal is usually directly proportional to the activity of the enzyme within the sample. For instance, Hsu et al, 2019, discloses a suitable FRET assay. Alternatively, the presence and functional integrity of the protease may be assessed through immunoblotting techniques using antibodies specific to the intact enzyme or its processed substrates, providing qualitative and semi-quantitative data regarding protease expression and activity. In another alternative, mass spectrometry-based proteomics may be employed to identify and quantify proteolytic processing events and enzyme expression levels .

It is evident to the skilled person in view of the present disclosure that the inventive mechanism generally works with all kinds of NA present in the cytoplasm, not only mRNA. For instance, tRNAs are essential for protein synthesis, carrying amino acids to ribosomes based on codon recognition. Dysregulation of tRNA expression or function has been implicated in various diseases, such as cancer and neurodegenerative disorders. NAM-based (in particular PNA- based) strategies targeting specific tRNAs can specifically target cells with aberrant tRNA expression. Other important RNA population present in the cytoplasm are microRNAs (miRNA) , which are small, non-coding RNAs that regulate gene expression e.g., by binding to target mRNAs and inhibiting their translation or leading to their degradation. More specifically, miRNAs are small (about 19-25 nts) non-coding RNAs that regulate gene expression at the post-transcriptional level by binding to one or more target mRNAs causing mRNA cleavage, translational inhibition or destabilization of the mRNA by shortening the poly (A) tail. MiRNA genes are often located in unstable regions of the genome (fragile chromosomal sites and other cancer-associated regions) . More than 2300 miRNAs have been identified in humans so far (see Fromm et al, 2020; Fromm et al, 2022) . Dysregulation of miRNA expression is associated with various diseases, including cancer, cardiovascular diseases, neurodegenerative disorders, and autoimmune diseases. In some types of cancers e.g. breast or cervical cancer, the following miRNAs are known to be overexpressed: miR-21 and miR-155 (Sunyoung Park et al, 2017; Mengdi Shang et al, 2022) .

Taken together, the target NA molecule may therefore preferably also be a tRNA or miRNA. The target NA molecule may also be deoxyribonucleic acid (DNA) such as extrachromosomal plasmid DNA or viral DNA.

In another preferred embodiment, the target NA molecule is viral, bacterial, archaeal, or eukaryotic, preferably vertebrate, more preferably mammalian, especially human. This allows for targeting and inhibiting proliferation of cells containing specific NA molecules that are associated with various infectious diseases or genetic disorders in different organisms. Using SP-NAMs (in particular SP-PNAs) that specifically recognize and bind to viral, bacterial, archaeal, or eukaryotic NA molecules as the target NA molecule provides a valuable tool for medical and non-medical uses.

In yet another preferred embodiment, the cells are diseased cells. This allows for targeting and inhibiting specific NA molecules that are associated with various pathological conditions or diseases in different cell types or tissues. Diseased cells can have altered gene expression patterns, genomic instability, or epigenetic modifications that can contribute to their abnormal phenotype or behavior. Therefore, using SP-NAMs (in particular SP-PNAs) that are cytotoxic specifically for these cells is a promising therapeutic approach. Examples of such diseases include cancer, neurodegenerative disorders, infectious diseases, cardiovascular diseases, metabolic disorders, autoimmune diseases, and genetic disorders. Further diseases are discussed hereinbelow.

Use in cancer (which is also discussed below in detail) is particularly preferred. Accordingly, the cells are preferably neoplastic, preferably malignant, especially cells of a solid tumor or a haematological malignancy.

In embodiments, the delivering step in the inventive method may be preceded by a step of identifying the target NA, e.g. by a search in publicly available literature or databases, or by performing routine screens.

For instance, a target tumor-associated mRNA can be identified through a literature review or by utilizing public databases containing genomic, transcriptomic, proteomic, and clinical data for numerous cancer types. More specifically, the HER2/neu (ErbB2) gene is overexpressed in 20-30% of breast cancers and plays a critical role in tumorigenesis . A peptide construct may be provided with NAM (in particular PNA) complementary to the HER2/neu mRNA sequence. Alternatively, public databases such as The Cancer Genome Atlas (TCGA) , Gene Expression Omnibus (GEO) , or the International Cancer Genome Consortium (ICGC) may be utilized to gather information on gene expression patterns, mutations, and alterations in various cancers .

Viral or bacterial target RNAs or DNAs (which are present in the cytoplasm of infected host cells) may be identified by consulting specialized databases such as the National Center for Biotechnology Information (NCBI) GenBank, UniProt, or the Universal Virus Database. These resources provide information on virus genome organization, gene expression patterns, and protein functions. For example, SARS-CoV-2, the causative agent of COVID-19, expresses various structural and non- structural proteins during its life cycle, including the spike (S) protein responsible for host cell entry. By searching these databases, one can identify viral RNA or DNA located in the cytoplasm of host cells, with known functions in infection and replication cycles or that are critical for virus survival and propagation as potential target RNA or DNA.

In another alternative embodiment, the target RNA may be identified by obtaining a biopsy sample from an individual (e.g., who has a disease such as cancer) , such as a tumor biopsy sample, and analysing the genome, transcriptome or proteome of cells (or a subpopulation thereof) contained in this sample.

Upon having obtained a biopsy sample from an individual, such as a tumor biopsy sample, various methods can be employed to analyze the genome, transcriptome, or proteome of cells contained within this sample to identify the target RNA.

Genome Analysis: The genome of cells in the biopsy sample can be analyzed using techniques such as next-generation sequencing (NGS) or polymerase chain reaction (PGR) to identify gene mutations, deletions, insertions, or other alterations that may e.g., lead to the overexpression of specific RNAs . By identifying these genomic aberrations, a target RNA can be pinpointed based on its relevance to the disease .

Transcriptome Analysis: The transcriptome of cells in the biopsy sample can be analyzed using techniques such as RNA sequencing (RNA-seq) or microarray analysis to determine which RNAs are overexpressed compared to normal cells. By comparing the transcriptomes of diseased and healthy cells, a target RNA can be identified based on its differential expression patterns .

Proteome Analysis: The proteome of cells in the biopsy sample can also be analyzed using techniques such as mass spectrometry or protein arrays to identify differentially expressed proteins that may be associated with specific disease pathways. By analyzing these proteomic data, a target mRNA can be identified based on its relevance to the disease and its role in regulating the expression of specific proteins involved in disease pathways.

Upon having identified the target mRNA through genome, transcriptome, or proteome analysis, a peptide construct with the SP sequence and a NAM sequence (in particular a PNA sequence) complementary to a sequence fragment of the target RNA can be provided and delivered to cells (in vivo or ex vivo) using a suitable delivery vehicle.

With this approach, a peptide construct specifically tailored to a cell population harboured by the individual can be provided, allowing specific inhibition of cell proliferation in this population. This approach offers a promising strategy for minimizing off-target toxicity and side effects, ultimately leading to effective therapeutic interventions for diseases such as cancer.

In certain embodiments, combining peptide constructs for several target NAs leads to even higher specificity by exploiting the synergistic effects of multiple target-specific NAM sequences (in particular PNA sequences) . This allows more effective inhibition of protein translocation in cells which harbour several of the target NAs (e.g., diseased cells which are co-expressing several mRNAs at a higher level than healthy cells) . The first step involves providing peptide constructs specific to each target NA by identifying unique NAM sequences complementary to the target NA sequences (as e.g., described herein) . Each peptide construct comprises an SP sequence and a NAM sequence specific to one target NA molecule. Once individual peptide constructs have been provided, they can be combined into a single composition e.g., by mixing them. This multipronged approach further reduces off-target events. The set of peptide constructs can be delivered to cells using a suitable delivery vehicle. Various techniques, such as LNPs, GPPs, or electroporation, can be employed to introduce the peptide constructs into target cells effectively.

In the course of the present invention, the capability of SP-PNAs in particular to significantly reduce the viability of cells harboring respective target NA was discovered (see also experimental examples below) . In addition, the absence of apparent off-target effects confirms the exceptionally high specificity of the inventive approach. Consequently, the SP- PNA of the present invention (and, more generally, also the SP-NAM of the present invention) is considered as a widely potent and highly customizable agent, useful for a plethora of non-medical and medical applications (e.g., treatment of Covid-19, AIDS, malaria, cancer, etc.) .

For instance, SP-NAMs (in particular SP-PNAs) can be provided which recognize viral RNA/DNA present in infected eukaryotic cells. Such cells will have their Sec61 translocation system blocked by the SP-NAM bound to a viral RNA/DNA by virtue of sequence complementarity. For example, SP-NAMs directed against the E and M proteins of SARS-CoV-2 can be expected to stop the spreading of the infection.

The first effect of an antiviral SP-NAM (in particular SP- PNA) is a significant diminution in the production of viral particles, specifically of enveloped viruses. These particles are usually assembled by utilizing the eukaryotic translocon machinery. In the presence of specific SP-NAMs and subsequent blockage of the translocon system, the production of viral particles is stalled . Furthermore , the blockage of Sec61 complexes generally leads to a reduction of essential endogenous membrane proteins in the ER- and subsequently the cell membrane of infected cells . At some point , crucial cell functions cannot be suf ficiently maintained, and the cell dies . Thus , infected cells can be expected to be selectively removed from the body and do not contribute to the production and spread of viral material anymore .

Although antiviral SP-NAMs ( in particular SP-PNAs ) may enter all cells of treated tissue , only those cells containing viral mRNA will be af fected . It is further worth mentioning that in the course of viral infection and subsequent forced production of new viral material as well as the assembly of viral particles , infected cells usually die from exhaustion or in the course of the violent release of massive amounts of viral particles . Anti-viral SP-NAMs will thus only remove cells that would either way die from viral infection but in doing so , will also prevent the infected cells from expressing viral components . Treatment with speci fic SP-NAMs will ef ficiently suppress viral production and spread, lowering the viral load in a patient , and consequently also lowering the burden on the patient ' s body . This ef fect will moreover allow the immune system of the patient to catch up with the viral infection .

The emphasis in the design of respective NAM sequences ( in particular PNA sequences ) for antiviral SP-NAMs ( in particular SP-PNAs ) should be on selecting ( a ) highly speci fic and at the same time (b ) highly conserved sequences , to guarantee selectivity as well as to prevent the target virus from escaping treatment via rapid evolution/mutation .

Furthermore , SP-NAMs ( in particular SP-PNAs ) of fer a rapid route to answer emerging resistance to treatment . Conventional antiviral substances usually take multiple years from the first promising results in basic research to success ful phase 3 trials and introduction to the market . Once resistance against an antiviral compound develops (usually via selective pressure ) , the nature of nearly all conventional anti-viral compounds as small molecule substances requires changes in the chemical make-up of such substance. In turn, this commands a „return to the drawing board" - also known as a return to basic efficacy tests. In any case, altered chemistry of the active molecule requires vigorous safety testing and very likely leads to new Phase 1 to Phase 3 studies. In summary, once resistance against an anti-viral substance starts to spread among the targeted virus population, the respective compound will lose its efficacy and there is no quick way to adapt it.

In contrast, SP-NAMs (in particular SP-PNAs) can be (i) easily adapted once resistance among a respective target virus species is detected. This adaption can take the form of limited modifications of the NAM sequence (in particular PNA sequence) to „follow" the emerging mutations in the target. Alternatively, an altogether different target sequence can be chosen, circumventing the selective pressure that was built up against the first sequence. Yet, in the first place, SP-NAMs (in particular SP-PNAs) (ii) are chosen to target highly conserved and thus essential sequence stretches, as explained before. Hence, the virus' evolutionary plasticity to evade SP- NAM treatment is substantially limited.

Viruses can be roughly classified into DNA and RNA viruses, where either DNA or RNA serves as the media to store the genetic information. The DNA or RNA can occur as a single or as a double strand. In any case, to produce viral proteins and subsequently more viral particles, the viral genes need to be transcribed to mRNA either utilizing viral or host enzymes. Hence, viral mRNA can be expected to be present in large numbers in the course of an infection, acting as the target for specific antiviral SP-NAMs (in particular SP-PNAs) . Yet, even the primary viral DNA or RNA is usually released into the cytoplasm of an infected cell as well as multiplied to be packed into newly synthesized viral particles. SP-NAMs (in particular SP-PNAs) can also be directed against this primary DNA or RNA as it is not protected by the nucleus. Furthermore, the type of DNA or RNA - be it single-stranded or doublestranded - does not make much difference for the affinity of SP-NAMs (in particular SP-PNAs) , since NAMs (in particular PNAs) generally have the ability to invade double-stranded DNA or RNA efficiently (see e.g., Peffer et al, 1993 and Aiba et al, 2022) .

An SP-NAM (in particular SP-PNA) can be provided which recognizes any specific viral mRNA or primary DNA/RNA of the pathogens relevant to human health, e.g. of viruses causing hemorrhagic fever (Ebola, Marburg virus - both ssRNA viruses or Machupo-, Junin- and Crimean-Congo virus) , Hanta viruses, the bird flu virus, Lassa virus and Dengue fever, to name just a few. Further suitable targets are furthermore any viruses affecting the respiratory system, like MERS, SARS, SARS-CoV2 and influenza viruses.

Viruses with the ability to enter the so-called lysogenic cycle are another suitable target for SP-NAMs (in particular SP-PNAs) . In the course of entering the lysogenic cycle, a virus incorporates its genetic information in the DNA of the host cell, effectively hiding among the endogenous genetic material. This way, the virus may lie dormant and undetected for a long period. Usually, the viral infection re-appears when the immune system of the patient is challenged and the virus "senses" a chance to multiply and spread efficiently again. Examples of such viruses highly relevant to human health are HIV or viruses causing herpes or warts (herpes simplex virus, varicella zoster virus) . In the course of the lysogenic cycle, the genetic material of the virus as well as resulting mRNAs are widely absent from the cytoplasm and will thus not be detected or dealt with by most medical screens or potential treatments. Nevertheless, to stay dormant and undetected by the host's immune system, lysogenic viruses usually need to keep expressing certain immune systemmodifying proteins at low levels. SP-NAMs (in particular SP- PNAs) may be provided to specifically bind these kinds of mRNAs, thus enabling the successful removal of cells harboring lysogenic viruses. Using highly efficient delivery methods, treatment with specialized SP-NAMs (in particular SP-PNAs) can even lead to the complete clearance of such cells from a patient's body, erasing all traces of dormant viruses and curing the disease completely. Additionally, specific SP-NAMs (in particular SP-PNAs) can be expected to be effective in the treatment of viral infections in animals (e.g., livestock, pets, and even wild species) , further broadening their applicability. Exemplary and highly relevant targets are African Swine Fever Virus (ASFV) , Pseudorabies (Suid herpesvirus 1), Avian flu (Influenza A virus) , Bluetongue-Virus (BTV) , Bovine viral diarrhea (BVD) , Classical Swine Fever virus (CSFV) , Foot and Mouth Disease virus (FMDV) , Cowpox virus (CPXV) , among many others .

For delivery, in case of viruses usually replicating in the respiratory tract, SP-NAMs (in particular SP-PNAs) can be enclosed in lipid particles (see e.g., Cipolla et al, 2013) or polymers as a delivery vehicle and delivered to the alveoli in the lung or the upper respiratory tract via inhalation devices or nasal sprays.

Infections localized to the digestive tract may be reached by encapsulating SP-NAMs (in particular SP-PNAs) in a matrix that releases the agent at a desired pH as a delivery vehicle. This way, various targets like rotaviruses, noroviruses, adenoviruses, or reoviruses can be targeted. For example, this will help to fight Rotaviruses and Noroviruses which are commonly causing strong diarrheal disease in infants and small children, in the case of rotaviruses leading to above 100,000 deaths due to diarrhea in 2019 (see e.g., Janko et al, 2022) .

Systemic infections of viruses spreading to multiple organs of a patient via the blood system may be addressed by application of encapsulated SP-PNA (e.g. by delivery vehicles such as lipid nanoparticles, polymers, or peptides) , similar to methods used for mRNA vaccine delivery (see e.g., Nitika et al, 2022) . There are emerging approaches utilizing polymorphism of lipid nanoparticles for drug delivery. For instance, lamellar LNPs may be used as delivery vehicles but also nanoparticles with inverted hexagonal phase (HIT) , as well as bicontinous cubic phase (QII) . The latter two were reported to be most efficient in fusing with the target cellular membrane (Zheng et al, 2023) . In case of skin infections (e.g., warts - caused by the human papilloma virus, HPV; hand-foot-mouth disease, HEMD - caused by human enteroviruses or coxsackieviruses) , encapsulated SP-NAMs (in particular SP-PNAs) specific for viral NA may be delivered in the form of a cream or lotion, effectively covering the infected tissue.

Another application of the present invention is the treatment of bacterial (or fungal) infections which are resistant to conventional treatments.

Antibiotic-resistant bacteria are a growing concern for human health, with the potential to severely cripple humanity' s success in fighting the cause of many deadly diseases with easy to produce, effective agents. Likewise, in the last decades, as the number of novel agents has plummeted, the demand to successfully release a new active antibiotic compound has risen dramatically. Consequently, the count of so called "antibiotics of last resort", still effective against resistant pathogens, is dwindling steadily. Among others, bacteria relevant in this regard are: methicillin-resistant Staphylococcus aureus (MRSA) , Pseudomonas species, Escherichia coil (EHEC) and Mycobacterium tuberculosis .

Yet, many more diseases are caused by bacteria and are thus highly relevant to public health: pertussis / whooping cough (Bordetella pertussis) , gonorrhea (Neisseria gonorrhoeae) , pseudomonas infection (Pseudomonas aeruginosa) , typhoid fever (Salmonella typhi) , several food transmitted infections (Salmonella enterica, Campylobacter jejuni, Helicobacter pylori, Clostridium perfringens , Clostridium botulinum, Clostridium difficile) , tuberculosis (Mycobacterium tuberculosis - MBT) , pneumonia (Streptococcus pneumoniae) or infections with Enterobacter faecalis .

SP-NAMs (in particular SP-PNAs) tailored against bacterial mRNA/DNA have the potential to inhibit the bacterial translocation machinery and ultimately kill targeted pathogens. In effect, patients treated with specific SP-NAMs (in particular SP-PNAs) can be expected to see quick recovery, as the spread of pathogens in the course of their infection will be severely limited, allowing their immune system to catch up with the infection. Furthermore, the ability of SP- NAMs (in particular SP-PNAs) to specifically recognize and invade even double-stranded DNA (as discussed earlier) enables targeting chromosomal bacterial DNA or even extra-chromosomal DNA (plasmids) , both of which are not protected by a nuclear membrane in bacterial cells. Consequently, the design of a respective PNA sequence is not limited to being complementary to an mRNA destined for protein production. It is even possible to target specific genes of antibiotic-resistant bacteria, which are usually provided on extra-chromosomal DNA, also used to spread the resistance in a process termed horizontal gene transfer. Administering SP-NAM (in particular SP-PNA) against bacteria harboring such resistance plasmids can thus be considered as an additional treatment to be combined with classical antibiotics, compensating for any loss in effectiveness of these antibiotics due to apparent resistance in the pathogens.

Another relevant benefit of specific SP-NAMs (in particular SP-PNAs) as a novel class of antibiotically active compounds is that they only affect the respective pathogen spreading within the patient, leaving symbiotic bacterial species unhampered. In contrast, classical antibiotics usually show a very broad efficacy against bacteria, generally suppressing or killing these organisms negligent of their type and relevance to the body. Consequently, using specific SP- NAMs (in particular SP-PNAs) will e.g., render the necessity to build up one's gut microbiome after treatment with classical antibiotics unnecessary.

Furthermore, SP-NAMs (in particular SP-PNAs) offer a rapid route to answer emerging resistance to antibiotic treatment. Conventional antibiotic substances usually take multiple years from first promising results in basic research to successful phase 3 trials and introduction to the market. Once resistance against an antibiotic develops, the nature of nearly all conventional antibiotic compounds as small molecule substances requires changes in the chemical make-up of such substance. In turn, this commands a "return to the drawing board"- also known as a return to basic activity tests. In any case, an altered chemistry of the active molecule requires vigorous safety testing and very likely leads to new Phase 1 to Phase 3 studies. In summary, once resistance against an antibiotic starts to spread among the targeted bacteria population, the respective antibiotic compound will lose its efficacy and there is no quick way to adapt it.

In contrast, SP-NAMs (in particular SP-PNAs) can be (i) easily adapted once resistance among respective target species is detected. This adaption can take the form of limited modifications of the PNA sequence to „follow" the emerging mutations in the respective target. Alternatively, an altogether different target sequence of the bacterial species can be chosen, circumventing the selective pressure that was built up against the first target. Yet, in the first place, SP-NAMs (in particular SP-PNAs) are (11) chosen to target highly conserved and thus essential sequence stretches, as explained before. Hence, the bacteria's evolutionary plasticity to evade SP-NAM (in particular SP-PNA) treatment is substantially limited. Specific SP-NAMs (in particular SP- PNAs) may also target fungal infections, which are usually divided into local or systemic infections. Examples of local mycosis, affecting only a limited area of the patients' body, are infections of the skin, nails, vagina or mouth by Candida species. Examples of systemic mycosis, spreading among multiple organs of the patient (e.g. lungs, eyes, liver or even the brain) , are (i) opportunistic fungal infections (aspergillosis, candidosis, mucormycosis) in immunodeficient patients and (ii) primary fungal infections (histoplasmosis, blastomycosis, sporotrichosis, coccidioidomycosis, etc.) , among others .

As with SP-NAMs directed against viral NA, the choice of delivery of SP-NAMs (in particular SP-PNAs) should be adapted to the infection which is being treated. E.g. in case of infections of the digestive tract, the need for encapsulation and pH-dependent release may occur. Otherwise, when in course of a bacterial infection or mycosis a patients' lung is affected, SP-NAMs (in particular SP-PNAs) enclosed in lipid particles may be delivered via an inhalation device. Analog to viral skin infections, bacterial or fungal skin infections (e.g. caused by Staphylococcus and Streptococcus species) may be treated with encapsulated SP-NAMs (in particular SP-PNAs) delivered as a cream or lotion, effectively covering the infected tissue.

Furthermore, similar to the design of respective NAM sequences (in particular PNA sequences) in case of viral targets, emphasis should again be put on selecting (a) highly specific and at the same time (b) highly conserved sequences, to guarantee selectivity as well as to prevent the target bacteria or fungi to escape treatment via rapid evolution / mutation .

As in the case of viral targets (see above) , SP-NAMs (in particular SP-PNAs) can also be expected to be effective in the treatment of bacterial or fungal infections in animals (e.g. livestock, pets and even wild species) , further broadening their applicability. Examples of relevant bacterial infections are: botulism (Clostridium botulinum) , tuberculosis (Mycobacterium tuberculosis - MBT) , brucellosis (Brucella species) , Campylobacter species, Glanders (Burkholderia mallei) , parrot fever (Chlamydia psittaci) and Streptococcus suis, among others. Likewise, there are many relevant fungal infections affecting animals: histoplasmosis, cryptococcosis, blastomycosis, sporotrichosis, candidiasis, aspergillosis, oomycosis, among others.

As a particular example of application, specific SP-NAMs (in particular SP-PNAs) may be used as anti-malarial agents. Antimalarial drug resistance is a global threat to malaria control efforts. The disease has been eradicated about 50 years ago across most European countries so the current malaria cases in Europe may have been infections acquired by travelers. Nevertheless, the anopheline vectors are still present (see Bertoia et al, 2022)

The malaria parasite Plasmodium falciparum faces may for instance be targeted by SP-NAM (in particular SP-PNA) directed against its aquaglyceroporin (PfAQP) mRNA. The genome of P. falciparum does not harbor another gene for an aquaglyceroporin (see Hansen et al, 2002) . Since the parasite faces drastic osmotic changes during kidney passages and is engaged in the massive biosynthesis of glycerolipids during its development in the blood-stage, PfAQP is essential. Significantly, the two canonical Asn-Pro-Ala (NPA) motifs that mammalian aqua ( glycero ) porins exhibit in their pore region are changed to (NLA and NPS, respectively) in PfAQP. Thus, PfAQP has crucial differences in its mRNA sequence, enabling the development of specific NAMs, which, if targeted via SP-NAM (in particular SP-PNA) to the translocon, can be expected to be effective antimalaria drugs.

SP-NAM (in particular SP-PNA) may also be used to fight other parasites. Treatment of Leishmaniasis is one example. Targeting Leishmania' s major aquaglyceroporin LmAQPTl (see Mukhopadhyay et al, 2011) by SP-NAM (in particular SP-PNA) is also a feasible approach.

Cancer cells differ from their host tissue e.g., by misexpression of oncogenic genes or miRNAs, chromosomal translocations that lead to fusion genes, or mutations that occur in tumor suppressor genes or oncogenes. These changes lead to enhanced growth, invasion of the surrounding tissue, and sometimes spreading to other parts of the body. By "tricking" the immune system, cancer cells remain undetected.

Mutations that are found in cancer cells can generally be divided into driver mutations, which transform normal cells into cancerous cells, and passenger mutations, which are acquired during the abnormal growth of cancer cells and do not necessarily have a functional significance for the tumor. Nevertheless, all of these mutations distinguish cancer cells from their healthy counterparts. Personalized medicine allows researchers and doctors to find these differences in the genetic profile of the cancerous specimen and respond with a specific treatment. A growing list of cancer fusion genes and mutations that have been discovered can be found on COSMIC (https : //cancer . sanger . ac . uk/ cosmic/ fusion) .

Aberrations in cancer cells also involve miRNAs (as reviewed e.g., by Smolarz et al, 2022) . Since passenger mutations also occur in miRNA genes, certain patients carry cancer-specific miRNAs that can be targeted by SP-NAMs (in particular SP-PNAs) . Galka-Marciniak et al, 2019 and Urbanek- Trzeciak et al., 2020, analyzed a set of over 10,000 somatic mutations in miRNA genes in different cancer studies. All of these are potential targets for SP-NAM (in particular SP-PNA) treatments .

For example, the most frequently mutated miRNA gene found in the Pan-Cancer Analysis of Whole Genomes (Aaltonen et al, 2020) and The Cancer Genome Atlas (Weinstein et al, 2013) is MIR142, which usually serves as a tumor suppressor. Mutations in MIR142 occur predominantly in acute myeloid leukemia, chronic lymphocytic leukemia, follicular lymphoma, and diffuse large B-cell lymphoma, yet are also detected in solid tumors (glioblastoma, breast cancer, endometrial cancer, and bladder cancer) .

Taken together, SP-NAMs (in particular SP-PNAs) represent a powerful tool for personalized medicine, as any mutations or fusions that produce RNA (e.g. mRNA, miRNA) differing in sequence and/or expression level (i.e., is overexpressed) from healthy cells may serve as a specific target for cancer treatment. By way of example, local application of specific SP-NAMs, in particular SP-PNAs, (e.g. injection at the relevant site, inhalation for lung cancer, creams and lotions for skin cancer) can be expected to clear the patient's body from the malignant cells harboring the target NA sequence and may thus completely eradicate the cancer. Yet even when not all cancer cells can be destroyed, treatment with SP-NAMs (in particular SP-PNAs) can be a powerful tool in concert with other forms of treatment, helping to secure removal of malignant cells as much as possible. Once more, a striking advantage of SP-NAMs (in particular SP-PNAs) is their specificity by means of sequence complementarity. Healthy cells are typically unaffected, thus, the toll on the patient may be expected to be negligible. In addition, when cancer cells die as a result of SP-NAM (in particular SP-PNA) treatment, immunogenic cell death may occur, triggering the innate immune system and allowing the patients' body to recognize and eliminate untreated cancer cells. This stands in stark contrast to conventional therapies, like chemotherapy, where many healthy tissues of a patient are affected parallel to the targeted tumor cells.

SP-NAMs (in particular SP-PNAs) may also be used against diseases caused by somatic mutations, in particular against senescent cells. There is increasing evidence suggesting that somatic mutations acquired during aging lead to non-cancerous disorders later in the life of an individual. Such single mutations may have no effect on the phenotype, yet some mutations may lead to an increase in the fitness of the cell relative to its wild-type neighbors, giving it the potential to clonally expand (Olafsson & Anderson, 2021; Jaiswal & Ebert, 2019) .

One example is the relationship between clonal hematopoiesis and cardiovascular disease. Hematopoietic stem cells (HSCs) give rise to lymphoid, myeloid, erythroid, and platelet cells. Aging of the hematopoietic system has been a target of research for some time, as many diseases of the elderly are associated with changes in immune effector cells. Clonal hematopoiesis of indeterminate potential (CHIP) describes cancer-associated mutations in blood cells of individuals without malignancies or other clonal entities, such as monoclonal B-lymphocytosis (MBL) or paroxysmal nocturnal hemoglobinuria (Steensma et al, 2015) . The term "indeterminate potential" indicates the uncertainty of their effect on the individual's phenotype, yet some of these mutations have already been described as being associated with an increased risk of developing cardiovascular disease, blood cancer, and death. In more than 10% of people over the age of 70, mutations in a CHIP-associated gene lead to a clonal expansion of these cells, which eventually account for more than 4% of blood cells. Many of these genes affect transcriptional regulation. Since HSCs give rise to cells of the immune system, an effect on the patient's immune response is expected. Increased inflammation in atherosclerosis, allergies, graf t-versus-host disease, and age-related increases in systemic inflammation have already been linked to CHIP-associated mutations (see e.g, Natarajan et al, 2018, Leoni et al, 2017, Frick et al, 2019, Franceschi & Campisi, 2014) .

These observations serve as a basis for SP-NAM-based treatments (in particular SP-PNA-based treatments) tailored for the "rejuvenation of the immune system" of affected individuals, thus combating age-related diseases caused by the clonal expansion of individual mutated HSCs .

SP-NAMs (in particular SP-PNAs) may also be used to specifically target senescent cells. Senescence is defined as a state of infinite growth arrest and is induced by sub-lethal stresses. Being important and beneficial in embryonic development of an individual, tissue remodeling, wound healing, and also tumor suppression in youth, it has adverse effects when cells accumulate in the elderly, leading to age- related diseases like atherosclerosis, insulin resistance, chronic obstructive pulmonary disease (COPD) and even Alzheimer's and Parkinson's disease.

For instance, Casella et al., 2019, identified by RNA sequencing unique biomarkers that are universally expressed in various senescent cell models, among those commonly expressed 68 RNAs . These RNAs are suitable as target NAs for compositions of the present invention.

Patients suffering from autoimmune diseases like systemic lupus erythematosus (SLE) , multiple sclerosis (MS) , and Crohn's disease (CD) , rheumatoid arthritis, type 1 diabetes, and pemphigus, produce autoreactive B cells and autoantibodies directed against their own antigens leading to organ damage. B cell depletion therapies (BCDTs) are state-of-the-art treatment strategies helping to suppress the damaging effect of the immune system. These drugs are antibodies that are directed against specific surface proteins of B cells (mainly CD19 and CD20) and rely on the presence of effector cells that mediate antibody-induced cellular toxicity. Unfortunately, depletion of B cells does not seem to be complete, as they can also be found in niches of tissues, where there is a lack of effector cells, leading to a relapse of the disease after the treatment is discontinued. Moreover, long-term BCDT increases the risk of infections , as serum IgG levels are low ( Schett et al , 2023 ) .

BCDT are directed against the surface antigens CD19 and CD20 , which can be found in a subset of B cells di f ferentiation stages , from pro-B cell to B-cell derived plasmablasts , but are lacking in long-living plasma cells in the bone marrow that are the main source for autoantibodies in autoimmune diseases . Such cells express other characteristic surface molecules including CD38 and CD138 . Drugs targeting CD38 ( anti-CD38 antibodies ) have already been tested in some autoimmune diseases with limited success . Future research will also provide information on novel genes that are speci fically expressed in cells involved in autoimmune responses . For example , T-bet positive B cells may play an important role in autoimmune diseases as stated by Lee et al . , 2021 .

Since all of these strategies directed against the disease-causing immune cells are based on antibodies , they all depend on the presence of ef fector cells to ful fill their task .

Using SP-NAM-based ( in particular SP-PNA-based) elimination of CD18- or CD20-expressing B cells in combination with targeting CD38- or CD138-positive plasma cells , or other targets speci fically expressed in auto-immunogenic cells , provides an ef ficient tool in the fight against autoimmune diseases . After all , their toxic action mainly relies on the presence of speci fic mRNAs encoding the surface molecules , without the need for the help of other cells .

Additionally, SP-NAMs ( in particular SP-PNAs ) can be used to target immune cells expressing auto-immune antibodies directly, once the antibodies ( and subsequently their respective mRNA sequence ) responsible for the autoimmune reaction have been identi fied . This way, a patients ' body could be cleared completely of only such auto-immunogenic cells that actually cause the disease , rendering relapse (usually caused by the antibody-producing cells still present even long after an inflammation event ) nearly impossible . All other immune cells would be kept unhampered . This is a tremendous benefit in comparison to conventional auto-immune treatments , where whole classes of immune cells are either removed or „shut down" in order to mitigate the harmful autoimmune ef fects of a few members of this class of cells . Conventionally, the immune system is hampered on a global level and thus the patient is left vulnerable to infections and other threats which are usually dealt with via the full might of the immune system . In contrast , treatment with speci fic SP-NAM ( in particular SP-PNA) directed against the source of the auto-immune disease will have no negative ef fect on the patients ' immune system .

In another embodiment , SP-NAMs ( in particular SP-PNAs ) may be provided to speci fically target microbial , amebic, or insect pests , relevant to agriculture . A striking example is the varroa mite ( Varroa Destructor) , a pest af fecting beehives all over the world, leading to a mean accumulated loss of 10 to 15 % of all bee colonies every year and thus having a signi ficant impact on profits in agriculture ( degrade in pollination of crops ) and honey production . Current treatment for the varroa mite includes incubation with mild acids ( formic acid or oxalic acid) either by dropping the solution onto af fected bees , application of sprays , or evaporation in the beehive . The acids are taken up by the mites via porous tissue on their feet . Bees lack these tissues and are generally more resistant to the application and are thus only mildly af fected . Alternative treatments usually involve toxic substances that can only be applied at certain moments in the course of honey production, as the agents will end up in the end product as a potentially harmful contaminant .

Encapsulated SP-NAMs ( in particular SP-PNAs ) speci fically targeting mRNA of the varroa mites can be applied to the bees and the beehive as a spray . This leads to the direct incorporation of the agent by the mites via their porous tissue . Alternatively, the SP-NAM agents ( in particular SP-PNA agents ) will be taken up by the bees and distributed in their bodies ( lymphoid fluid) . Mites feeding on the bees ' bodies will consequently take up the SP-NAMs ( in particular SP-PNAs ) . It is expected that the ensuing cell death following the uptake of SP-NAMs ( in particular SP-PNAs ) in the mites ' bodies will lead to adverse effects on the organism and, subsequently, its death. As the SP-NAMs (in particular SP- PNAs) are provided to be specific against the respective pest, the bees are not at all affected by the treatment. Furthermore, it is expected that the agent will degrade over time and thus not end up in the honey produced. Yet, even if traces of the agent can be found in the honey, the SP-NAMs (in particular SP-PNAs) are generally non-toxic and are not anymore present in a form that can be taken up by human cells (no encapsulation/delivery system) . Lastly, the designed specificity towards the Varroa Mites excludes any off-target effects in other organisms.

Similar invasive bee pests already pose severe problems are the small hive beetle (Aethina tumida, originally sub- Saharan Africa) and the Tropilaelaps mite (originally from south-east asia) , among others.

Bees are also affected by bacterial or fungal infections, which may specifically be targeted by respective SP-NAMs (in particular SP-PNAs) . Treatment may include repeated application of aerosols to significantly reduce the number of such pathogens or as a preventive measure.

Invasive species are a growing concern for endogenous flora and fauna. Their appearance can lead to (i) competition for limited space and food sources in a habitat, (ii) endangerment of prey species that are not adequately adapted to the new predators, and (iii) the spread of novel diseases that use the invasive species as hosts. Striking examples of relevant mammal species are the common raccoon dog (Nyctereutes procyonoides) _r muskrat (Ondatra zibethicus) _r raccoon (Procyon lotor) _r squirrel species (Sciurus carolinensis, Sciurus niger, Tamias sibiricus _r Callosciurus erythraeus Callosciurus finlaysonii) and Javan mongoose ( Urva j avanica) . Furthermore, rats and wild cats are responsible for the decline of many endogenous bird, amphibic and reptilic species in ecosystems worldwide, especially on isolated islands .

In embodiments, encapsulated SP-NAMs (in particular SP- PNAs) directed against specific mRNAs of such invasive species, meant to be eaten and to take full effect in the digestive tract of the targets, may be delivered as spiked bait. Such bait can be widely distributed in a habitat, increasing the chance of target individuals spreading among a vast area to find and eat it. The application is similar to poisonous bait currently in use to reduce the number of certain invasive species or general pests (rats, mice, etc.) , which has to be distributed in large quantities as well. In contrast to poison, which will harm any animal that eats the bait, the specificity of SP-NAMs (in particular SP-PNAs) will reduce the off-target effects, thus minimizing any adverse ramifications to endogenous species.

In another preferred embodiment, the peptide construct (SP-NAM, in particular SP-PNA) further comprises a label, preferably a fluorescent label. This allows for visualizing and tracking the localization, distribution, and uptake of the SP-NAM in cells or tissues, which can provide valuable information for optimizing its delivery and activity. It also allows the use of the SP-NAM as a research tool, e.g. in molecular biology laboratories. For example, the label may be conjugated to the C-terminus of the peptide construct, e.g. via a flexible linker or spacer; it can be covalently or non- covalently bound to the peptide construct. The label, if fluorescent, may be chosen based on its spectral properties, brightness, photostability, and compatibility with the other components of the composition. Fluorescent labels are especially useful for imaging applications.

In yet another preferred embodiment, the delivery vehicle is non-covalently or covalently bound to the peptide construct (SP-NAM, in particular SP-PNA) . This allows for different modes of interaction between the delivery vehicle and the SP- NAM, depending on the specific application and properties of each component. Non-covalent binding can be achieved through electrostatic interactions, hydrophobic forces, or affinitybased methods (such as biotin-streptavidin or antibodyantigen) . Covalent binding can be achieved through chemical conjugation or crosslinking reactions, which can provide a more stable and specific linkage between the delivery vehicle and the SP-NAM.

In a particularly preferred embodiment, the delivery vehicle is selected from the group of lipid particles, viral particles, polymer particles, lipid-polymer particles, dendrimers, cell-penetrating peptides (GPPs) , and lipid moieties. This allows for a wide range of options for delivering the SP-PNA to cells, depending on the specific application and target cells. Viral particles can be engineered to target specific cell types or tissues. Selecting an appropriate delivery vehicle for the SP-NAM (in particular SP-PNA) is expected to improve its efficiency, safety, and versatility in inhibiting protein translocation in cells harboring a target NA molecule.

Examples of lipid moieties are cationic lipid groups attached to one or more of the nucleobases of the NAM (in particular the PNA) . For PNAs, such modifications are also disclosed e.g., in WO 2009/113828 A2 and WO 2018/122610 Al, both of which are incorporated herein by reference.

In another particularly preferred embodiment, the delivery vehicle is a lipid particle selected from liposomes, LNPs, and exosomes .

LNPs have recently come into focus because LNP-based mRNA vaccines against SARS-CoV-2 (primarily elasomeran marketed under Spikevax® by Moderna Inc., and tozinameran marketed under Comirnaty® by Biontech SE/Pfizer Inc) were administered to hundreds of millions of individuals.

In general, LNPs with SP-NAM cargo (or payload) comprise a lipid layer as well as microdomains of lipid and encapsulate the SP-NAM (in particular SP-PNA) . They have a median diameter between 10 nm to 1000 nm (e.g. as determined by dynamic light scattering, DLS) and may adopt e.g. a spherical or polyhedral shape. They may be multilamellar , dependent on their specific lipid composition. The LNPs comprise cationic lipids (in particular lipids which are protonated at low pH, i.e. when in an endosome, also called ionizable lipids) . Typically, the LNPs further comprise stabilizers such as polyethylenglycol ( PEG) -decorated lipids which decrease LNP aggregation. In addition, LNPs usually comprise other types of lipids (often termed "helper lipids") , such as phosphatidylcholines or phosphatidylethanolamines, to improve properties such as delivery efficacy, tolerability, or biodistribution. Finally, LNPs may contain cholesterol or other sterols to modulate membrane integrity and rigidity. Suitable LNPs are for instance also disclosed in US patents US 7,404,969, US 8,058,069, US 9,364,435, WO 2020/061284 Al, WO 2020/219941 Al, WO 2021/123332 Al and US 9,404,127, all incorporated herein by reference .

In contrast to LNPs, liposomes are spherical lipid bilayer vesicles surrounding an aqueous space. They are carriers for the administration of drugs, vaccines, genes, proteins, small molecules, antibiotics, and nutrients. Liposomes are made of (i) phospholipids, mainly phosphatidylcholine, but may also include other lipids, like phosphatidylethanolamine, and (ii) may include cholesterolor other components of bio-membranes such as sphingolipids. Liposomes are generated by a large number of different methods (reviewed e.g. by van Hoogevest, 2017; Szoka et al., 1980) , for example by dispersing phospholipids in aqueous medium, e.g. using mechanical treatment (e.g. in a homogenizer, preferably by high pressure homogenization) or sonication. They vary between 0.02 to 10 pm in diameter. Liposomal delivery systems are disclosed, for example, in, U.S. Pat. No. 6,429,200; U.S. Patent Application No. 2003/0026831; and U.S. Patent Application Nos. 2002/0081736 and 2003/0082103.

In another preferred embodiment, the delivery vehicle is a cell-penetrating peptide (CPP) selected from the group consisting of TAT peptides, penetratins, arginine-rich peptides, transportans , pH-low insertion peptides, and SynB- based peptides. GPPs are short peptides that facilitate the uptake of various types of cargo, such as nucleic acids, proteins, or nanoparticles, into cells by overcoming the plasma membrane barrier. They have been widely used for delivering therapeutic agents to specific cell types or tissues, either alone or in combination with other delivery vehicles. The GPPs listed in this embodiment are some of the most commonly used and well-studied GPPs, which have different properties and mechanisms of action. For example, TAT peptides are derived from the HIV-1 Tat protein and can bind to negatively charged membranes or proteins; penetratins are derived from homeodomain proteins and can form complexes with nucleic acids or other charged molecules; arginine-rich peptides contain multiple arginine residues and can interact with heparan sulfate proteoglycans on the cell surface; transportans are chimeric peptides derived from neuropeptides and can form pores or channels in membranes. Therefore, selecting an appropriate GPP as the delivery vehicle for the SP-NAM (in particular SP-PNA) improves its efficiency, specificity, and safety in inhibiting protein translocation in cells harboring a target NA molecule.

Further suitable examples of GPPs are disclosed in WO 2015/127094 Al (incorporated herein by reference) , in particular in Table 2 thereof.

The GPP (in particular the ones disclosed above) may be covalently conjugated to the peptide construct (SP-NAM, in particular SP-PNA) .

In a preferred embodiment, the composition of the present invention is a pharmaceutical composition. Such a composition is preferably provided with at least one excipient. Excipients suitable for the pharmaceutical composition of the present invention are known to the person skilled in the art, upon having read the present specification, for example water (especially water for injection) , saline, Ringer's solution, dextrose solution, buffers, Hank solution, 5% dextrose in saline, substances that enhance isotonicity and chemical stability, buffers and preservatives. This pharmaceutical composition can (as a drug) be administered via appropriate procedures known to the skilled person (upon having read the present specification) to a patient or individual in need thereof (i.e. a patient or individual having or having the risk of developing the diseases or conditions mentioned herein) . The preferred route of administration of said pharmaceutical composition is parenteral administration, in particular through intraperitoneal, subcutaneous, intramuscular and/or intravenous administration. Other preferred routes are oral administration or topical administration. The dosage and method of administration depends on the individual patient or individual to be treated. Said pharmaceutical composition can be administered in any suitable dosage known from other biological dosage regimens or specifically evaluated and optimized for a given individual. For example, the peptide construct (e.g., encapsulated into the delivery vehicle) may be present in the pharmaceutical composition in an amount from 1 mg to 10 g, preferably 50 mg to 2 g, in particular 100 mg to 1 g. Usual dosages can also be determined on the basis of kg body weight of the patient, for example preferred dosages are in the range of 0.1 mg to 100 mg/kg body weight, especially 1 to 10 mg/kg body weight (per administration session) . The administration may occur e.g. once daily, once every other day, once per week or once every two weeks. As the preferred mode of administration of the inventive pharmaceutical composition is parenteral administration, the pharmaceutical composition according to the present invention is preferably liquid or ready to be dissolved in liquid such sterile, de-ionised or distilled water or sterile isotonic phosphate-buff ered saline (PBS) . Preferably, 1000 pg (dry-weight) of such a composition comprises 0.1-990 pg, preferably l-900pg, more preferably 10- 200pg compound, and optionally 1-500 pg, preferably 1-100 pg, more preferably 5-15 pg (buffer) salts (preferably to yield an isotonic buffer in the final volume) , and optionally 0.1-999.9 pg, preferably 100-999.9 pg, more preferably 200-999 pg other excipients. Preferably, 100 mg of such a dry composition is dissolved in sterile, de-ionised/distilled water or sterile isotonic phosphate-buff ered saline (PBS) to yield a final volume of 0.1-100 ml, preferably 0.5-20 ml, more preferably 1- 10 ml.

The inventive (pharmaceutical) composition may be administered to an individual in need thereof, preferably by intravenous administration, transdermal administration, intradermal administration, intramuscular administration, intraosseous administration, intravitreal administration, intraperitoneal administration, intrathecal administration, oral administration, topical ( in particular dermal ) administration or by inhalation . This generally allows for delivering the SP-NAMs ( in particular SP-PNAs ) to the target cells or tissues in a safe and ef fective manner . The choice of administration route depends on various factors , such as the nature of the target NA molecule , the type of cells or tissues , the disease indication, the patient population, the dosage regimen, and the safety profile . Intravenous administration is a common route for systemic delivery of therapeutics , which can achieve high bioavailability and rapid onset of action . Transdermal and intradermal administrations are non-invasive routes that can provide local or systemic ef fects , depending on the formulation and device used . Intramuscular administration is a convenient route for long- acting depot formulations . Intraosseous administration is an emergency route for accessing the central circulation when other routes are not available . Intravitreal administration is a local route for treating ophthalmic diseases . Intraperitoneal and intrathecal administrations are regional routes for treating abdominal or neurological diseases , respectively . Oral administration is a convenient route for chronic treatments , which can achieve patient compliance and convenience . Inhalation is a non-invasive route for delivering therapeutics to the lungs or systemically, depending on the formulation and device used .

Herein, entries in the UniProt databases are identi fied by their accession codes ( referred to herein e . g . as "UniProt accession code" or briefly as "UniProt" followed by the accession code ) , usually a code of six alphanumeric letters ( e . g . "Q1HVF7" ) . I f not speci fied otherwise , the accession codes used herein refer to entries in the Protein Knowledgebase (UniProtKB ) of UniProt . I f not stated otherwise , the UniProt database state for all entries referenced herein is of 1 March 2023 (UniProt/UniProtKB Release 2023_01 ) .

The term "preventing" or "prevention" as used herein means to stop a disease state or condition from occurring in a patient or subject completely or almost completely or at least to a (preferably significant) extent, especially when the patient or subject or individual is predisposed to such a risk of contracting a disease state or condition.

The present invention further relates to the following embodiments :

Embodiment 1. A composition for inhibiting protein translocation in cells harbouring a target nucleic acid (NA) molecule, the composition comprising

- a peptide construct, comprising

- a protein secretion signal amino-acid sequence ("SP sequence") , and

- a peptide nucleic acid (PNA) sequence comprising 5 to 30 consecutive bases preferably complementary to a sequence of the target NA molecule; and

- a delivery vehicle for delivering the peptide construct to cells.

Embodiment 2. The composition of embodiment 1, wherein the delivery vehicle is selected from the group of lipid particles, viral particles, polymer particles, lipid-polymer particles, dendrimers, cell-penetrating peptides (GPPs) and lipid moieties.

Embodiment 3. The composition of embodiment 1 or 2, wherein the delivery vehicle is a lipid particle selected from liposomes, lipid nanoparticles (LNPs) and exosomes.

Embodiment 4. The composition of any one of embodiments 1 to

3, wherein the delivery vehicle is a cell-penetrating peptide selected from the group consisting of TAT peptides, penetratins, arginine-rich peptides, transportans , pH-low insertion peptides, and SynB-based peptides.

Embodiment 5. The composition of any one of embodiments 1 to

4, wherein the SP sequence has a length of 10 to 40 amino acids, preferably 11 to 35 amino acids, more preferably 12 to 30 amino acids, even more preferably 13 to 27 amino acids, yet even more preferably 14 to 26 amino acids , especially 15 to 25 amino acids or even 16 to 24 amino acids .

Embodiment 6 . The composition of any one of embodiments 1 to

5 , wherein the SP sequence targets the peptide construct to a translocon, preferably a Sec61 translocon or a secYEG translocon .

Embodiment 7 . The composition of any one of embodiments 1 to

6 , wherein the translocon is a bacterial , archaeal , fungal , animal or plant translocon, preferably wherein the translocon is a vertebrate translocon, especially a mammalian translocon such as a human translocon .

Embodiment 8 . The composition of any one of embodiments 1 to

7 , wherein the SP sequence first enters the translocon followed by PNA sequence , preferably wherein the SP starts at an N-terminus of the peptide construct .

Embodiment 9 . The composition of any one of embodiments 1 to

8 , wherein the SP sequence comprises a positively charged N- terminal segment , a hydrophobic segment and optionally a C- terminal cleavage site .

Embodiment 10 . The composition of any one of embodiments 1 to

9 , wherein the SP sequence does not comprise a cleavage site for signal peptidase I .

Embodiment 11 . The composition of embodiment 10 , wherein the SP sequence does not comprise a cleavage site for any signal peptidase .

Embodiment 12 . The composition of any one of embodiments 1 to

11 , wherein the cells have FtsH activity .

Embodiment 13 . The composition of any one of embodiments 1 to

12 , wherein the cells have ZMPSTE24 or Ste24 activity .

Embodiment 14 . The composition of any one of embodiments 1 to

13 , wherein the SP sequence is covalently linked to the PNA sequence . Embodiment 15. The composition of any one of embodiments 1 to

14, wherein the SP sequence is linked to the PNA sequence by a linker peptide.

Embodiment 16. The composition of any one of embodiments 1 to

15, wherein at least 10, preferably at least 11 or even at least 12, more preferably at least 13 or even at least 14, even more preferably at least 15 or even at least 16, yet even more preferably at least 17 or even at least 18, especially at least 19 or even at least 20 consecutive amino acids of the SP sequence have a predicted translocon recognition probability of at least 0.4, preferably at least 0.6, more preferably at least 0.7, yet even more preferably at least 0.8, especially at least 0.9.

Embodiment 17. The composition of any one of embodiments 1 to

16, wherein at most 3, preferably at most 2, more preferably at most 1, especially no amino acids of the SP sequence have a predicted cleavage probability by a signal peptidase of at least 0.3, preferably at least 0.4, more preferably at least 0.5, yet even more preferably at least 0.6, especially at least 0.7.

Embodiment 18. The composition of any one of embodiments 1 to

17, wherein the delivery vehicle is non-covalently or covalently bound to the peptide construct.

Embodiment 19. The composition of any one of embodiments 1 to

18, wherein the peptide construct further comprises a label, preferably a fluorescent label.

Embodiment 20. The composition of any one of embodiments 1 to

19, wherein the target NA molecule is desoxyribonucleic acid (DNA) such as extrachromosomal plasmid DNA or viral DNA, or RNA, preferably messenger RNA (mRNA) , transfer RNA (tRNA) , viral RNA or microRNA (miRNA) .

Embodiment 21. The composition of any one of embodiments 1 to

20, wherein the target NA molecule is viral, bacterial, archaeal or eukaryotic, preferably vertebrate, more preferably mammalian, especially human. Embodiment 22 . The composition of any one of embodiments 1 to

21 , wherein the cells are bacterial , archaeal or eukaryotic, preferably vertebrate , more preferably mammalian, especially human .

Embodiment 23 . The composition of any one of embodiments 1 to

22 , wherein the cells are diseased cells .

Embodiment 24 . The composition of any one of embodiments 1 to

23 , wherein the cells are neoplastic, preferably malignant , especially cells of a solid tumor or a haematological malignancy .

Embodiment 25 . The composition of any one of embodiments 1 to

24 , wherein the composition is a pharmaceutical composition, preferably comprising pharmaceutically acceptable excipients .

Embodiment 26 . A ( therapeutic or non-therapeutic, e . g . in- vitro ) method for inhibiting protein translocation in cells harbouring a target NA molecule , comprising the step of :

- delivering to the cells a peptide construct , comprising

- an SP sequence , and

- a PNA sequence comprising 5 to 30 consecutive bases complementary to a sequence of the target NA molecule ; preferably wherein SP sequence is as defined in any one of embodiments 1 to 25 ; and/or preferably wherein the cells are as defined in any one of embodiments 1 to 25 ; and/or preferably wherein the target NA molecule is as defined in any one of embodiments 1 to 25 .

Embodiment 27 . The composition of any one of embodiments 1 to 25 for use in therapy, preferably for use in the method of embodiment 26 .

Embodiment 28 . The composition for use according to 27 , for use in prevention or treatment of an infection .

Embodiment 29 . The composition for use according to 27 , for use in prevention or treatment of a neoplasm, preferably a malignant neoplasm, especially a solid tumor or a haematological malignancy .

Embodiment 30 . The composition for use according to 27 , for use in prevention or treatment of a somatic mutation .

Embodiment 31 . The composition for use according to 27 , for use in prevention or treatment of an autoimmune disease or an inflammatory disease .

Embodiment 32 . The composition for use according to 27 , for use in treatment of cell senescence .

Embodiment 33 . The composition for use according to any one of embodiments 27 to 32 , wherein the composition is administered to an individual in need thereof , preferably by intravenous administration, transdermal administration, intradermal administration, intramuscular administration, intraosseous administration, intravitreal administration, intraperitoneal administration, intrathecal administration, oral administration, topical administration or by inhalation .

Embodiment 34 . Use of the composition or method of any one of the embodiments 1 to 33 for treating a plant or fungus .

Embodiment 35 . Use of the composition or method of any one of the embodiments 1 to 33 for pest control .

Embodiment 36 . Use of the composition or method of any one of the embodiments 1 to 33 for non-human animal population control or control of an invasive species .

Embodiment 37 . A method for producing a peptide construct for inhibiting protein translocation in cells harbouring a target NA molecule , the method comprising the steps of

- providing a peptide comprising an SP sequence ,

- providing a PNA comprising at least 5 consecutive bases complementary to a sequence of the target NA molecule ,

- linking the peptide to the PNA to obtain the peptide construct . Embodiment 38 . The method of embodiment 37 , wherein the peptide further comprises a donor site recogni zed by a sortase and the PNA further comprises an acceptor site recogni zed by the sortase , wherein the linking comprises contacting the peptide with the PNA and the sortase to ligate the donor site to the acceptor site .

Embodiment 39 . The method of embodiment 37 , wherein SP sequence is as defined in any one of embodiments 1 to 25 .

Embodiment 40 . The method of embodiment 37 , wherein the cells are as defined in any one of embodiments 1 to 25 .

Embodiment 41 . The method of embodiment 37 , wherein the target

NA molecule is as defined in any one of embodiments 1 to 25 .

Embodiment 42 . A method for producing a composition for inhibiting protein translocation in cells harbouring a target NA molecule , comprising performing the method of any one of embodiments 37 to 41 , and further comprising the step of

- binding the peptide construct to a delivery vehicle for delivering the peptide construct to cells or encapsulating the peptide construct in a delivery vehicle for delivering the peptide construct to cells , to obtain the composition .

Embodiment 43 . The method of embodiment 42 , wherein the delivery vehicle is as defined in any one of embodiments 1 to 25 .

Embodiment Al . A composition for inhibiting protein translocation in cells harbouring a target nucleic acid (NA) molecule , the composition comprising

- a peptide construct , comprising

- a protein secretion signal amino-acid sequence ("SP sequence" ) , preferably wherein the SP sequence targets the peptide construct to a Sec61 translocon or a secYEG translocon, and

- a peptide nucleic acid ( PNA) sequence comprising 5 to 30 consecutive bases complementary to a sequence of the target NA molecule ; and - a delivery vehicle for delivering the peptide construct to cells , preferably selected from the group of lipid particles , viral particles , polymer particles , lipid-polymer particles , dendrimers , cell-penetrating peptides ( GPPs ) and lipid moieties .

Embodiment A2 . The composition of embodiment Al , wherein the SP sequence has a length of 10 to 40 amino acids , preferably 11 to 35 amino acids , more preferably 12 to 30 amino acids , even more preferably 13 to 27 amino acids , yet even more preferably 14 to 26 amino acids , especially 15 to 25 amino acids or even 16 to 24 amino acids .

Embodiment A3 . The composition of embodiment Al or A2 , wherein the SP sequence does not comprise a cleavage site for signal peptidase I .

Embodiment A4 . The composition of any one of embodiments Al to A3 , wherein the SP sequence is covalently linked to the PNA sequence .

Embodiment A5 . The composition of any one of embodiments Al to A4 for use in therapy .

Embodiment A6 . The composition for use according to embodiment A5 , for use in prevention or treatment of an infection, of a neoplasm, preferably a malignant neoplasm, especially a solid tumor or a haematological malignancy, or of a somatic mutation, or of an autoimmune disease or of an inflammatory disease , or of cell senescence .

Embodiment A7 . A method for inhibiting protein translocation in cells harbouring a target NA molecule , comprising the step of :

- delivering to the cells a peptide construct , comprising

- an SP sequence , and

- a PNA sequence comprising 5 to 30 consecutive bases complementary to a sequence of the target NA molecule .

Embodiment A8 . Use of the composition of any one of embodiments Al to A4 or the method of embodiment A7 for treating a plant or fungus . Embodiment A9 . A method for producing a peptide construct for inhibiting protein translocation in cells harbouring a target NA molecule , the method comprising the steps of

- providing a peptide comprising an SP sequence ,

- providing a PNA comprising at least 5 consecutive bases complementary to a sequence of the target NA molecule ,

- linking the peptide to the PNA to obtain the peptide construct .

Embodiment A10 . A method for producing a composition for inhibiting protein translocation in cells harbouring a target NA molecule , comprising performing the method of embodiment A9 , and further comprising the step of

- binding the peptide construct to a delivery vehicle for delivering the peptide construct to cells or encapsulating the peptide construct in a delivery vehicle for delivering the peptide construct to cells , to obtain the composition .

The present invention is further illustrated by the following figures and examples, without being restricted thereto .

Fig. 1: How the Sec61 translocon works. Binding of the SB primes the translocon for translocation (upper cartoon) . Once translocated, the mature protein is cleaved from the SP and released into the ER lumen (middle cartoon) . In case of membrane protein insertion, the substrate protein is released into the lipid phase (lower cartoon) . The auxiliary complexes essential for the complete translocation / insertion are not presented for clarity. The principal scheme holds also for the SecYEG complex, yet at the (inner) bacterial membrane instead of the ER membrane.

Fig. 2: Comparison of the backbone composition of DNA and PNA. In DNA (left) , desoxyriboses are connected via phosphate bonds. In PNA (right) , amide bonds are responsible for the backbone connections, similar to the backbone of peptides. The amide bond cannot be broken by nucleases, which renders PNA molecules immune to digestion by such enzymes. PNA is also resistant to proteolytic digestion, as cellular proteases require specific amino acid side chains in the cleavage region, which PNA lacks. Furthermore, specific PNA-DNA or PNA- RNA interaction leads to stable double strands with significantly raised melting temperatures as compared to the NA-NA interaction.

Fig. 3: Application of PNA probes as biosensors (see also Das & Pradhan, 2021) .

Fig. 4: The bi-functional molecule SP-PNA. The molecule recognizes and inserts into the mammalian Sec61 translocon via its signal sequence (SP) module. The SP is connected to the peptide nucleic acid (PNA) module via a flexible loop. Once SP-PNA is internalized by the cell, it is free to specifically bind its unique mRNA target.

Fig. 5: The working principle of the SP-PNA molecule in mammalian cells. (A) In healthy cells, the SP-PNA construct does not jam the Sec61 translocon and may be released by other incoming protein substrate complexes. (B) In cells harboring specific mRNA target sequences in the cytoplasm, the PNA module of the SP-PNA construct tightly binds to the matching mRNA stretch. The translocon is jammed by the SP-PNA-mRNA complex. With its membrane system of protein translocation / insertion severely hampered, the cell is not able to maintain crucial functions and dies.

Fig. 6: Structure of protein Gasl with the signal sequence (SP) highlighted in orange (AlphaFold prediction AF-P22146- Fl) . The rest of the molecule was not part of the exemplary construct .

Fig. 7: Evaluation of the original Gasl signal recognition (probability score) by the bacterial SecYEG (A) and eukaryotic Sec61 (B) translocon (red lines) . Furthermore, sites for endogenous signal sequence cleavage are marked (yellow line) . For gram-positive bacteria (A) other colors represent recognition probabilities for other translocons (Tat and Lipo SP) . These values are close to zero, which means that the SP will not be recognized by other translocation systems. Analysis performed with SignalP5.0. Details on the usage of SignalP5.0 see in the following Figure.

Fig. 8: Comparison of selected signal sequences. (A) Probability for recognition by the Sec61 translocon (full lines) as well as cleavage site signal strength (dotted lines) . Analysis performed with SignalP5.0. (B) Sequence alignment of the Gasl SP variants as well as prion and insulin SPs in the context of the full SP-MBP construct sequences. Mutations in Gasl are marked in orange (W) or blue (N) . A cystein (C, orange) was introduced in the linker segment to enable labeling of the constructs. The 6 amino acid-long stretch in blue letters marks the sortase site necessary for later assembly of SP-PNA constructs, as well as the part of the poly-histidine tag (purple) used in SP-MBP purification. MBP: Maltose-binding protein.

Fig. 9: Comparison of recognition probabilities of all chosen signal sequences. Results for eukaryotic Sec61 (full line) and bacterial SecYEG (dotted line) . Analysis performed with SignalP5.0. For analysis, the 38-42 amino acid-long N-terminal sequence of the SP-MBP constructs was used to minimize the border effects.

Fig. 10: Construct design of SP-MBP variants with the 6x-His- tag (purple) following the sortase site (A) or being placed right after the SP component (B) .

Fig. 11: 1^st Purification Step Immobilized Metal Ion Chromatography (IMAC) . Purification of SP-MBP variants were done by IMAC. A general schematic representation of IMAC can be seen in (A) . (B) Nickel ions are immobilized via an NTA support to agarose resin. The Polyhistidine tag binds to Ni and is therefore retained by the agarose resin. (C) Schematic representation of an SP-MBP construct with its polyhistidine tag in purple. (D) representative SDS-PAGE of a successful purification of SP-MBP (here Gaslwt-MBP) . Whole cell lysate of overexpressing E.coli cells (Lys, lanel) , unbound fraction (UB, lane 2 ) ) and Eluatesl-5 (lanes 4-8) are separated via SDS- PAGE. Purified Gaslwt-MBP can be seen as a prominent band at a size of roughly 40 kDa . As size reference Precision Plus Protein Standard (Bio-Rad) has been used.

Fig. 12: 2^nd Purification Step - Size Exclusion Chromatography (SEC) . After labeling of the SP-MBP on the Cystein residue depicted in Fig.llC (black) with a fluorescent maleimide dye (AlexaFluor647 ) , a second purification step is performed using SEC. A general schematic representation of SEC can be seen in (A) . (B) At a run volume of 14-16, 5ml peaks for tryptophan absorption (black, 280nm) and fluorophore absorption (red, 650nm) , with a peak at 15,2ml for both channels, can be seen. These peaks correspond to fluorescently labeled Gaslwt-MBP.

(C) SDS-PAGE showing 5 fractions that cover run volume 14,5- 16ml .

Fig. 13: Analysis of purity and labeling efficiency. After the SEC step, the Gasl-MBP variants with the His-tag located prior and after the sortase site were analyzed on SDS PAGE in regular (left) and fluorescent (right) modes. Labelling with AlexaFluor488 appears somewhat more efficient as with AlexaFluor647 , however the position of the His-tag does not affect the labeling efficiency. Fig. 14: Principle of electrophysiological (EP) measurements. (A) Schematic representation of the current, I, through the lipid bilayer containing one SecYEG-Gasl-MBP complex (one fluctuating channel) . The average number of channels, Nr, in the membrane is defined as the average current measured through the membrane over time t, <Y>_t, divided by the current corresponding to a single SecYEG-Gasl-MBP complex, AJ^. When more complexes are present in the bilayer, the characteristic "skyline" current pattern is observed. Inset: in all experiments, the SecYEG-vesicles were added prior to the addition of the corresponding Gasl-MBP construct. (B) Only vesicles containing SecYEG-Gasl-MBP complexes fuse with the lipid bilayer. In this example, one should observe two opened SecYEG channels that were delivered into the planar bilayer. Above, a scheme of the patch-clamp amplifier connected to two Ag/AgCl electrodes is shown. The command electrode is immersed into the hypertonic compartment on the left side of the chamber. This compartment also contains the SecYEG-vesicles and Gasl-MBP constructs.

Fig. 15: SP potency as assessed by the SignalP5.0 scoring and electrophysiology. The average number, N, of open SecYEG channels (right Y-axis) is compared to the SP recognition probability by the translocon, P, (left Y-axis) calculated with SignalP5.0. N follows the trend predicted for P of the eukaryotic translocon (bars with solid white frames) and the prokaryotic translocon (broken frames) . GaslW-His shows the highest P and N values, followed by Gaslwt-His and the GaslN- His mutant with the lowest values.

Fig. 16: Mode of construct delivery. Lipid-enclosed SP-MBP constructs may either fuse directly with the plasma membrane (left) or may first enter the cell via endocytosis, followed by endosomal escape. In both cases, the SP-MBP constructs end up in the cytosol of the HEK293 cells.

Fig. 17: Expected results of HEK293 transfection with SP-MBP. (Left) HEK293 cells expressing membrane protein AQP4-eGFP are transfected with SP-MBP variants. (Middle) Due to endogenous proteases, the blockage of the Sec61 translocon is only temporal. (Right) A temporal decrease in membrane localization of AQP4-eGFP is expected, yet cell survival is expected to be on par with mock transfected cells.

Fig. 18: Effect of proteases on the SP-MBP constructs. (A) General scheme of proteolytic digest. (B) SDS-PAGE of cellular extracts. Coomassie-stain (left) of HEK293 cells expressing Aqp4-eGFP transfected with no (mock) or one of our SP-MBP constructs Gaslwt-/ GaslN-/ GaslW-/ prion-MBP. Fluorescent image (right) of the same SDS-PAGE gel showing fluorescence of AlexaFluor647-labeled SP-MBPs in red. Full-length SP-MBP constructs run at 40 kDa, while digested products can be seen at lower molecular weights.

Fig. 19: Localization of AQP4-GFP in mock transfected cells (no SP-MBP variants) . As AQP4 is a membrane protein, AQP4-eGFP is likewise localized to the cell membrane of HEK293 cells.

Fig. 20: Effect of SP-MBP variants on GFP membrane localization in AQP4-eGFP expressing HEK293 cells. Since not all cells take up the SP-MBP constructs, AQP4-GFP will localize to the plasma membrane of such cells (left images) . Cells harboring SP-MBP constructs as seen by the presence of AlexaFluor647 signals, membrane localization of AQP4-GFP is diminished if not completely missing (middle and right images) .

Fig. 21: Effect of SP-MBP variants on GFP expressing HEK293 cells. (A) Normalized percentage of GFP expressing cells. A reduction of 32 up to 52 % was observed for the cells transfected with respective SP-MBP variants. (B) Normalized percentage of cell viability of the same cells. Cell survival is unaffected by transfection with SP-MBP variants.

Fig. 22: Specific binding of respective PNAs to their respective target. Cy3-labeled DNA with specific target sequences of PNAfp, PNAe and PNActrl were either incubated with mock sample (negative control - ) , or respective PNA molecules. Cy3-DNA exclusively shifts when its corresponding PNA molecule is present, thus excluding off-target effects in subsequent experiments.

Fig. 23: Generating the SP-PNA fusion constructs. (A) Workflow of the SortaseA-based fusion of the SP to the respective PNA. (B) SDS-PAGE evaluation of the SortaseA reaction (coomassie stain - left; fluorescence image - right) , showing bands for the starting product (SP-MBP - top) , the SP-SrtA intermediate (middle) and the desired SP-PNA end product (bottom) . A marker (red bands) has been added to allow for size evaluation. Lanes 1 and 8 Precision Plus Protein marker (BioRad) , lane 2 SP-MBP control, lane 3 SP-MBP and SortaseA, lanes 4-7 SP-MBP, SortaseA and increasing amounts of PNA.

Fig. 24: Principal scheme of electrophysiology experiments with Gasl constructs. The experiments were the same as described in Fig.13. Here we used the SP Gasl in all samples: once followed with MBP, then by the PNA, and lastly by PNA with bound complementary RNA.

Fig. 25: Different Gasl conjugates interact with SecYEG identically in vitro. (A) Schematic image of Gasl (SP) constructs used for the experiment. (B) The average number of SecYEG channels activated by the constructs per bilayer (similar to the right axis on Fig.15) , as well as the single channel conductivity (SSC) is comparable for all three constructs used. SSC is a parameter used to estimate the size of the conductive pore.

Fig. 26: Expected results of HEK293 transfection with SP-PNAs. (A) Cells transfected with non-specific SP-PNAs show no major effect, since the SP-PNA will be released from the Sec61 translocon due to a missing binding partner. (B) Transfection of specific SP-PNAs will lead to reduction of fluorescence and cell death since the SP-PNA will target the mRNA of AQP4-eGPF expressed in these cells.

Fig. 27: SP-PNAs are not toxic for mammalian HEK293 cells. (A) Scheme of the experimental procedure. HEK293 cells are transfected with either Gasl-AF647-PNAfp or Gasl-A488-PNAfp . After a day of incubation, the cells are inspected via fluorescence microscopy and the cell survival is assessed. (B) Gasl-PNAfps are efficiently delivered to the HEK293 cells. (C) Cell survival of cells transfected with Gasl-PNAfp, or with Gasl-PNAm (complementary to the mRNA region of SARS-CoV-2 M protein) in absence of target mRNA is on par with mock transfected cells. Fig. 28: SP-PNActrl and eGFP can co-exist in HEK293 cells. (A) Scheme of the experimental procedure. HEK293 cells are first transfected with a plasmid coding for eGFP, followed by transfection with Gasl-AF647-PNActrl . After incubation, the cells are inspected via fluorescence microscopy, and the cell survival is assessed. The inspection reveals cells that have not been transfected with either plasmid or SP-PNActrl, cells that have been transfected with only one of the transfection agents, and cells that have been transfected with both. (B) Gasl-PNActrl does not target the mRNA of eGFP present in the cytoplasm of respective cells. (C) Gasl-PNActrl (left) and eGFP (middle) are mutually present (right) in cells successfully transfected with both, eGFP expression plasmid and Gasl-PNActrl.

Fig. 29: SP-PNAfp and eGFP are mutually exclusive in HEK293 cells. (A) Scheme of the experimental procedure. HEK293 cells are first transfected with a plasmid coding for eGFP, followed by transfection with Gasl-AF647-PNAfp . After incubation, the cells are inspected via fluorescence microscopy and cell survival is assessed. Inspection reveals cells that have not been transfected with either, eGFP plasmid or SP-PNAfp, and cells that have been transfected with only one of the transfection agents. As SP-PNAfp and eGFP are mutually exclusive, fluorescence microscopy is expected to show the lack of cells that have been transfected with both. (B) The scheme of the labelled Gasl-PNAfp-GFP mRNA complex. Gasl-PNAfp does target the mRNA of eGFP present in the cytoplasm of respective cells. (C) Gasl-PNAfp (left) and eGFP (middle) are mutually exclusive (right) in transfected HEK293 cells.

Fig. 30: Effect of PNAfp on HEK293 cells expressing eGFP (light green) or sfGFP (dark green) . 24 h post transfection, a significant reduction in the number of GFP expressing cells is apparent when SP-PNA targeting eGFP/sfGFP is being used. In contrast, SP-PNActrl only leads to a marginally reduced number of GFP expressing cells.

Fig. 31: Effect of SP-PNAs Gaslwt-PNActrl, Gaslwt-PNAfp, GaslN-PNAfp, and prion-PNAfp on Aqp4-GFP localization and cell phenotype. GFP fluorescence left, SP-PNA labeled with AlexaFluor647 in middle, and merged images in right columns (A-D) . (A) Gaslwt-PNActrl and Aqp4-GFP co-exist within individual cells with no change to Aqp4-GFP localization to the plasma membrane. (B) example of cells where Aqp4-GFP translocation to the plasma membrane is lost in the presence of Gaslwt-PNAfp . (C) example of cell detachment upon transfection with GaslN-PNAfp. (D) example of cell clumping in the presence of prion-PNAfp.

Fig. 32: Effect of SP-PNAfp targeting Aqp4-GFPmRNA on the number of GFP-expressing cells and cell viability. (A) Normalized percentage of cells expressing GFP as detected by automated cell counting (left graph) . As opposed to Gaslwt- PNActrl transfected cells, a decrease in GFP-expressing cells (plasma membrane and cytoplasmic localization were not distinguished) is observed in cells transfected with all SP- PNAfp constructs. (B) The effect of SP-PNAfp constructs on normalized viability is even more pronounced.

Fig. 33: Comparison of Gasl-PNA affinity to match or mismatch target sequences. (A) The respective melting temperature of mismatching or matching target (GFP) mRNA. (B) Normalized number of cells after Gasl-PNAfp transfection of HEK293 harboring mismatch or matching mRNA. Cell viability is not affected in cells harboring mismatch mRNA compared to mock- transfected cells, whereas the number of cells decreased in cells with matching GFP mRNA (right) .

Fig. 34: Absolute expression levels of genes RORA (A) and IQUB (B) according to GENEVES T I GATOR . RORA and IQUB mRNA show medium to high for RORA and low expression levels for IQUB for untreated HEK293 cells.

Fig. 35: Comparison of Gasl-PNAe affinity to endogenous target mRNA sequences of RORA or IQUB. (A) Respective melting temperature of RORA or IQUB target mRNA. Melting temperature of perfect base-pairing to the original target: 50.3°C (B) Normalized number of cells after Gasl-PNAe transfection of HEK293 harboring mRNA of endogenous genes RORA and IQUB. Both, untransfected and pEGFP-Aqp4 transfected cells, show a decrease in cell numbers. Fig. 36: Reciprocal translocation of chromosomes 22 and 11 in Ewing Sarcomas. Chromosomes 22 and 11, encoding for the genes EWSR1 and FLU, respectively, are rearranged in Ewing Sarcoma cells leading to the fusion of EWS-FLI1 genes. See Xiao et al, 2018.

Fig. 37: Breakpoint region of EWS-FLI1 in A673 cells. Green letters indicate the sequence encoded by gene EWSR1 while red letters show the sequence originating from FLU. The possible target sequence for a specific PNA is underlined.

Examples

Example 1: Materials & Methods

The following materials and methods were used in subsequent examples.

PNA sequence selection and production

PNAs were designed to be complementary to selected DNA/mRNA sequences of their target genes. Purine content was chosen to be less than 60 % and purine stretches were chosen not to exceed a length of 4. The length chosen for PNAs was 12 to 14 bases but shorter or longer sequences are possible, depending on target sequences and melting temperature, Tm, if necessary. Self-complementary sequences such as inverse repeats, hairpins, and palindromic sequences were avoided. Sequences were run against the human transcriptome using primer BLAST (https://www.ncbi.nlm.nih.gov/tools/primer- blast/) and candidate sequences were analyzed with the PNA tool (https://www.pnabio.com/support/PNA_Tool.htm) to obtain information on melting temperature Tm and selfcomplementarity. All PNAs were synthesized as peptide- conjugated PNAs with an N-terminal GGK peptide preceding the PNA. Peptide-conj ugated PNAs were ordered from PANAGENE Inc., South Korea (http://www.panagene.com/) , and sb-PEPTIDE, France (https://www.sb-peptide.com/) , and used at HPLC purified purity ( >90% ) .

SP-maltose-binding protein (MBP) construct cloning

The Gasl ( Saccharamyces cerevisiae, UniProt: P22146) , prion (Mesocricetus auratus, UniProt: P04273) and insulin (Homo sapiens, UniProt: P01308) signal sequences, as well as the linker harboring a cystein for labeling, the 6x-His-tag and the MBP sequences were cloned in-frame into the E. coli expression vector pET21a using the SLICE restriction-less cloning method or by adding short stretches via kissing primer PGR. Single point mutations or other limited changes were introduced via mutational PGR with kissing primers. The validity of the respective sequences was confirmed using a sequencing service (Eurofins Genomics Europe Shared Services GmbH, Germany) .

SP-MBP construct production

Overexpression of SP-MBP variants was achieved by clonal expansion of transfected E. coll C43 in LB medium containing appropriate antibiotics and induction with 1 mM IPTG for 4 h at 37°C. Cells were harvested and kept frozen at -20°C till further usage. Cell pellets were resuspended in Lysis Buffer (50 mM TrisHCl pH 7.5, 150 mM NaCl, 10 mM CaCl 10% glycerol and complete protease inhibitors (Roche) ) and lysed 3x with 20,000 psi on EmulsiFlex homogenizer. After a 10 min centrifugation step at 8,000 rpm and 10°C, another centrifugation step was performed for 30 min at 18,000 x g at 4°C. The lysate was applied to 2 ml equilibrated Ni-NTA resin and incubated for 1.5 h at 4 °C. After removing unbound proteins via gravity flow, the Ni-NTA resin was washed using Wash Buffer 1 (50 mM TrisHCl pH 7.5, 150 mM NaCl, 10 mM CaCl, 10% glycerol) and Wash Buffer 2 (50 mM TrisHCl pH 7.5, 150 mM NaCl) before elution with 5 x 1 ml Elution Buffer (50 mM TrisHCl pH 7.5, 150mM NaCl, 500 mM Imidazole, and 10% glycerol) . Protein concentration of purified SP-MBP was determined using Bradford Assays.

If applicable, purified SP-MBP was labeled ON at 4°C with maleimide dyes. On the next day, the buffer was exchanged to Srt Buffer (50 mM TrisHCl pH 7.5, 150 mM NaCl, 10% glycerol) by repeated centrifugation steps with centrifugal filter devices at 4,000 rpm and 8°C. This process also removed unbound maleimide dye. Alternatively, size exclusion chromatography on an Akta Pure system was utilized for buffer change, excess dye removal and further sample purification (Cytiva, Superdex200 Increase 10/300 GL) . Again, Bradford Assays were used to determine protein concentrations, which were adjusted to about 1 mg/ml prior to aliquoting of samples in liquid nitrogen. The resulting aliquots were stored at - 80°C. The quality of protein purifications and staining reactions were evaluated using SDS-PAGE. Sortase A (SrtA) purification

The expression plasmid encoding His-tagged SortaseA from St. aureus, pET30b-7M Srt was purchased from Addgene, USA (https://www.addgene.org/) and transformed into competent E. coll BL21 (DES) . Overexpression was induced with 0.5 mM IPTG in 1 L LB-Amp at mid-log phase for 16 h at 25°C. The collected pellet was resolved in ice-cold lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 5 mM MgC12, 10 mM imidazole, and 10 % glycerol) in the presence of complete protease inhibitor (Roche) and cells were lysed with an Emulsiflex homogenizer (Avestin) . Lysate was cleared by centrifugation at 20,000 x g at 4°C for 30 minutes and SortaseA was purified performing affinity chromatography on Ni-NTA- Agarose (Qiagen) , followed by size exclusion chromatography on an Akta Pure system (Cytiva, Superdex200 Increase 10/300 GL) . Purified SrtA was stored in aliquots at -80°C in Sortase Buffer (50 mM Tris pH 7.5, 150 mM NaCl, 10 % glycerol) . Concentration of SrtA was determined by Bradford Assay and quality of protein purifications were evaluated using SDS-PAGE.

SP-PNA fusion (Sortase reaction)

To link SP modules and PNA-modules, Sortase reactions were performed as follows: 130 nMol of labeled or unlabeled SP-MBP was mixed with 1 pMol peptide-conj ugated PNA and 200 nMol SrtA in 50 pl Sortase buffer (50 mM Tris pH 7.5, 150 nM NaCl) . Reactions were incubated for 2-3 h at room temperature. Samples were separated on SDS-PAGE including no-PNA and no- SrtA controls in a MES buffer system to increase resolution of low-molecular-weight proteins. Using a gel documentation device (Bio-Rad Laboratories, USA) the position of the SP-PNA reaction products was determined and gel slices were recovered from the SDS gel. Gel slices were crushed into small pieces and incubated with Sortase buffer containing 10 % glycerol at 30°C overnight on a rocking platform. After a 10-minute centrifugation, the supernatant was aliquoted and fresh-frozen to be stored at -80°C. Concentrations of labeled SP-PNA constructs were determined using fluorescence correlation spectroscopy . SecYEG purification and reconstitution

SecYEG Purification

The purification of SecYEG (wild type containing a mutation for labeling - SecY M142C) was performed as previously described (Knyazev et al, 2014; Knyazev et al, 2013) . Briefly, SecYEG variants were overexpressed for 4 h in E. coli C43 (DE3) cells from a pBAD22-derived expression vector using 2 L 2xYT media and subsequently induced with 2 g/L arabinose. The collected cell pellet was solved in extraction buffer (basic buffer - BB : 300 mM NaCl, 20 mM Tris pH7.5, 10% glycerol, HALT protease inhibitor cocktail as well as AEBSF (both ThermoFisherScientif ic) or solely complete protease inhibitor (Roche) ) and the cells lysed with an Emulsiflex homogenizer (Avestin) . The membrane fraction was pelleted at 100,000 x g and solvated in BB containing 1% (w/v) Dodecyl-malto-pyranoside (DDM, Anatrace) . Labeling with 10 pl of 10 mM AlexaFluor647 maleimide (Fluoroprobes) or Atto488 (Atto tec) was performed in course of affinity chromatography with Ni-NTA- Agarose (Qiagen) . Prior to labeling overnight at 4 °C, the bead-bound sample was treated with TCEP containing wash buffer (BB + 20 mM imidazole and 200 pM TCEP) to reduce the designated labeling cysteine (M142C) and thus increase labeling efficiency. After successful affinity purification, size exclusion chromatography on an Akta Pure system (Cytiva, Superdex200 Increase 10/300 GL) was performed to improve sample purity, to check for complex stability, approximate size estimation and to remove aggregates. The quality of protein purifications and staining reactions were evaluated using SDS-PAGE.

SecYEG Reconstitution into Lipid Vesicles

SecYEG was reconstituted into E. coli polar lipid extract (Avanti Polar Lipids) vesicles pre-dissolved in deoxy-BigChap (Anatrace) as previously described (Saparov et al, 2007) . A mass ratio of protein to lipid of 1:200 was used. Biobeads SM2 (Biorad) were added to remove the excess detergent and the resulting turbid suspension was pelleted at 100,000 g. The pellet was resuspended in reconstitution buffer (50 mM Hepes pH7.0, 10% (v/v) glycerol) and extruded through a 100 nm filter. 25 pl aliquots were flash frozen with liquid nitrogen and stored at -80°C for later usage.

Electrophysiology

The approach is described in previous publications (Saparov et al, 2007; Knyazev et al, 2013) . In brief, Ag/AgCl reference electrodes were immersed into the buffer solutions on both sides of the planar bilayers (Fig. 13, Right) . The command electrode of the patch clamp amplifier (model EPC9, HEKA electronics, Germany) is localized in the cis compartment, and the ground electrode in the trans compartment. The recording filter for the transmembrane current was a 4-pole Bessel with a -3dB corner frequency of 0.1 kHz. The raw data were analyzed using the TAG software package (Bruxton Corporation, Seattle, WA) . Gaussian filters of 12 Hz were applied to reduce noise. The data were further processed using Origin (OriginLab, Massachusetts, USA) .

Transfection of HEK293 cells

HEK293 cells were grown in DMEM (PAS) containing 10 % fetal calf serum (FCS) and Pen/Strep under normal cell culture conditions (37°C, 5 % CO₂) .

Plasmid transfections were performed using 293tran (Origene) according to the manufacturer's manual at a cell confluency from 50-70 %, when used the following day for SP- MBP or SP-PNA transfection.

For protein and peptide transfections of SP-MBP and SP-PNA Pierce protein transfection reagent was chosen. SP-MBP was used at a concentration of 50 nM, while for SP-PNA only 0,5 nM were used for transfections of cells at a density of 70-80 %. Fluorescence microscopy

Confocal images were acquired using an inverted laser scanning microscope (Zeiss LSM 510) with a water-immersion objective (Plan-Apochromat, 40x, NA 1.2) . 488 nm and 633 nm laser light was directed on to the samples via a dichroic beam splitter (488/561/633) . The signals were detected by means of an avalanche photodiode using a band-pass filter (BP 505-610 nm) or a long-pass filter (LP 650 nm) , respectively. Images were analyzed with Zeiss ZEN V3.6 software and with Image J VI .53q.

Fluorescence images were acquired using a SAFe 180 singlemolecule localization microscope (Abbelight) connected to an inverted microscope (1X83, Evident Europe) . Diode lasers with 488nm and 640nm (OXXIUS) were used to illuminate the sample via a dichroic beam splitter (405/488/532/640) . A digital CMOS-camera (C14440-20UP, Hamamatsu) was used to detect the signal which was analyzed using Fiji (ImageJ, version 2.14.0) . Cell counting

For the determination of cell numbers, GFP positive cells, and cell viability, a cell counting device (CellDropFL, DeNovix) was used. Settings for HEK293 cells were as follows:

Cell counting and viability counting were done using the Trypan Blue App on CellDropFL. Cells were detached from cell culture plates by a two-minute Trypsin/EDTA treatment after carefully rinsing cells with PBS. Trypsin treatment was stopped by adding FluoroBrite DMEM (Gibco) supplemented with 10 % FCS, Pen/Strep. Samples were treated according to the manufacturer and triplets were counted using the settings listed in Table 1.

Table 1: Settings for DeNovix CellDropFL Trypan Blue App and GFP App .

Numbers were normalized to control samples (mock transfected or Gaslwt-PNActrl ) . Example 2 : Choosing SP sequences and testing the concept of clogging the translocon with SP-MBP fusions

Choosing SP sequences

SPs do not have a fixed sequence - the natural SPs diverge in length and charge of the N-terminal charged region, and in the length and hydrophobicity of the hydrophobic span. To give an estimation of the given sequence potency as a signal sequence, we used the online statistics-based tool SignalP 5.0 (Almagro Armenteros et al, 2019) . This tool allows the prediction of the probability, P, of a respective SP for interacting with both, eukaryotic and prokaryotic translocons. To minimize the artifacts due to border effects, we used not only the SP itself for calculating the SP-score but the longer SP-linker sequence. The original sequence, Gaslwt, was chosen as an adequately potent SP for both eu- and prokaryotes (Fig. 6-7) , whose potency could be significantly increased or decreased by single amino-acid substitutions. Gasl is a 1,3- beta-glucanosyltransf erase from Saccharomyces cerevisiae (Fig. 8-9) . Note that the Gaslwt differs from the original Gasl sequence in the SP protease-site (AGA changed to SAC) . We then performed single amino-acid substitutions in different positions of the SP and chose the SP versions with the maximum score (GaslW) and the minimum score (GaslN) . The three SP versions of Gasl, Gaslwt, -N and -W , were then overexpressed and purified as parts of the Gasl-MBP constructs. The SPs of prion and insulin served as a standard of potent SPs.

Construct design of SP-MBP to evaluate SP impact

To test the SignalP5.0 predictions, we used Gasl-MBP (monofunctional) constructs (Fig. 10A) . They contained the SP version of interest, and MBP, a part common to all constructs. The linker between SP and MBP consists of: a single cysteine for fluorescent labelling, the sortase site enabling fusion of the anterior N-terminal part with the PNA module, and the His- tag for purification of the SP-MBP construct via affinity chromatography. The 42.5 kDa-large water-soluble MBP domain was added for the ease of construct purification. We produced two versions of Gasl-MBP constructs: with the His-tag anterior (Fig. 10A) and posterior (Fig. 10B) to the sortase site. The SP-MBP constructs were later used for creating bi-functional constructs .

We used the 20 a-a— long version of the Gasl SP (as described in Fig. 8) , or insulin's and prion's SPs. A flexible peptide linker GASA flanked the cysteine to provide accessibility .

SP-MBP production

As an alternative to solid-phase peptide synthesis and subsequent purification by reversed-phase HPLC, peptides can be expressed as fusion proteins and purified using different tags. For the overexpression, different host organisms can be used. The fusion domains are added to enhance the fusion proteins solubility and can also vary. Different affinity tags can be added for consecutive fusion protein purification. Two consecutive purification steps, like e.g. immobilized metal ion affinity chromatography (IMAC) and size exclusion chromatography (SEC) , enhance the purity of the fusion product .

For our SP-MBP production we chose E. coll as the expressing host organism, MBP as the soluble fusion partner of our signal peptides, and a hexa-Histidine tag for IMAC purification (Fig. 11) . The IMAC purification was followed by a SEC step (Fig. 12) .

In IMAC, the His-tag of the fusion construct SP-MBP coordinates Nickel ions that are chelated by a Ni-NTA resin. While all other proteins can be washed off the resin, His- tagged proteins are stably bound to the matrix and can be eluted with high concentrations of imidazole. Furthermore, bound protein can be labeled with fluorescence markers, as was done with SP-MBP (Fig. 13) .

Possible contaminations and unwanted higher order complexes or aggregates from the IMAC eluates can be excluded using SEC, which further separates the samples by size due to the different retention volumes of the particles. SP-MBP variants open SecYEG in-vitro

To test the prediction of the construct potency assessed by SignalP5.0, we performed in-vitro experiments where we demonstrated the probability of the construct to prime Sec translocons. We used the monofunctional construct Gasl-MBP (Fig. 10B) , where the versions of Gasl were either wt, GaslW or GaslN (Fig. 8B) . The SecYEG from E. coli was used as a target translocon. SecYEG was overexpressed in E. coli, purified and reconstituted into lipid vesicles, which were fused with free-standing planar bilayers formed by folding lipid monolayers from E. coli polar lipid extract (Avanti Polar Lipids) in the ~150 pm wide aperture of a PTFE film (Goodfellow) .

We added SecYEG vesicles in the presence of Gasl-MBP construct. If the construct bound to SecYEG, the osmotic gradient-driven fusion of SecYEG-vesicles with the bilayer was efficient (cf . Knyazev et al, 2014) . Successful fusion events of the vesicles containing SecYEG-Gasl-MBP complex with the bilayer manifested themselves in an abrupt increase of electric current flowing through the bilayer (Fig. 14) . To simultaneously apply the transmembrane potential and measure the current through the bilayer, we used a patch-clamp amplifier HEKA10 (HEKA electronics, Germany) .

The average current, <I>t, through the bilayer over 1 minute of observation under a transmembrane potential of 50 mV was transformed into the average number, N, of SecYEG-Gasl-MBP complexes in the bilayer by dividing the measured current by the current, AIx, conducted by a single complex. This number of complexes was the parameter we used to assess the potency of the constructs.

Electrophysiology data obtained on SecYEG correspond reasonably well with SignalP5.0's predictions for the eukaryotic Sec61 and the prokaryotic SecYEG (Fig. 15) . The quantitative correlation between N and P for prokaryotic cells may be further improved by including motor protein SecA in the experiments. SecA participates in the substrate protein presentation to SecYEG in-vivo - and that is the situation reflected by the topology database SignalP5.0.

Delivery of SP-MBP into mammalian HEK293 cells

Different methods are available for the delivery of peptides and proteins into the cytoplasm of eukaryotic cells.

(1) Cell-penetrating peptides (CPPs) can be covalently or non- covalently attached to their cargo and are taken up by cells through direct penetration, endocytosis, or via the formation of intermediate inverted micelles. (2) Lipid-based delivery methods (Fig. 16) rely on either direct fusion of the liposome carrying the cargo with the plasma membrane, or the uptake of the liposome via endocytosis and fusion with the membrane of the endosome. In all cases, the cargo is released into the cytoplasm of the cells. (3) pHlip is a pH-sensitive peptide that integrates into the membrane of cells upon low pH and therefore delivers cargo attached to its C-terminus via disulfide bonds into the cytoplasm. Since the cytoplasm is a reducing environment, disulfide bonds are broken, and the cargo is released.

To test the effect of SP-MBP constructs in eukaryotic cells, we chose the well-characterized human embryonic kidney cell line HEK293 for our experimental setup. Using lipid-based delivery, we were able to study the effect of different SP-MBP constructs on the expression level of cytoplasmatic GFP as well as GFP fused to a membrane protein (Aqp4-eGFP) , and the viability of cells.

Expectations

In the course of the experiment, HEK 293 cells expressing the membrane protein AQP4 fused to eGPF (AQP4-eGFP) were transfected with variants of the mono-functional SP-MBP construct (Fig. 17 - left) . AQP4-eGFP localizes to the cell membrane, as should be clearly visible upon inspection of the cells with fluorescence microscopy. Upon transfection, we expected a swift insertion of SP-MBPs into the Sec61 translocon, which should lead to a visible decrease in AQP4- eGEP membrane localization, as the corresponding pathway for its transport to the cell membrane is occupied (Fig. 17 - right) . Differences in the strength of the effect should align with prior described affinities of SP variants to the translocon. In contrast to SP-PNAs (see Example 3) , SP-MBPs are not protected from proteases (Fig. 17 - middle) . Hence, we expected these endogenous enzymes to gradually remove all SP- MBPs blocking the translocons. SP-MBPs are thus expected to only show a temporal effect on the distribution of AQP4-eGFP. Thus, the effect on the translocation of other membrane proteins is also expected to be temporal. We expected high cell survival rates of SP-MBP transfected cells on par with mock-transfected samples.

Our expectations were generally confirmed by the results described below.

Proteolytic digestion of SP-MBP

Signal Peptidase I cleaves the pre-protein after the SP region (Fig. 18) , usually recognizing the AXA sequence, where A is alanine, and X is any other residue (see Auclair et al, 2012) . This results in a cleaved-off SP and the mature protein. To avoid cleavage, in the Gasl construct, the original ATA motif in position 23-26 in our construct was substituted with SAC.

If the substrate construct cannot be cleaved by a signal peptidase, the likelihood that Sec translocons will stay jammed or be degraded is further increased. In both cases, cell viability will be strongly compromised.

AQP4-eGFP localization

To verify that our system can be used for studying the effects of mono-functional SP-MBP and bi-functional SP-PNA transfections, HEK293 cells were first transfected with the expression vector pEGFP-Aqp4. Transfected cells show GFP fluorescence in the plasma membrane. After 24 h cells were transfected with empty lipid nanocontainers to create mock controls for SP-construct experiments. Fluorescent imaging revealed no changes in Aqp4-eGFP localization after another incubation time of 24 h in mock transfected cells (Fig. 19) . Effects of SP-MBP variants on AQP4-eGFP harboring cells HEK293 cells expressing Aqp4-eGFP were transfected with AlexaFluor647-labeled SP-MBP-containing lipid nanocontainers (see Example 1) . 24 h later, fluorescent images were taken, showing that not all cells have taken up SP-MBP. The nontransfected cells serve as internal reference for comparison of GFP fluorescence of cells showing positive signals at 647nm, which indicates the uptake of SP-MBPs. As can be seen in Fig. 20, changes in intensity as well as complete loss of plasma membrane fluorescence (green) was observed in cells showing SP-MBP uptake (red) .

SP-MBP variants do not show an effect on cell survival

Transfection of Aqp4-eGFP expressing HEK293 cells with SP- MBP constructs showed a change in Aqp4-eGFP intensity and localization after 24 h, as seen in the fluorescent images (Fig. 20) . Automated cell counting revealed a reduction in the number of GFP-expressing cells ranging from 32 to 52 % when normalized to mock transfected cells (Fig. 21A) . This number still includes cells that show mis-localization of Aqp4-eGFP, thus, even a higher percentage of affected cells is expected.

Staining of dead cells with Trypan Blue showed low toxicity of SP-MBP constructs, with viability values ranging from 86 to 98 % as compared to mock-transfected cells (Fig. 21B) . Since SP-MBP fusions can be targeted by cellular proteases that are activated upon Sec61 blockage, we assume that SP-MBP constructs block translocons only temporally, the blockage is removed by proteases. To test this hypothesis, we harvested cellular extracts of our samples and ran them on SDS-PAGE (Fig. 18B) . Indeed, aside from full-length SP-MBP fusion proteins, also lower molecular weight degradation products became apparent on the fluorescent image, pointing to a digestion of the constructs by proteases.

Conclusions

In our HEK293 cell-based experiments, we show the effect of different SP-MBP constructs on Aqp4-eGFP fluorescence and cell viability. All experiments include a mock control as a reference, which is set to 100 % when comparing cell counts. As opposed to the mock control, all cells transfected with SP-MBP constructs show a decrease in GFP expression level while viability is not affected. Fluorescence imaging reveals a decrease in membrane localization of Aqp4-eGFP in the presence of SP-MBPs. The effect seems to be more pronounced with constructs GaslN-MBP (-53 %) and prion-MBP (-42 %) , while GaslW-MBP seems to reflect the effect of Gaslwt-MBP (-33 % and -32 %, respectively) .

Viability counts of the same cells range from 86 % (Gaslwt-MBP) , via 91 % (GaslN-MBP) , up to 97 % (prion-MBP) and 98 % (GaslW-MBP) , proving that these mono-functional SP-MBP constructs are not toxic for the cells.

Example 3: SP-PNAs successfully and specifically decrease the viability of cells harboring target NAs

PNA sequences

PNAs were designed based on the corresponding DNA/mRNA sequence of their target genes. The purine content was chosen to be less than 60 %, and purine stretches were chosen not to exceed a length of 4. The length chosen for the PNAs was 12 to 14 bases, but shorter or longer sequences are possible, depending on target sequences and Tm, if necessary. Self- complementary sequences such as inverse repeats, hairpins, and palindromic sequences were avoided. Sequences were run against the human transcriptome using primer BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) , and candidate sequences were analyzed with the PNA tool (https://www.pnabio.com/support/PNA_Tool.htm) to obtain information on melting temperature Tm and selfcomplementarity.

PNA sequences are listed in Table 2 below. Table 2 : Sequences and characteristics of PNA modules used in the experiments .

We chose Aqp4-eGFP as our target for PNAfp, since this fusion membrane protein can be easily monitored by its fluorescence , and the targeted sequence in the GFP moiety of the mRNA is unique in HEK293 cells .

On the other hand, PNAe is complementary to endogenous genes IQUB and RORA, both having a relatively high expression level in HEK293 cells ( see Targeting of Endogenous Genes ) .

As a control , we designed PNActrl , which has no complementary mRNA in the human genome and, therefore , serves as a negative (no target ) control in our experiments .

All PNAs were synthesi zed as peptide-conj ugated PNAs with an N-terminal GGK peptide preceding the PNA. These three amino acid-long peptides serve as nucleophile reaction partner for the enzyme SortaseA that is used to covalently link the signal peptides to the PNA molecules ( see Example 1 ) .

Speci ficity of PNA sequences chosen

To demonstrate the speci ficity of our designed PNAs , a gel-shi ft assay was performed using complementary DNA oligonucleotides covalently linked to the fluorescence dye Cy3 . Upon binding to PNA, a shi ft in the migrating Cy3-DNA can be observed . As seen in Figure 22 , all PNAs bound exclusively to their corresponding DNA molecules .

Generating the SP-PNA fusion constructs

To covalently link the signal peptides of the SP-MBP constructs ( see also Example 2 ) to our peptide-conj ugated (GGK-) PNAs, Sortase-catalyzed ligations were performed (Figure 23A) . The LPETG motif (Sortase site) found in SP-MBP constructs is detected by the enzyme SortaseA and serves as an acyl-donor. The enzyme forms an intermediate state with the signal peptide bearing N-terminus of SP-MBP attached via LPET, while leaving behind the glycine of the motif and the following C-terminal MBP. The N-terminal glycine of the peptide-conj ugated PNA serves as the nucleophilic acyl- acceptor in this reaction and is therefore ligated to the LPET motif of the signal peptide by SortaseA. Thus, a site-specific trans-peptidation leads to the desired SP-PNA constructs.

For the experiments in Example 2, the sequence Gaslwt (disclosed in Fig. 8B) was used as SP sequence in the peptide construct. In the other examples, other sequences disclosed in Fig. 8B were used as well as SP sequences.

As an example, we show Sortase reaction mixtures for the creation of Gaslwt-PNAfp . The samples were separated on SDS- PAGE (Figure 23B) and show fluorescent signals for Gaslwt-MBP, the Gaslwt-SortaseA intermediate and the Gaslwt-PNAfp end product. Subsequently, Gaslwt-PNAfp was purified by gel extraction. The Gasl-PNA constructs open SecYEG in-vitro

We performed similar experiments to the ones described in relation to Figs. 14-15 to clarify whether the Gasl-based construct still interacts with the Sec translocon when the proteinaceous MBP domain is substituted with the PNA module. The PNA module did not compromise Gasl-SP interaction with the translocon, neither does the annealing of complementary RNA (Figs . 24-25) .

Delivery of SP-PNAs into mammalian HEK293 cells

The delivery of SP-PNAs into mammalian HEK293 cells was performed as described in Example 2 ("Delivery of SP-MBP into mammalian HEK293 cells") .

SP-PNAs are not toxic for mammalian HEK293 cells

In order to study the toxicity of SP-PNA constructs, HEK293 cells were transfected with different SP-PNA constructs (Figure 27A) . 24 hours after transfection of the cells, SP-PNA localization was examined by fluorescence imaging and the cell viability was assessed using Trypan Blue (TB) staining.

Fluorescence (Figure 27B) images show cytoplasmic localization of SP-PNA Gaslwt-PNAfp, labeled with Atto488 and AlexaFluor647 , respectively. Cell counting of TB negative (live) and TB positive (dead) cells show no toxicity of SP-PNA constructs in the absence of target mRNA (Figure 27C) . Expectations

In course of the experiment, HEK 293 cells expressing the membrane protein AQP4 fused to eGPF are transfected with nonspecific variants of the SP-PNA construct (Figure 26A) . As shown before, AQP4-eGFP localizes to the cell membrane. Upon transfection, we expect a swift insertion of the SP-PNAs into the Sec61 translocon. As there is no interaction with a corresponding RNA molecule, the SP-PNA blocks translocon transiently. Consequently, we do not expect to see a strong effect on AQP4-eGFP localization or cell survival. Furthermore, in fluorescence imaging, one can observe cells that contain non-specific SP-PNAs and AQP4-eGFP in parallel.

In contrast, transfection with specific SP-PNAs (Figure 26B) leads to a strong interaction with target RNA present in the cytoplasm and subsequently to blockage of the Sec61 translocon. The artificial nature of the PNA renders SP-PNA immune to endogenous proteases and nucleases, which would otherwise gradually remove the blockage. With their translocon machinery blocked, the cells will not be able to maintain crucial cellular functions. Thus, we expect cells that express AQP4-eGFP (target mRNA present) to die, as AQP4-eGFP and specific SP-PNAs are mutually exclusive. This will lead to a clear reduction in cells expressing AQP4-eGFP as well as a clear reduction in general cell survival.

Our expectations were generally confirmed by the results described below.

Co-existence of SP-PNActrl and eGFP

To assess the effect of a non-target SP-PNA construct (Gaslwt-PNActrl ) on cells expressing cytoplasmic eGFP, HEK293 cells were transfected with expression plasmid pEGFP-Nl and Gaslwt-PNActrl 24 h later (Figure 28A) . After another 24 h, cell images were taken.

Upon expression of eGFP, its mRNA, as well as matured protein, can be found in the cytoplasm (Figure 28B) . Likewise, after transfection by lipid nanoparticles, Gaslwt-PNActrl is located in the cytoplasm. Since PNActrl has no complementary counterpart, Gasl-PNActrl will not block Sec61. Hence, eGFP mRNA and Gasl-PNActrl can co-exist in HEK293 cells without affecting cell survival or eGFP expression. As seen in Figure 28C, cells can harbor both eGFP (green) and Gaslwt-PNActrl (red) . eGFP-specif ic SP-PNAfp and eGFP are mutually exclusive

Using the same conditions of HEK293 cell transfection as above, we studied the effect of an SP-PNA construct (Gaslwt- PNAfp) specifically targeting a sequence in the eGFP mRNA (Figure 29A) .

Again, eGFP mRNA and protein can be found in the cytoplasm of cells transfected with the expression plasmid peGFP-Nl (Figure 29B) . After delivery of Gaslwt-PNAfp to the cytoplasm of the cells, PNAfp can bind to eGFP mRNA, creating a Gaslwt- PNAfp-mRNAGFP complex that jams Sec61 translocons at the ER membrane, leading to blockage of the Sec61 translocation pathway. Since efficient nuclease or protease activity on SP- PNA-mRNA conjugate is unlikely, cells will start lacking crucial membrane and ER luminal proteins and eventually die.

Consequently, the presence of pEGFP-Nl plasmid (as seen by GFP fluorescence) and Gaslwt-PNAfp (red) in a single cell were mutually exclusive. Figure 29C shows that HEK293 cells still expressing eGFP do not contain Gaslwt-PNAfp, while cells transfected with Gaslwt-PNAfp do not show any eGFP expression. Gasl-PNAfp specifically reduces the number of GFP-expressing cells

When normalizing the percentage of GFP expressing cells to mock-transfected controls, it became evident that Gaslwt-PNAfp specifically eliminates GFP-expressing cells (HEK293 cells transfected with pEGFP or pEGFP-Aqp4) , reducing their abundance by 70%, while Gaslwt-PNActrl only has a minor effect (23%) (Figure 30) . The reduction of GFP expression in the case of Gaslwt-PNActrl transf ectants can be explained by the temporal blockage of the Sec translocon, which showed no effect on their survival, as can be seen in Figure 27.

Similar effects were obtained when transfecting HEK293 cells with another expression vector that encodes superfolder GFP (pcDNA3-protE-sfGFP) , also a target for Gaslwt-PNAfp (Figure 30) .

Co-existence of AQP4-GFP and Gaslwt-PNActrl

Since turn-over rates of cytoplasmic proteins might differ from those of membrane proteins, we turned our attention on the effect of SP-PNAs on the transmembrane fusion protein Aqp4-GFP used earlier for experiments with Gasl-MBP fusion protein. Moreover, Aqp4 follows the Sec61 pathway to be translocated to the plasma membrane, thus, we expect that Aqp4-GFP membrane insertion will be compromised upon Sec61 j amming .

To show that jamming is not induced by an unspecific (arbitrary) Gaslwt-PNA construct, we transfected Aqp4-GFP expressing cells with the non-template construct Gaslwt- PNActrl. Figure 31A shows the presence of both, Aqp4-GFP mRNA and Gaslwt-PNActrl, in HEK293 cells. Strikingly, the membrane abundance of Aqp4-GFP was not affected.

SP-PNAfp constructs specifically affect AQP4-GFP localization and cell phenotype

Transfecting Aqp4-GFP expressing HEK293 cells with the SP- PNAfp constructs specifically targeting Aqp4-GFP mRNA, Gaslwt- PNAfp, GaslN-PNAfp, and prion-PNAfp, affected the Aqp4 membrane localization and/or phenotype of transfected cells. Some cells lost their ability to insert Aqp4-GFP into their plasma membrane in the presence of both, Aqp4-GFP mRNA and Gaslwt-PNAfp (Figure 31B) . Other cells detached from the glass surface and exhibited a roundish phenotype, as seen in Figure 31C in the presence of GaslN-PNAfp and its target mRNA. Prion- PNAfp transfection lead to detachment of transfected cells, and the expression of Aqp4-GFP seems to be limited to nontransfected cells (Figure 31D) . SP-PNAs specifically targeting Aqp4-GFPmRNA affect the percentage of GFP-expressing cells and their viability

Cell counting of samples taken from above experiments show a clear effect on the number of Aqp4-GFP-expressing cells and their viability. Numbers were normalized to control transfections (Gaslwt-PNActrl ) (Figure 32) .

A clear decrease in the number of GFP-expressing cells was observed, ranging from -17 % (GaslN-PNAfp) and -22 % (Gaslwt- PNAfp) down to -51 % (prion-PNAfp) . Even more pronounced is the effect on the viability of the cells. The viability was reduced to 63 % in the presence of GaslN-PNAfp, while for Gaslwt-PNAfp and prion-PNAfp it dropped to 35 % and 32 %, respectively. Thus, SP-PNAs specifically targeting an mRNA within a cell show enhanced apoptosis and decrease their target gene expression.

Compromised targeting of mismatched mRNA

According to literature (see, e.g., Jensen et al, 1997) , single mismatches between PNAs and their target mRNA reduce the melting temperature by 10 to 20°C, depending on the nucleotide substitution and the position within the complementary stretch.

To test Gaslwt-PNAfp on a mismatched target mRNA, 3 mismatches were introduced into the sequence stretch covering the PNAfp target site of an expression vector (pTracer NaVMs- GFP) with which HEK293 cells were transfected . As a comparison, the cells were transfected with an expression vector encoding a fully complementary GFP sequence. The next day, cells were transfected with Gaslwt-PNAfp or mock treated.

The estimated Tm for the mismatched PNAfp-mRNA pair is below 37°C, which should lead to weak or no binding of the SP- PNA construct under normal cell culture conditions (37°C, 5 % CO2 ) in cells expressing the mismatched mRNA. In contrast, the fully complementary mRNA sequence leads to a Tm far above 37 °C (estimated 65.4°C) and cells should be affected (Figure 33A) .

24 h after SP-PNA transfection, cells were counted and normalized to mock transfected cells. No reduction in cell count was seen in the presence of the mismatch, whereas cell numbers significantly go down when the PNA is fully matching its mRNA target (Figure 33B) .

In conclusion, SP-PNAs are highly specific to their target NA.

Targeting of endogenous genes

PNAe has complementary stretches with only a single mismatch in mRNAs of the endogenous genes RORA and IQUB as suggested by primer BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) . Both genes are expressed in untreated HEK293 cells according to GENEVES T I GATOR (see Hruz et al, 2008) with medium to high expression levels for RORA, and low expression levels for IQUB (Figure 34A and B) . The single mismatches found in their respective mRNA sequences lower the melting temperature, which is still above the culturing conditions of the HEK293 cells (>37°C) , enabling a stable binding of PNAe to the mRNAs (Figure 35) .

HEK293 cells were transfected with the expression vector for Aqp4-GFP (pEGFP-Aqp4) and after 24 h empty (mock) or Gaslwt-PNAe-bearing lipid nanoparticles were added to the cells. Additionally, untreated cells without an expression vector were transfected with the SP-PNA construct. After another 24 h under normal cell culture conditions (37°C, 5 % C02 ) , cells were counted and normalized to mock transfected controls .

Cell numbers strikingly decreased for the cells transfected with Gasl-PNAe, in both control cells (no vector) and transfected cells (pEGFP-Aqp4) . Independent of the presence of the expression vector, Gaslwt-PNAe induced enhanced cell death in HEK293 cells (Figure 35B) . Conclusions

SP-PNAs are bi-functional molecules capable of targeting the Sec translocon with their SP module on the one hand, and a specific NA within a eukaryotic or prokaryotic cell via their designed PNA module. The resulting jamming of Sec translocons solely in the presence of this specific NA allows for the selective elimination of cells expressing such unique NAs . In the experiments described above, signal peptide modules were designed with various probabilities of recognition by eukaryotic Sec61 and bacterial SecYEG translocons. Using the software SignalP5.0, we selected signal peptides of Gasl, prion, and insulin. To remove cleavage sites that are used by signal peptidases to cut signal peptides off their proteins, amino acids were substituted to decrease respective cleavage probabilities below 40%. The probability of prion and insulin signal peptides being recognized by both Sec61 and SecYEG was high. Moreover, two constructs were designed, which varied solely in the amino acid present at position 13 of Gasl (Alanine in the wild type, with medium recognition probability) , which significantly affected recognition by the Sec translocons (increased recognition of construct Gasl A13W, and weak recognition of construct Gasl A13N) .

The signal modules were cloned into a bacterial expression vector in a frame with a linker region (cystein, LPETG, His- tag) followed by the soluble protein MBP. Transfected E. coll cells were utilized for the overexpression process, after which the SP-MBP fusion proteins were purified via IMAC and size-exclusion chromatography (SEC) . In addition, the SP module was labeled with a maleimide dye at the Cystein in the linker region.

To test binding capabilities of SP-MBP constructs, they were subjected to bacterial SecYEG translocons reconstituted into liposomes. Only binding of SP-MBPs can open the translocons, which then leads to spontaneous fusion of such liposomes to a planar lipid bilayer in the presence of an osmotic gradient. The number of open translocons in the planar bilayer was followed for the different SP-MBPs and correlated well with recognition probabilities calculated by SignalP5.0, with GaslN having the lowest number of channel openings, followed by Gaslwt-MBP, and GaslW-MBP with the highest number of open channels.

Cell experiments were done in HEK293 cells expressing Aqp4-GFP. Cells were imaged after transfection with SP-MBP for localization of fluorescent markers. Furthermore, cells containing GFP and dead cell stain Trypan Blue were counted. All SP-MBP constructs used (AlexaFluor647 labeled Gas lwt-MBP, Gas lN-MBP, Gas lW-MBP, and prion-MBP ) showed a decrease in GFP intensities in the cell membrane , with some cells showing no GFP expression at all .

These findings were also reflected in the decreased number of cells counted positive for GFP . Viability was found to be close to mock-trans fected cells , suggesting that SP-MBP is not toxic for the cells . Analysis of cell extracts revealed degradation of SP-MBP, probably due to cellular proteases that are activated by Sec61 translocon blockage .

Additionally, we designed PNAs that either target speci fic segments within various mammalian expression vectors ( full complementarity to the GFP encoding part of mRNAs derived from pEGFP, pcDNAS-protE-s fGFP, pEGFP-Aqp4 but containing 3 mismatches in pTracer NaVMs-GFP ) , or endogenously expressed genes (with single mismatches in RORA and IQUB mRNAs ) , or a viral protein M from SARS CoV, which acted as a negative control . All of these constructs were synthesi zed with a short glycine-rich peptide sequence ( GGK) at the N-terminus , which serves as the acceptor site in the subsequent transpeptidation reactions .

Speci ficity of PNAs was veri fied performing gel shi ft assays with labeled complementary DNA.

The coupling of SP modules and PNA modules was performed by the enzyme SortaseA which covalently links the Sortase site ( LPET/G) within the SP module to the N-terminal peptide ( GGK) of the PNA module , thereby producing the desired SP-PNA end product . Reaction samples were separated via SDS-PAGE , SP-PNAs were extracted out of the gel , and their concentration was determined by fluorescence correlation spectroscopy .

At this stage we were able to produce a variety of bifunctional SP-PNA types capable of targeting the Sec translocon with di f ferent probabilities , and binding to speci fic mRNAs . Fluorescence labeling enabled SP-PNA tracking .

Electrophysiological measurements confirmed that the binding capability of SP modules to the SecYEG translocon is not af fected by di f ferent fusion partners . No di f ference was observed in the number of SecYEG channels opened by Gaslwt- MBP, Gaslwt-PNA, or Gaslwt-PNA in complex with complementary RNA. Hence, replacing MBP with the PNA modules did not alter the probability of the SP module recognition by the translocon .

Transfection of a bi-functional SP-PNA (Gaslwt-PNAfp) had no toxic effect on HEK293 cells since no complementary target mRNA was present, and Gaslwt-PNA did not obstruct the translocon. Likewise, HEK293 cells expressing cytoplasmic eGFP demonstrate the coexistence of GFP mRNA expressed from the transfected vector and Gaslwt-PNActrl, which has no target in these cells.

In contrast, Gaslwt-PNAfp cannot be detected in cells expressing eGFP, presumably since eGFP mRNA binds to the SP- PNA, thus blocking the Sec61 translocon. The SP-PNA-mRNA complex bound to the translocon cannot be efficiently digested, which results in elimination of eGFP-expressing cells from the culture containing Gaslwt-PNAfp.

Counting Gaslwt-PNAfp-transf ected cells indicated a decrease in GFP-expressing cells by 70% compared to mock controls, while Gaslwt-PNActrl transfection resulted in only a slight reduction (23%) . Similar results were obtained when testing HEK293 cells expressing the superfolder version of GFP (sfGFP) .

The evaluation of SP-PNA effects on HEK293 cells expressing the membrane protein Aqp4-GFP showed a similar outcome. While Aqp4-GFP and control Gaslwt-PNActrl can coexist, SP-PNA constructs targeting GFP influence Aqp4-GFP abundance and the cell phenotype. In some cells, membrane staining of Aqp4-GFP vanishes, while others detach from the vessel surface and start rounding up. Additionally, cell clumping, indicative of sticky free DNA released by dying cells into the medium, is observed. Further experiments were conducted to investigate a mismatched target for Gaslwt-PNAfp. The mammalian expression construct pTracer-NaVMs-GFP utilizes slightly different codons, resulting in a mismatch of three nucleotides in the PNAfp recognition sequence. Jensen et al, 1997, reported that mismatches between PNA and its target RNA lead to a reduction in the melting temperature, destabilizing the PNA:RNA complex. According to that, the Tm from 65.4 °C is decreased to a temperature lower than 37 °C, which is the temperature at which HEK293 cells are grown. Thus, cells can survive the treatment since there is no binding of Gaslwt- PNAfp to NaVMs-GFP mRNA.

Mismatches resulting in a Tm above culture conditions (>37°C) lead to stable binding of SP-PNA to its target. PNAe was designed to target two different genes natively expressed by HEK293 cells (RORA and IQUB) with only a single mismatch leading to a Tm high enough to allow a stable PNA:RNA complex under normal culture conditions. When HEK293 cells were transfected with Gaslwt-PNAe, cell numbers decreased dramatically, regardless of the expression of a vector-derived mRNA.

The experiments collectively demonstrate that the SP- module on its own (SP-MBPs and unspecific SP-PNAs) does not have a toxic effect on HEK293 cells. Only the presence of an SP module linked to a PNA module that specifically targets an mRNA present within the cell can lead to Sec61 jamming. This is reflected by the disappearance of the membrane protein Aqp4-GFP and the impact on the cell phenotype, like the detachment from surfaces, a process typically prevented by the action of certain other membrane proteins.

Example 4 : SP-PNAs for use in mammalian individuals

SP-PNA for use in mammalian individuals (such as mice or humans) trials are synthesized by direct synthesis, which is expected to greatly enhance purity and yield of the desired SP-PNAs, omitting laborious purification steps.

Care is taken when selecting the length of the linker between the SP module at the N-terminus and the PNA module at the C-terminus. The reason is that the SP still needs to be able to place itself in the Sec61/SecYEG translocon, with sufficient linker space to (i) efficiently interact with the translocon channel and (ii) ensure PNA accessibility at the cytoplasmic entrance of the translocon. Consequently, the His- Tag and sortaseA site used in the previous examples are replaced with neutral amino acids that provide flexibility, yet will not lead to aggregation or unwanted interactions due to strong hydrophobic, polar, or electrostatic forces.

Typically, a flexible linker with a pattern of glycines followed by one or two serines is used (e.g. GGGS or GGSGS) . Multiple repeats of such a pattern may be used to span the desired length of the linker. For synthesis reasons, to avoid aggregation in the course of synthesis of too hydrophobic products ) , e.g., individual charged or polar residues may optionally be introduced to raise the solubility of the desired SP-PNAs. Various resources exist to help in linker design, such as SynLinker (Lie et al, 2015) , the Registry of Standard Biological Parts (http://parts.igem.org/Protein_domains/Linker) , or a review by Chen et al., 2013.

The SP component of the constructs is altered according to the application's needs. In the previous examples, we have shown the diversity of affinities towards Sec61 (eukaryote) and SecYEG (bacterial) of different wild-type signal sequences (Gasl, prion, insulin) as well as the effect of single point mutations. We have also shown that the online tool SignalP5.0 can be effectively utilized to predict SP affinities towards the translocon and the occurrence of unwanted cleavage sites. The SPs used in the previous experiments already show high affinities and are thus suitable for further application.

Lastly, the specific PNA sequence used is adapted to the respective needs of the application. SP-PNAs provides the necessary flexibility to perform these adaptations and consequently allows the SP-PNAs to be used in a plethora of scenarios .

Example 5: SP-PNAs in cell biology

SP-PNAs have the great advantage of leading to the death of cells expressing unique (m)RNAs, while showing no toxicity towards other cells . We have already shown that SP-PNAs can be easily trans fected into cells by means of liposomes .

In cell culture , one can exploit the selective nature of bi- functional SP-PNAs by fine-tuning the SP module and/or targeting speci fic (m) RNAs with the PNA-module .

By the modi fication of the SP-module , one can vary the recognition probability by the Sec translocon, creating constructs that are less likely to be or stay bound, achieving a partial blockage of the translocation process . Thus , tuning of the presence of membrane proteins that follow the Sec translocation pathway can be achieved .

Mixed or chimeric cell cultures obtained when culturing biopsies or tissues can be "puri fied" by eliminating cells that are not wanted, when targeting their uniquely expressed genes .

Knock-out of a defined set of cells in in-vi vo studies can help understanding their role in their natural environment .

Example 6 : SP-PNA for use in treatment of Ewing Sarcoma

Ewing Sarcoma is a highly malignant tumor of the bones mostly af fecting people in their second decade of li fe . Despite the development of new therapies , the survival rate for patients suf fering from such tumors is still less than 30% , due to its metastatic nature and high probability of relapse .

The Ewing sarcoma family of tumors (ESFT ) is characteri zed by reciprocal chromosomal translocations that lead to the fusion of two genes generating a novel gene , which acts as an aberrant oncogenic transcription factor . One fusion partner is EWSR1 (Ewing sarcoma breakpoint region 1 ) while the other fusion partner is a gene of the ETS family of transcription factors . In 80 % of cases the chromosomal rearrangement creates the hybrid gene EWS-FLI 1 , while EWS-ERG is found in 15 % of patients , and less abundant are fusions between EWS and ETV1 , E1AF and FEV . Breakpoint regions of oncogenic fusion genes can serve as targets for SP-PNAs, thus we examined the chromosomal fusion creating EWS-FLI1 in the Ewing sarcoma cell line A673 with the reciprocal translocation of chromosomes 22 (EWS) and 11 (FLU) , on mRNA level (Figs. 36-37) . Using the criteria mentioned earlier, a target sequence for SP-PNA was chosen (as shown in Fig. 37) and analyzed with primerBLAST, showing no significant similarity with other human mRNAs .

A SP-PNA with a sequence complementary to the target sequence is provided and tested in Ewing Sarcoma cell line A673 alone or in a mixture of A673 cells and other human cells to test its selective specificity.

If the tests are successful, a lipid formulation of SP-PNA (e.g., encapsulated in liposomes or LNPs) is administered to a mammalian individual.

Example 7 : An SP-NAM other than SP-PNA in cell biology

Based on the above experiments conducted with SP-PNAs, SP- LNAs (SP-locked nucleic acids) are also expected to exhibit a capacity to selectively induce death in cells expressing specific mRNA sequences while demonstrating negligible toxicity toward non-target cells.

In cell culture, SP-LNAs are transfected using liposomal delivery methods, as previously validated for SP-PNAs. The constructs allow for fine-tuning through modification of either the SP module or the LNA sequence. Adjustments to the SP component enable modulation of Sec translocon recognition probability, facilitating partial or complete inhibition of protein translocation. This feature provides a versatile tool for studying the dynamics of membrane proteins that rely on the Sec pathway, such as ion channels or receptors.

The specificity of SP-LNAs is particularly beneficial in mixed or chimeric cell cultures derived from biopsies or tissues. By targeting mRNA sequences uniquely expressed by undesired cells the constructs enable selective elimination, effectively "purifying" the culture to retain only desired cell types.

In experimental in vivo applications, this allows for selective knockout of defined cell subsets within complex tissues. The non-toxic nature of SP-LNAs will ensure that adjacent cells remain unaffected by the SP-LNAs themselves during experiments.

Example 8: Another SP-NAM for use in mammalian individuals

SP-PMOs ( SP-phosphorodiamidate morpholino oligomers) for use in mammalian individuals, such as mice or humans, are synthesized using direct synthesis methods.

In designing these constructs, careful consideration is given to the linker length between the SP module at the N- terminus and the PMO module at the C-terminus. The SP should maintain sufficient mobility to engage with the Sec61/SecYEG translocon complex while ensuring the PMO remains accessible at the cytoplasmic entrance of the channel. Therefore, flexible linkers composed of glycine-serine repeats (e.g., GGGS or GGSGS) may be employed, with multiple repeats used to adjust linker length as needed. To mitigate synthesis challenges associated with overly hydrophobic products, charged or polar residues may be selectively introduced to enhance solubility.

The SP component is optimized based on application requirements, based on prior observations of wild-type signal sequences (e.g., Gasl, prion, insulin) and single-point mutations for their affinities toward Sec61 (eukaryotic translocon) and SecYEG (bacterial translocon) , see above.

The specific PMO sequence is tailored to target complementary regions within the NA molecule of interest, with sequence design guided by thermodynamic analysis tools like UNAFold (Markham & Zuker, 2008) . The modular nature of SP-PMOs allows for adaptation across diverse scenarios , including therapeutic applications targeting oncogenic mRNAs or viral RNA. This approach ensures precise inhibition of protein translocation in target cells while maintaining minimal impact on non-target cell populations .

A lipid formulation of the SP-PMO ( e . g . , encapsulated in liposomes or LNPs ) may be produced for administration to a mammalian individual .

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Claims

1. A composition for inhibiting protein translocation in cells harbouring a target nucleic acid (NA) molecule, the composition comprising

- a peptide construct, comprising

- a protein secretion signal amino-acid sequence ("SB sequence") , and

- a nucleic acid mimic (NAM) sequence comprising 5 to 30 consecutive bases complementary to a sequence of the target NA molecule; and

- a delivery vehicle for delivering the peptide construct to cells.

2. The composition of claim 1, wherein the NAM is selected from the group consisting of peptide nucleic acids (PNAs) , phosphorodiamidate morpholino oligomers (PMOs) , locked nucleic acids (LNAs) , and threose nucleic acids (TNAs) ; preferably wherein the NAM is a PNA.

3. The composition of claim 1 or 2, wherein the delivery vehicle is selected from the group of lipid particles, viral particles, polymer particles, lipid-polymer particles, dendrimers, cell-penetrating peptides (GPPs) and lipid moieties .

4. The composition of claim 3, wherein the delivery vehicle is a lipid particle selected from liposomes, lipid nanoparticles (LNPs) and exosomes; or wherein the delivery vehicle is a CPP selected from the group consisting of TAT peptides, penetratins, arginine-rich peptides, transportans , pH-low insertion peptides, and SynB-based peptides.

5 . The composition of any one of claims 1 to 4 , wherein the SP sequence has a length of 10 to 40 amino acids , preferably 11 to 35 amino acids , more preferably 12 to 30 amino acids , even more preferably 13 to 27 amino acids , yet even more preferably 14 to 26 amino acids , especially 15 to 25 amino acids or even 16 to 24 amino acids .

6 . The composition of any one of claims 1 to 5 , wherein the SP sequence targets the peptide construct to a translocon, preferably a Sec61 translocon or a secYEG translocon .

7 . The composition of any one of claims 1 to 6 , wherein the translocon is a bacterial , archaeal , fungal , animal or plant translocon, preferably wherein the translocon is a vertebrate translocon, especially a mammalian translocon such as a human translocon .

8 . The composition of any one of claims 1 to 7 , wherein the SP sequence first enters the translocon followed by NAM sequence , preferably wherein the SP starts at an N-terminus of the peptide construct .

9 . The composition of any one of claims 1 to 8 , wherein the SP sequence comprises a positively charged N-terminal segment , a hydrophobic segment and optionally a C-terminal cleavage site .

10 . The composition of any one of claims 1 to 9 , wherein the SP sequence does not comprise a cleavage site for signal peptidase I .

11 . The composition of claim 10 , wherein the SP sequence does not comprise a cleavage site for any signal peptidase .

12. The composition of any one of claims 1 to 11, wherein the cells have FtsH activity.

13. The composition of any one of claims 1 to 12, wherein the cells have ZMPSTE24 or Ste24 activity.

14. The composition of any one of claims 1 to 13, wherein the SP sequence is covalently linked to the NAM sequence.

15. The composition of any one of claims 1 to 14, wherein the SP sequence is linked to the NAM sequence by a linker, preferably a linker peptide.

16. The composition of any one of claims 1 to 15, wherein at least 10, preferably at least 11 or even at least 12, more preferably at least 13 or even at least 14, even more preferably at least 15 or even at least 16, yet even more preferably at least 17 or even at least 18, especially at least 19 or even at least 20 consecutive amino acids of the SP sequence have a predicted translocon recognition probability of at least 0.4, preferably at least 0.6, more preferably at least 0.7, yet even more preferably at least 0.8, especially at least 0.9.

17. The composition of any one of claims 1 to 16, wherein at most 3, preferably at most 2, more preferably at most 1, especially no amino acids of the SP sequence have a predicted cleavage probability by a signal peptidase of at least 0.3, preferably at least 0.4, more preferably at least 0.5, yet even more preferably at least 0.6, especially at least 0.7.

18. The composition of any one of claims 1 to 17, wherein the delivery vehicle is non-covalently or covalently bound to the peptide construct.

19. The composition of any one of claims 1 to 18, wherein the peptide construct further comprises a label, preferably a fluorescent label.

20. The composition of any one of claims 1 to 19, wherein the target NA molecule is desoxyribonucleic acid (DNA) such as extrachromosomal plasmid DNA or viral DNA, or RNA, preferably messenger RNA (mRNA) , transfer RNA (tRNA) , viral RNA or microRNA (miRNA) .

21. The composition of any one of claims 1 to 20, wherein the target NA molecule is viral, bacterial, archaeal or eukaryotic, preferably vertebrate, more preferably mammalian, especially human.

22. The composition of any one of claims 1 to 21, wherein the cells are bacterial, archaeal or eukaryotic, preferably vertebrate, more preferably mammalian, especially human.

23. The composition of any one of claims 1 to 22, wherein the cells are diseased cells.

24. The composition of any one of claims 1 to 23, wherein the cells are neoplastic, preferably malignant, especially cells of a solid tumor or a haematological malignancy.

25. The composition of any one of claims 1 to 24, wherein the composition is a pharmaceutical composition, preferably comprising pharmaceutically acceptable excipients.

26. A method for inhibiting protein translocation in cells harbouring a target NA molecule, comprising the step of:

- delivering to the cells a peptide construct, comprising

- an SP sequence, and

- a NAM sequence comprising 5 to 30 consecutive bases complementary to a sequence of the target NA molecule.

27. The method of claim 26, wherein the NAM is selected from the group consisting of PNAs, PMOs, LNAs, and TNAs, preferably PNAs .

28. The method of claim 26 or 27, wherein the SP sequence is as defined in any one of claims 1 to 25.

29. The method of any one of claims 26 to 28, wherein the cells are as defined in any one of claims 1 to 25.

30. The method of any one of claims 26 to 29, wherein the target NA molecule is as defined in any one of claims 1 to 25.

31. The composition of any one of claims 1 to 25 for use in therapy, preferably for use in the method of any one of claims 26 to 30.

32. The composition for use according to 31, for use in prevention or treatment of an infection.

33 . The composition for use according to 31 , for use in prevention or treatment of a neoplasm, preferably a malignant neoplasm, especially a solid tumor or a haematological malignancy .

34 . The composition for use according to 31 , for use in prevention or treatment of a somatic mutation .

35 . The composition for use according to 31 , for use in prevention or treatment of an autoimmune disease or an inflammatory disease .

36 . The composition for use according to 31 , for use in treatment of cell senescence .

37 . The composition for use according to any one of claims 31 to 36 , wherein the composition is administered to an individual in need thereof , preferably by intravenous administration, transdermal administration, intradermal administration, intramuscular administration, intraosseous administration, intravitreal administration, intraperitoneal administration, intrathecal administration, oral administration, topical administration or by inhalation .

38 . Use of the composition or method of any one of the claims 1 to 37 for treating a plant or fungus .

39 . Use of the composition or method of any one of the claims 1 to 38 for pest control .

40 . Use of the composition or method of any one of the claims 1 to 39 for non-human animal population control or control of an invasive species .

41 . A method for producing a peptide construct for inhibiting protein translocation in cells harbouring a target NA molecule , the method comprising the steps of

- providing a peptide comprising an SP sequence ,

- providing a NAM comprising at least 5 consecutive bases complementary to a sequence of the target NA molecule ,

- linking the peptide to the NAM to obtain the peptide construct .

42 . The method of claim 41 , wherein the NAM is selected from the group consisting of PNAs , PMOs , LNAs , and TNAs .

43 . The method of claim 41 or 42 , wherein the NAM is a PNA.

44 . The method of claim 43 , wherein the peptide further comprises a donor site recogni zed by a sortase and the PNA further comprises an acceptor site recogni zed by the sortase , wherein the linking comprises contacting the peptide with the PNA and the sortase to ligate the donor site to the acceptor site .

45 . The method of any one of claims 41 to 44 , wherein the SP sequence is as defined in any one of claims 1 to 25 .

46 . The method of any one of claims 41 to 45 , wherein the cells are as defined in any one of claims 1 to 25 .

47 . The method of any one of claims 41 to 46 , wherein the target NA molecule is as defined in any one of claims 1 to 25 .

48 . A method for producing a composition for inhibiting protein translocation in cells harbouring a target NA molecule , comprising performing the method of any one of claims 41 to 47 , and further comprising the step of

- binding the peptide construct to a delivery vehicle for delivering the peptide construct to cells or encapsulating the peptide construct in a delivery vehicle for delivering the peptide construct to cells , to obtain the composition .

49 . The method of claim 48 , wherein the delivery vehicle is as defined in any one of claims 1 to 25 .