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WO2025219570A1 - Composition pour inhiber la translocation de protéines et procédés l'utilisant - Google Patents

Composition pour inhiber la translocation de protéines et procédés l'utilisant

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Publication number
WO2025219570A1
WO2025219570A1 PCT/EP2025/060733 EP2025060733W WO2025219570A1 WO 2025219570 A1 WO2025219570 A1 WO 2025219570A1 EP 2025060733 W EP2025060733 W EP 2025060733W WO 2025219570 A1 WO2025219570 A1 WO 2025219570A1
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Prior art keywords
cells
composition
sequence
target
pna
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Inventor
Denis KNYAZEV
Peter Pohl
Christine SILIGAN
Nikolaus GÖSSWEINER-MOHR
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JOHANNES KEPLER UNIVERSITAT LINZ
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JOHANNES KEPLER UNIVERSITAT LINZ
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • C07K14/003—Peptide-nucleic acids (PNAs)

Definitions

  • 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 .
  • NA nucleic acid
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • 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 .
  • mRNA messenger RNA
  • 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 .
  • SecA a cytoplasmic ATPase that provides the energy needed for protein translocation .
  • 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.
  • compounds that generally inhibit protein translocation can be classified as toxins.
  • Eeyarestatin I 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 .
  • mycolactone produced by Mycobacterium ulcerans , which is the causative agent of a necrotizing skin disease known as Buruli ulcer (Deband & 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.
  • mycolactone 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 & Debal, 2024.
  • 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) .
  • diseased cells e.g., cells infected by a virus, cells harboring an undesired genomic mutation, or tumor cells
  • 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.
  • 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.
  • SP sequence protein secretion signal amino-acid sequence
  • NAM nucleic acid mimic
  • 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) .
  • PNAs peptide nucleic acids
  • PMOs phosphorodiamidate morpholino oligomers
  • LNAs locked nucleic acids
  • TAAs threose nucleic acids
  • the NAM is a PNA.
  • the peptide construct is also called "SP-NAM” herein.
  • 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.
  • 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.
  • a linker peptide or bioconjugation tools for instance, click-chemistry tools.
  • 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) .
  • the inventive composition is provided for use in therapy, in particular for use in the inventive method.
  • 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 .
  • 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 .
  • 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 .
  • a target NA molecule in the cytoplasm
  • NAM preferably PNA, PMO, LNA or TNA; in particular a PNA
  • 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 .
  • 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 ) .
  • SP-PNA is not toxic in the absence of target NA.
  • the SP-NAM in particular SP-PNA
  • the SP-NAM forms a larger complex with the target NA and binds to the Sec translocon via the SP module .
  • inventive concept is also applicable to cells without a nucleus such as bacterial cells , thereby opening a new avenue for highly targeted antibiotics .
  • 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 .
  • 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.
  • a signal peptide SP
  • is generally sufficient for binding the substrate protein to the translocon see Simon & Blobel, 1992 and Vorhees & Hedge, 2016.
  • 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.
  • 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) .
  • DNA serves as the primary carrier of genetic information.
  • 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.
  • 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.
  • nucleobases within DNA and RNA have been leveraged in artificial molecules engineered to mimic this behavior (see e.g., Duffy et al, 2020) .
  • NAMs also called NA analogs or xeno NAs
  • nucleobases are positioned along an artificial backbone structure.
  • 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.
  • NAMs incorporate one or more of the standard (or canonical) nucleobases: adenine, guanine, cytosine, thymine and uracil.
  • 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.
  • 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.
  • the NAM is preferably selected from the group consisting of PMOs, LNAs, TNAs and PNAs.
  • 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.
  • PMOs 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.
  • FDA US Food and Drug Administration
  • DMD Duchenne muscular dystrophy
  • 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.
  • TNA TNA
  • TNA TNA
  • LNAs 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.
  • glycineserine repeats e.g., GGGS or GGSGS
  • Bioconjugation strategies may vary by NAM chemistry:
  • 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;
  • 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 .
  • PNAs 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.
  • PNA can be synthesized with relative ease using regular peptide synthesis methods with base building blocks (see e.g., Nandhini et al, 2023) .
  • 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) .
  • 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.
  • nucleobases of PNAs 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.
  • 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.
  • PNAs are 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) .
  • rRNA ribosomal RNA
  • 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.
  • 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.
  • PPR pentatricopeptide repeat
  • 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 .
  • the peptide construct 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.
  • a non-target cell e.g., a healthy cell such as a non-cancerous cell
  • SP-NAM in particular SP-PNA
  • the SP sequence 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.
  • 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 .
  • 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 .
  • 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 .
  • 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 .
  • 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 .
  • 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.
  • 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 .
  • 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 .
  • SP sequences are disclosed in WO 2015/127094 Al (incorporated herein by reference) , in particular in Table 1 thereof.
  • 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.
  • 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.
  • the (targeted) cells have FtsH activity (e.g., if they are bacterial) , or ZMPSTE24 or Ste24 (if they are eukaryotic, e.g. mammalian) activity.
  • FtsH activity e.g., if they are bacterial
  • 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.
  • FRET Förster Resonant Resonant Resonant Resonant Resonant Resonant Resonant Resonant Resonant Resonant Resonant Resonant Resonant Resonant Resonant Resonant Resonant Resonant Resonant Resonant Resonant Resonant Resonant Resonant Resonant Resonant Resonant Resonant Reduction mediated by 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.
  • mass spectrometry-based proteomics may be employed to identify and quantify proteolytic processing events and enzyme expression levels .
  • 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.
  • 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) .
  • 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.
  • DNA deoxyribonucleic acid
  • 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.
  • SP-NAMs in particular SP-PNAs
  • 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.
  • 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.
  • the cells are preferably neoplastic, preferably malignant, especially cells of a solid tumor or a haematological malignancy.
  • 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.
  • 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.
  • 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 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.
  • NCBI National Center for Biotechnology Information
  • S spike
  • 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.
  • 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.
  • RNA analysis 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 .
  • NGS next-generation sequencing
  • PGR polymerase chain reaction
  • RNA sequencing RNA-seq
  • microarray analysis microarray analysis to determine which RNAs are overexpressed compared to normal cells.
  • a target RNA By comparing the transcriptomes of diseased and healthy cells, a target RNA can be identified based on its differential expression patterns .
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.) .
  • SP-NAMs in particular SP-PNAs
  • 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.
  • 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 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 .
  • antiviral SP-NAMs 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 .
  • NAM sequences in particular PNA sequences
  • SP-PNAs antiviral SP-NAMs
  • 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 .
  • SP-NAMs in particular SP-PNAs
  • SP-PNAs SP-PNAs
  • 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 mecanicreturn to the drawing board" - also known as a return to basic efficacy tests.
  • altered chemistry of the active molecule requires vigorous safety testing and very likely leads to new Phase 1 to Phase 3 studies.
  • the respective compound will lose its efficacy and there is no quick way to adapt it.
  • SP-NAMs in particular SP-PNAs
  • 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 brieflyfollow" 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.
  • the viral genes need to be transcribed to mRNA either utilizing viral or host enzymes.
  • 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) .
  • SP-PNAs specific antiviral SP-NAMs
  • 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
  • SP-PNAs can also be directed against this primary DNA or RNA as it is not protected by the nucleus.
  • 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) .
  • SP-NAMs in particular SP-PNAs
  • 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.
  • 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.
  • viruses highly relevant to human health are HIV or viruses causing herpes or warts (herpes simplex virus, varicella zoster virus) .
  • SP-NAMs in particular SP- PNAs
  • SP- PNAs may be provided to specifically bind these kinds of mRNAs, thus enabling the successful removal of cells harboring lysogenic viruses.
  • SP-NAMs in particular SP-PNAs
  • specific SP-NAMs in particular SP-PNAs
  • 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 .
  • ASFV African Swine Fever Virus
  • Pseudorabies Purid herpesvirus 1
  • Avian flu Influenza A virus
  • BTV Bluetongue-Virus
  • BTV Bluetongue-Virus
  • BTV Bluetongue-Virus
  • BTV Bluetongue-Virus
  • BTV Bluetongue-Virus
  • BTV Bluetongue-Virus
  • BTV Bluetongue-Virus
  • BTV Bluetongue-Virus
  • BVD Bovine viral diarrhea
  • CSFV
  • SP-NAMs 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.
  • lipid particles see e.g., Cipolla et al, 2013
  • 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.
  • SP-NAMs in particular SP-PNAs
  • 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) .
  • encapsulated SP-PNA e.g. by delivery vehicles such as lipid nanoparticles, polymers, or peptides
  • delivery vehicles such as lipid nanoparticles, polymers, or peptides
  • HIT inverted hexagonal phase
  • QII bicontinous cubic phase
  • 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 civilization' s success in fighting the cause of many deadly diseases with easy to produce, effective agents.
  • MRSA methicillin-resistant Staphylococcus aureus
  • EHEC Escherichia coil
  • Mycobacterium tuberculosis a growing concern for human health, with the potential to severely cripple civilization' s success in fighting the cause of many deadly diseases with easy to produce, effective agents.
  • MRSA methicillin-resistant Staphylococcus aureus
  • EHEC Escherichia coil
  • Mycobacterium tuberculosis Mycobacterium tuberculosis .
  • SP-NAMs tailored against bacterial mRNA/DNA have the potential to inhibit the bacterial translocation machinery and ultimately kill targeted pathogens.
  • patients treated with specific SP-NAMs in particular SP-PNAs
  • SP-NAMs in particular SP-PNAs
  • the ability of SP- NAMs (in particular SP-PNAs) to specifically recognize and invade even double-stranded DNA enables targeting chromosomal bacterial DNA or even extra-chromosomal DNA (plasmids) , both of which are not protected by a nuclear membrane in bacterial cells.
  • 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.
  • SP-NAMs in particular SP-PNAs
  • SP-PNAs SP-PNAs
  • SP-PNAs a novel class of antibiotically active compounds
  • 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.
  • SP-NAMs in particular SP-PNAs
  • SP-PNAs SP-PNAs
  • Conventional antibiotic substances usually take multiple years from first promising results in basic research to successful phase 3 trials and introduction to the market.
  • an altered chemistry of the active molecule requires vigorous safety testing and very likely leads to new Phase 1 to Phase 3 studies.
  • the respective antibiotic compound will lose its efficacy and there is no quick way to adapt it.
  • SP-NAMs in particular SP-PNAs
  • SP-NAMs 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 summonfollow" the emerging mutations in the respective target.
  • an altogether different target sequence of the bacterial species can be chosen, circumventing the selective pressure that was built up against the first target.
  • SP-NAMs in particular SP-PNAs
  • SP-NAMs are (11) chosen to target highly conserved and thus essential sequence stretches, as explained before.
  • the bacteria's evolutionary plasticity to evade SP-NAM (in particular SP-PNA) treatment is substantially limited.
  • SP-NAMs may also target fungal infections, which are usually divided into local or systemic infections.
  • local mycosis affecting only a limited area of the patients' body, are infections of the skin, nails, vagina or mouth by Candida species.
  • systemic mycosis spreading among multiple organs of the patient (e.g.
  • opportunistic fungal infections asspergillosis, candidosis, mucormycosis
  • primary fungal infections histoplasmosis, blastomycosis, sporotrichosis, coccidioidomycosis, etc.
  • SP-NAMs directed against viral NA
  • the choice of delivery of SP-NAMs 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.
  • SP-NAMs in particular SP-PNAs
  • 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.
  • 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.
  • SP-NAMs 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.
  • SP-NAM in particular SP-PNA
  • PfAQP aquaglyceroporin
  • 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.
  • NAM canonical Asn-Pro-Ala
  • SP-NAM in particular SP-PNA
  • SP-NAM 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.
  • COSMIC https : //cancer . sanger . ac . uk/ cosmic/ fusion
  • RNAs 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 .
  • MIR142 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) .
  • SP-NAMs 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.
  • RNA e.g. mRNA, miRNA
  • 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
  • SP-NAMs in particular SP-PNAs
  • SP-PNAs a striking advantage of SP-NAMs
  • a striking advantage of SP-NAMs 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.
  • SP-NAM in particular SP-PNA
  • 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 may also be used against diseases caused by somatic mutations, in particular against senescent cells.
  • 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) .
  • HSCs Hematopoietic stem cells
  • MBL monoclonal B-lymphocytosis
  • MBL paroxysmal nocturnal hemoglobinuria
  • 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.
  • 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.
  • SP-NAMs 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.
  • atherosclerosis a chronic obstructive pulmonary disease (COPD)
  • COPD chronic obstructive pulmonary disease
  • 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.
  • B cell depletion therapies 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.
  • B cells 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
  • 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 .
  • 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 .
  • 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 .
  • SP-NAMs in particular SP-PNAs
  • 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 .
  • SP-NAMs 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 .
  • varroa mite 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 .
  • mild acids formic acid or oxalic acid
  • 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 .
  • 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 ) .
  • SP-NAMs in particular SP-PNAs
  • the bees are not at all affected by the treatment.
  • the agent will degrade over time and thus not end up in the honey produced.
  • the SP-NAMs in particular SP-PNAs
  • the SP-NAMs are generally non-toxic and are not anymore present in a form that can be taken up by human cells (no encapsulation/delivery system) .
  • the designed specificity towards the Varroa Mites excludes any off-target effects in other organisms.
  • 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.
  • SP-NAMs in particular SP-PNAs
  • 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 .
  • 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.
  • the specificity of SP-NAMs in particular SP-PNAs
  • the peptide construct (SP-NAM, in particular SP-PNA) further comprises a label, preferably a fluorescent label.
  • a label preferably a fluorescent label.
  • 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.
  • the delivery vehicle is non-covalently or covalently bound to the peptide construct (SP-NAM, in particular SP-PNA) .
  • SP-NAM peptide construct
  • SP-PNA peptide construct
  • 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.
  • 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.
  • GPPs cell-penetrating peptides
  • lipid moieties 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.
  • lipid moieties are cationic lipid groups attached to one or more of the nucleobases of the NAM (in particular the PNA) .
  • 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.
  • 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.
  • SARS-CoV-2 primarily elasomeran marketed under Spikevax® by Moderna Inc., and tozinameran marketed under Comirnaty® by Biontech SE/Pfizer Inc
  • LNPs with SP-NAM cargo 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) .
  • the LNPs further comprise stabilizers such as polyethylenglycol (PEG) -decorated lipids which decrease LNP aggregation.
  • stabilizers such as polyethylenglycol (PEG) -decorated lipids which decrease LNP aggregation.
  • 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.
  • helper lipids such as phosphatidylcholines or phosphatidylethanolamines
  • 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 .
  • 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.
  • 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.
  • 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.
  • CPPs cell-penetrating peptide
  • 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.
  • 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.
  • SP-NAM in particular SP-PNA
  • GPPs are disclosed in WO 2015/127094 Al (incorporated herein by reference) , in particular in Table 2 thereof.
  • the GPP may be covalently conjugated to the peptide construct (SP-NAM, in particular SP-PNA) .
  • the composition of the present invention is a pharmaceutical composition.
  • 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.
  • 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.
  • the peptide construct e.g., encapsulated into the delivery vehicle
  • 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.
  • 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) .
  • PBS sterile isotonic phosphate-buff ered saline
  • 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.
  • 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.
  • PBS sterile isotonic phosphate-buff ered saline
  • 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 .
  • 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” ) .
  • 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 ) .
  • preventing or prevention 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.
  • Embodiment 1 A composition for inhibiting protein translocation in cells harbouring a target nucleic acid (NA) molecule, the composition comprising
  • SP sequence protein secretion signal amino-acid sequence
  • PNA peptide nucleic acid
  • - 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.
  • 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.
  • 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 4.
  • 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.
  • 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 4.
  • 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
  • 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 8.
  • 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
  • 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
  • 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
  • Embodiment 13 The composition of any one of embodiments 1 to
  • the cells have ZMPSTE24 or Ste24 activity .
  • Embodiment 14 The composition of any one of embodiments 1 to
  • Embodiment 15 The composition of any one of embodiments 1 to
  • Embodiment 16 The composition of any one of embodiments 1 to 4.
  • 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 4.
  • Embodiment 18 The composition of any one of embodiments 1 to 4.
  • Embodiment 19 The composition of any one of embodiments 1 to 4.
  • the peptide construct further comprises a label, preferably a fluorescent label.
  • Embodiment 20 The composition of any one of embodiments 1 to 4.
  • 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) .
  • DNA desoxyribonucleic acid
  • RNA preferably messenger RNA (mRNA) , transfer RNA (tRNA) , viral RNA or microRNA (miRNA) .
  • Embodiment 21 The composition of any one of embodiments 1 to 4.
  • 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
  • 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
  • the cells are diseased cells .
  • Embodiment 24 The composition of any one of embodiments 1 to
  • 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
  • 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 :
  • 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 .
  • 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
  • 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
  • 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
  • SP sequence a protein secretion signal amino-acid sequence
  • SP sequence targets the peptide construct to a Sec61 translocon or a secYEG translocon
  • 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 .
  • PNA peptide nucleic acid
  • 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 .
  • 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 :
  • 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
  • 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
  • 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.
  • DNA desoxyriboses are connected via phosphate bonds.
  • PNA PNA
  • 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.
  • 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.
  • SP signal sequence
  • 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.
  • SP signal sequence
  • PNA peptide nucleic acid
  • 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) .
  • IMAC Immobilized Metal Ion Chromatography
  • 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.
  • D representative SDS-PAGE of a successful purification of SP-MBP (here Gaslwt-MBP) .
  • Fig. 12 2 nd Purification Step - Size Exclusion Chromatography (SEC) .
  • SEC Size Exclusion Chromatography
  • 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.
  • Fig. 13 Analysis of purity and labeling efficiency.
  • 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 ⁇ .
  • Nr The average number of channels in the membrane
  • AJ ⁇ the characteristic "skyline" current pattern is observed.
  • 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.
  • 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) .
  • 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.
  • 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.
  • 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) .
  • 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.
  • 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.
  • 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% ) .
  • MBP SP-maltose-binding protein
  • the Gasl Saccharamyces cerevisiae, UniProt: P22146
  • prion Mesocricetus auratus, UniProt: P04273
  • 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) .
  • 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.
  • purified SP-MBP was labeled ON at 4°C with maleimide dyes.
  • 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.
  • 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) .
  • 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.
  • 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.
  • SecYEG wild type containing a mutation for labeling - SecY M142C
  • 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) .
  • 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
  • Biorad Biorad
  • 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.
  • HEK293 cells were grown in DMEM (PAS) containing 10 % fetal calf serum (FCS) and Pen/Strep under normal cell culture conditions (37°C, 5 % CO 2 ) .
  • PAS DMEM
  • FCS fetal calf serum
  • Pen/Strep under normal cell culture conditions (37°C, 5 % CO 2 ) .
  • 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.
  • 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) .
  • a cell counting device (CellDropFL, DeNovix) was used.
  • Settings for HEK293 cells were as follows:
  • Table 1 Settings for DeNovix CellDropFL Trypan Blue App and GFP App .
  • 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.
  • 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.
  • P probability
  • 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) .
  • the Gaslwt differs from the original Gasl sequence in the SP protease-site (AGA changed to SAC) .
  • AGA changed to SAC
  • 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.
  • Gasl-MBP constructs (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.
  • the SP-MBP constructs were later used for creating bi-functional constructs .
  • peptides can be expressed as fusion proteins and purified using different tags.
  • 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 .
  • IMAC immobilized metal ion affinity chromatography
  • SEC size exclusion chromatography
  • the His-tag of the fusion construct 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) .
  • 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.
  • Cell-penetrating peptides 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.
  • 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.
  • 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.
  • HEK 293 cells expressing the membrane protein AQP4 fused to eGPF 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.
  • 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.
  • 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.
  • 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) .
  • 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.
  • 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.
  • PNA sequences are listed in Table 2 below.
  • Table 2 Sequences and characteristics of PNA modules used in the experiments .
  • 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 .
  • PNAe is complementary to endogenous genes IQUB and RORA, both having a relatively high expression level in HEK293 cells ( see Targeting of Endogenous Genes ) .
  • PNActrl which has no complementary mRNA in the human genome and, therefore , serves as a negative (no target ) control in our experiments .
  • 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 ) .
  • 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.
  • SortaseA a site-specific trans-peptidation leads to the desired SP-PNA constructs.
  • 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
  • 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.
  • TB Trypan Blue
  • HEK 293 cells expressing the membrane protein AQP4 fused to eGPF are transfected with nonspecific variants of the SP-PNA construct (Figure 26A) .
  • AQP4-eGFP localizes to the cell membrane.
  • the SP-PNA blocks translocon transiently. Consequently, we do not expect to see a strong effect on AQP4-eGFP localization or cell survival.
  • fluorescence imaging one can observe cells that contain non-specific SP-PNAs and AQP4-eGFP in parallel.
  • 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.
  • eGFP 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
  • eGFP mRNA and protein can be found in the cytoplasm of cells transfected with the expression plasmid peGFP-Nl ( Figure 29B) .
  • 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.
  • FIG. 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
  • FIG. 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
  • 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.
  • the fully complementary mRNA sequence leads to a Tm far above 37 °C (estimated 65.4°C) and cells should be affected ( Figure 33A) .
  • SP-PNAs are highly specific to their target NA.
  • 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) .
  • 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 .
  • 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 .
  • 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.
  • 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.
  • a linker region cyst, LPETG, His- tag
  • 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) .
  • the SP module was labeled with a maleimide dye at the Cystein in the linker region.
  • SP-MBP constructs 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.
  • 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 .
  • GGK short glycine-rich peptide sequence
  • 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 .
  • 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.
  • Tm from 65.4 °C is decreased to a temperature lower than 37 °C, which is the temperature at which HEK293 cells are grown.
  • cells can survive the treatment since there is no binding of Gaslwt- PNAfp to NaVMs-GFP mRNA.
  • 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.
  • 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.
  • GGGS or GGSGS a pattern of glycines followed by one or two serines.
  • Multiple repeats of such a pattern may be used to span the desired length of the linker.
  • individual charged or polar residues may optionally be introduced to raise the solubility of the desired SP-PNAs.
  • the SP component of the constructs is altered according to the application's needs.
  • 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.
  • SP-PNAs provides the necessary flexibility to perform these adaptations and consequently allows the SP-PNAs to be used in a plethora of scenarios .
  • SP-PNAs have the great advantage of leading to the death of cells expressing unique (m)RNAs, while showing no toxicity towards other cells .
  • SP-PNAs can be easily trans fected into cells by means of liposomes .
  • Knock-out of a defined set of cells in in-vi vo studies can help understanding their role in their natural environment .
  • Ewing Sarcoma is a highly malignant tumor of the bones mostly af fecting people in their second decade of li fe .
  • 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 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 .
  • EWS-FLI 1 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.
  • lipid formulation of SP-PNA e.g., encapsulated in liposomes or LNPs
  • LNPs liposomes
  • Example 7 An SP-NAM other than SP-PNA in cell biology
  • SP- LNAs SP-locked nucleic acids
  • 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.
  • SP-LNAs 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.
  • SP-PMOs SP-phosphorodiamidate morpholino oligomers
  • mammalian individuals such as mice or humans
  • SP-PMOs SP-phosphorodiamidate morpholino oligomers
  • 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.
  • wild-type signal sequences e.g., Gasl, prion, insulin
  • 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 .
  • RNA 29, 434-445 (2023) .

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Abstract

La présente invention concerne une composition pour inhiber la translocation de protéines dans des cellules hébergeant une molécule d'acide nucléique (NA) cible telle qu'une molécule d'acide ribonucléique (ARN), la composition comprenant une construction peptidique, comprenant une séquence d'acides aminés signal de sécrétion de protéine ("séquence SP"), et une séquence mimétique d'acide nucléique (NAM) telle qu'une séquence d'acide nucléique peptidique (PNA) comprenant de 5 à 30 bases consécutives complémentaires d'une séquence de la molécule NA cible ; et un véhicule d'administration pour administrer la construction peptidique à des cellules. La présente invention concerne en outre un procédé d'inhibition de la translocation de protéines dans des cellules hébergeant une molécule NA cible, comprenant une étape consistant à administrer aux cellules une construction peptidique, comprenant une séquence SP, et une séquence NAM telle qu'une séquence PNA comprenant de 5 à 30 bases consécutives complémentaires d'une séquence de la molécule NA cible. La présente invention concerne en outre des utilisations de telles compositions et des méthodes thérapeutiques pour être humain ou animal, le traitement de plantes et la lutte contre les organismes nuisibles, entre autres utilisations.
PCT/EP2025/060733 2024-04-17 2025-04-17 Composition pour inhiber la translocation de protéines et procédés l'utilisant Pending WO2025219570A1 (fr)

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