Alexander Klipp


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Klipp

First Name

Alexander

ORCID

0000-0003-2442-322X

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2022 - 20257
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Publications 1 - 7 of 7
  • The intracellular journey of DNA delivered with the protein-based transfection system TFAMoplex and Lipofectamine
    Item type: Journal Article
    Greitens, Christina; Honrath, Steffen; Maurer, Philip; et al. (2025)
    International Journal of Pharmaceutics
    In non-viral DNA delivery, assessing the cellular trafficking of DNA is crucial to understand how transfection systems interact with the cellular machinery. Using a combination of innovative and established imaging assays and flow cytometry, we characterize DNA delivery in HeLa cells with three different transfection systems: (i) the original protein-based TFAMoplex, using the mitochondrial transcription factor A (TFAM) for DNA compaction, (ii) a modified version of the TFAMoplex incorporating a basic leucine zipper (bZIP) domain, (TFAMoplex-bZIP), and (iii) Lipofectamine. Particularly, DNA cell association, uptake, endosomal rupture, cytoplasmic delivery and reporter gene expression are assessed. TFAMoplex-bZIP mediated delivery of Cy3-labeled DNA shows significantly higher cell association compared to the original TFAMoplex and Lipofectamine (15.6, 6.6. and 0.53 Cy3 objects per cell, respectively). However, the highest DNA internalization efficiency is achieved with Lipofectamine (21.8 % vs. ∼15 % for TFAMoplex-based systems). All transfection systems induce endosomal rupture and the formation of barrier-to-autointegration factor (BAF)-positive nucleoprotein structures, termed BAF clusters. With TFAMoplex-bZIP, BAF clusters are numerous, and significantly more cells show reporter gene expression (46.7 %) compared to Lipofectamine (8.7 %). However, reporter protein intensity is highest with Lipofectamine. The described high-precision characterization tools of DNA delivery might be applied to other non-viral transfection agents to identify their bottlenecks in detail.
  • Crystal structure of the α1B-adrenergic receptor reveals molecular determinants of selective ligand recognition
    Item type: Journal Article
    Deluigi, Mattia; Morstein, Lena; Schuster, Matthias; et al. (2022)
    Nature Communications
    α-adrenergic receptors (αARs) are G protein-coupled receptors that regulate vital functions of the cardiovascular and nervous systems. The therapeutic potential of αARs, however, is largely unexploited and hampered by the scarcity of subtype-selective ligands. Moreover, several aminergic drugs either show off-target binding to αARs or fail to interact with the desired subtype. Here, we report the crystal structure of human α1BAR bound to the inverse agonist (+)-cyclazosin, enabled by the fusion to a DARPin crystallization chaperone. The α1BAR structure allows the identification of two unique secondary binding pockets. By structural comparison of α1BAR with α2ARs, and by constructing α1BAR-α2CAR chimeras, we identify residues 3.29 and 6.55 as key determinants of ligand selectivity. Our findings provide a basis for discovery of α1BAR-selective ligands and may guide the optimization of aminergic drugs to prevent off-target binding to αARs, or to elicit a selective interaction with the desired subtype.
  • VWA7 – A Putative Human Phosphatidylcholine-specific Phospholipase C
    Item type: Journal Article
    Klipp, Alexander; Greitens, Christina; Leroux, Jean-Christophe; et al. (2025)
    Journal of Molecular Biology
    For over four decades, the existence of mammalian phosphatidylcholine-specific phospholipase C (pc-PLC) has been reported. However, the gene encoding this enzyme has not been unambiguously determined to date. This study presents human von Willebrand factor A domain-containing protein 7 (VWA7) as putative pc-PLC and provides some biochemical insights into its potential function. Structural comparison of the modelled N-terminal region of VWA7 with Bacillus cereus pc-PLC revealed high similarity and, remarkably, a conserved active site architecture. The localization and activity of different VWA7 versions were investigated in mammalian cells. Eventually, pc-PLC activity was confirmed for VWA7 variants produced in bacteria, with the most active variant reaching 733 mU/mg. These findings establish a basis for future investigations on the role of human pc-PLC as well as on the poorly studied human VWA7.
  • Modular Calcium-Responsive and CD9-Targeted Phospholipase System Enhancing Endosomal Escape for DNA Delivery
    Item type: Journal Article
    Klipp, Alexander; Greitens, Christina; Scherer, David; et al. (2025)
    Advanced Science
    Gene delivery systems must overcome multiple barriers, with endosomal escape representing a prominent obstacle. This work has previously shown that a bacterial phospholipase C (PLC) enabled endosomal escape of a non-viral protein-based DNA delivery system termed TFAMoplex. Building upon this, this work introduces a calcium-responsive system designed to enhance endosomal escape through non-covalent capturing of PLC to the TFAMoplex followed by its release within endosomes and nanobody-mediated targeting to the endosomal membrane. This approach leads to improved TFAMoplexes enabling transfection of HeLa cells in full serum with a half maximal effective concentration (EC50) of less than 200 ng DNA per mL serum, using only 5 nM PLC. Particularly, the modular capture, release and targeting system could potentially be adapted to other delivery agents previously constrained by poor endosomal escape. These findings present a promising strategy to achieve efficient endosomal escape, offering prospects for improved delivery of macromolecules, in particular nucleic acids.
  • Enzymatic absorption promoters for non-invasive peptide delivery
    Item type: Journal Article
    Bohley Steiger, Marilena; Steinauer, Angela; Gao, Daniel; et al. (2025)
    Journal of Controlled Release
    Peptide drugs offer considerable potential for treating a diverse range of diseases. Yet, their clinical application is generally restricted to injectable therapies. The main challenge hindering their broader use through globally accessible, patient-friendly, and non-invasive delivery routes such as oral or buccal, lies in their poor ability to cross biological barriers effectively. Here, we demonstrate that enzymes can be harnessed to transiently reduce these barriers and improve absorption. As a proof of concept, we employ a mucin-specific protease (mucinase) and a phospholipase to increase mucus diffusivity and epithelial cell membrane permeability, respectively. In a canine model, we show that enteric capsules containing both enzymes, and the peptide drug desmopressin achieved a relative bioavailability of 155 % compared to the drug alone. Additionally, a buccal patch loaded with phospholipase and semaglutide displayed a 5-fold higher bioavailability and lower variability (71.5 % reduction in the coefficient of variation) compared to the commercially available oral tablet. These results suggest that enzymatic modulation of biological barriers holds promise as a strategy to improve non-invasive delivery of peptides and potentially other macromolecular drugs.
  • Get out or die trying: peptide- and protein-based endosomal escape of RNA therapeutics
    Item type: Review Article
    Klipp, Alexander; Burger, Michael; Leroux, Jean-Christophe (2023)
    Advanced Drug Delivery Reviews
    RNA therapeutics offer great potential to transform the biomedical landscape, encompassing the treatment of hereditary conditions and the development of better vaccines. However, the delivery of RNAs into the cell is hampered, among others, by poor endosomal escape. This major hurdle is often tackled using special lipids, polymers, or protein-based delivery vectors. In this review, we will focus on the most prominent peptide- and protein-based endosomal escape strategies with focus on RNA drugs. We discuss cell penetrating peptides, which are still incorporated into novel transfection systems today to promote endosomal escape. However, direct evidence for enhanced endosomal escape by the action of such peptides is missing and their transfection efficiency, even in permissive cell culture conditions, is rather low. Endosomal escape by the help of pore forming proteins or phospholipases, on the other hand, allowed to generate more efficient transfection systems. These are, however, often hampered by considerable toxicity and immunogenicity. We conclude that the perfect enhancer of endosomal escape has yet to be devised. To increase the chances of success, any new transfection system should be tested under relevant conditions and guided by assays that allow direct quantification of endosomal escape.
Publications 1 - 7 of 7