Biology subjects

Pos, W.

Publications and source records attributed to Pos, W..

3 recordsLinked to original sources

bioRxiv2026

Development of a novel VHH intrabody targeting the N17 region of huntingtin exon 1 protein that prevents inclusion body formation.

Huntingtons disease (HD) is a progressive neurodegenerative disease caused by a mutation in the exon 1 of the huntingtin (HTT) gene, which leads to an extended polyglutamine (polyQ) tract in the mutant protein. As a result, mutant huntingtin (mHTT) exon 1 fragments aggregate in cells, which disrupts proper neuronal function and eventually induces cell death. The selective reduction of these toxic mHTT fragments without disturbing the wild-type full-length HTT function would be a potential therapeutic strategy to treat and prevent HD. Intracellular antibodies (intrabodies) have emerged as an attractive strategy to specifically target disease-related proteins, with VHH intrabodies being of high interest as they are much smaller than single-chain variable fragments (scFv). Here, we describe the identification and development of VHH 1 as a lead candidate intrabody targeting the first 17 amino acids of the mHTT protein, using a humanized VHH page-display library to screen against mHTT(Q46) exon 1 to identify potential binders. Next, we further optimized VHH 1 into VHH 1a to improve cytoplasmic solubility. Using immortalized mouse striatal cells that express inducible untagged mHTT exon 1 fragments, we investigated the effects of the intrabody on soluble and insoluble mHTT species via microscopy and biochemical assays. We showed that the VHH 1a intrabody reduces the levels of insoluble mHTT species, thereby effectively interrupting the aggregation process. This study highlights the potential for VHH intrabodies to specifically target mHTT fragments, enabling therapeutic strategies to delay and prevent HD pathology. HighlightsO_LIThree binders were down-selected from a phage-display library to bind HTT N17 C_LIO_LIVHH 1a intrabody is the most efficient at reducing mutant HTT exon 1 aggregation C_LIO_LIVHH 1a acts on soluble HTT exon 1 oligomers to block the transition to inclusion body C_LI

neuroscience↗

bioRxiv2025

AAV-Delivered Anti-PC-OxPL Antibody Fragments: A NovelTherapeutic Approach to Target ALS

Amyotrophic lateral sclerosis (ALS) is characterized by the progressive loss of motor neurons and premature death. The limited understanding of the mechanisms underlying selective motor neuron death has significantly hindered the development of disease-modifying treatments. Ferroptosis, a form of cell death dependent on iron accumulation, has been implicated in the selective degeneration of motor neurons in ALS. Oxidized phosphatidylcholines (PC-OxPL) have been identified as key effectors in the pathophysiological processes associated with this pathway. Preclinical observations revealed a distinct PC-OxPL profile in the cerebrospinal fluid (CSF) of sporadic ALS (sALS) patients and identified apolipoprotein E (APOE) particles as the primary carriers of PC-OxPL in the CSF. Furthermore, in ALS brain and spinal cord tissue sections, PC-OxPL was found to be predominantly associated with motor neurons. Exposure of iPSC-derived motor neurons to PC-OxPL led to transcriptomic changes in genes known to be linked to ALS, as well as the induction of significant TDP-43 pathology and motor neuron death. To counter this, we developed a single-chain antibody fragment (scFv) encoded by an AAV-delivered DNA construct that specifically targets PC-OxPL neoepitopes (PC-OxPL-VecTab(R)). PC-OxPL-VecTab(R) effectively neutralized PC-OxPL-induced neurotoxicity and TDP-43 aggregation in motor neurons, while preventing motor neuron death and deficits in a sALS CSF mouse model. When administered intrathecally to minipigs, PC-OxPL-VecTab(R) was distributed to both upper and lower motor neurons and expressed at levels predicted to be therapeutically effective. Our work identifies PC- OxPL as a critical pathological factor and a key inducer of TDP-43 pathology in ALS, providing the foundation for a novel therapeutic intervention modality for patients with sALS. Furthermore, it offers the exciting potential to be expanded to diseases characterized by PC-OxPL neurotoxicity.

neuroscience↗

bioRxiv2020

Implications for tetraspanin-enriched microdomain assembly based on structures of CD9 with EWI-F

Tetraspanins are ubiquitous eukaryotic membrane proteins that contribute to a variety of signaling processes by spatially organizing partner-receptor molecules in the plasma membrane. How tetraspanins bind and cluster partner receptors into so-called tetraspanin-enriched microdomains is unknown. Here we present crystal structures of the large extracellular loop of CD9 in complex with nanobodies 4C8 and 4E8; and, the cryo-EM structure of 4C8-bound CD9 in complex with its prototypical partner EWI-F. The CD9 - EWI-F complex displays a tetrameric arrangement with two centrally positioned EWI-F molecules, dimerized through their ectodomains, and two CD9 molecules, one bound to each EWI-F single-pass transmembrane helix through CD9-helices h3 and h4. In the crystal structures, nanobodies 4C8 and 4E8 bind CD9 at the C and D loop, in agreement with 4C8 binding at the ends of the CD9 - EWI-F cryo-EM complex. Overall, the 4C8 - CD9 - EWI-F - EWI-F - CD9 - 4C8 complexes varied from nearly two-fold symmetric (i.e. with the two CD9 - 4C8 copies in nearly anti-parallel orientation) to ca. 50{degrees} bent arrangements. Since membrane helices h1 and h2 and the EC2 D-loop have been previously identified as sites for tetraspanin homo-dimerization, the observed linear but flexible arrangement of CD9 - EWI-F with potential CD9 - CD9 homo-dimerization at either end provides a new concatenation model for forming short linear or circular assemblies, which may explain the occurrence of tetraspanin-enriched microdomains.

biophysics↗