Biology subjects

Kletzien, O. A.

Publications and source records attributed to Kletzien, O. A..

3 recordsLinked to original sources

bioRxiv2026

VE-cadherin NOT-gated CD93 CAR T cells discriminate between AML and healthy endothelial cells

BackgroundChimeric antigen receptor (CAR) T cell therapy has transformed the treatment of B cell malignancies, but translation to acute myeloid leukemia (AML) has been hindered by on-target, off-tumor (OTOT) toxicity. In particular, endothelial cell (EC)-specific toxicity has limited clinical translation of promising leukemia stem cell-enriched targets such as CD93. Innovative strategies to mitigate EC damage while preserving antileukemic efficacy are needed. MethodsWe hypothesized that a NOT-gated CAR T cell strategy could circumvent EC toxicity associated with CD93 targeting. Considering CAR target antigen density and the pro-inflammatory microenvironment of CAR T cells, we identified VE-cadherin (VC), a highly specific EC marker, as an optimal inhibitory CAR target. We engineered a novel VC-specific single chain variable fragment (scFv), confirmed EC specificity in the context of a VC-specific second-generation activating CAR, then evaluated VC/CD93 NOT-gated CAR T cells for EC protection and antileukemic activity in in vitro cytotoxicity assays and in a three-dimensional vascularized microphysiological system. ResultsVC/CD93 NOT-gated CAR T cells maintain potent cytotoxicity against AML across multiple effector-to-target ratios, but preserve EC integrity, including in a three-dimensional vascular model system. Importantly, prior AML exposure did not impair the EC-protective function of the VC-specific iCAR, indicating durable NOT-gate activity under inflammatory conditions. Conversely, EC-induced iCAR inhibitory functions did not limit downstream antileukemic cytotoxicity, confirming a reversibility of both activation and inhibitory signals. Conclusions: These findings establish NOT-gated CAR T cells as an effective strategy to overcome EC-specific OTOT toxicity. Our results underscore the importance of CAR target discovery and validation across a spectrum of inflammatory states that can influence antigen expression and available therapeutic windows. This approach expands the potential CAR target landscape for AML and may be more broadly applicable to other malignancies where OTOT toxicity limits clinical translation.

immunology↗

bioRxiv2024

The RNA-binding selectivity of the RGG/RG motifs of hnRNP U is abolished by elements within the intrinsically disordered region

The abundant nuclear protein hnRNP U interacts with a broad array of RNAs along with DNA and protein to regulate nuclear chromatin architecture. The RNA-binding activity is achieved via a disordered [~]100 residue C-terminal RNA-binding domain (RBD) containing two distinct RGG/RG motifs. Although the RNA-binding capabilities of RGG/RG motifs have been widely reported, less is known about hnRNP Us RNA-binding selectivity. Furthermore, while it is well established that hnRNP U binds numerous nuclear RNAs, it remains unknown whether it selectively recognizes sequence or structural motifs in target RNAs. To address this question, we performed equilibrium binding assays using fluorescence anisotropy (FA) and electrophoretic mobility shift assays (EMSAs) to quantitatively assess the ability of human hnRNP U RBD to interact with segments of cellular RNAs identified from eCLIP data. These RNAs often, but not exclusively, contain poly-uridine or 5-AGGGAG sequence motifs. Detailed binding analysis of several target RNAs reveal that the hnRNP U RBD binds RNA in a promiscuous manner with high affinity for a broad range of structured RNAs, but with little preference for any distinct sequence motif. In contrast, the isolated RGG/RG of hnRNP U motif exhibits a strong preference for G-quadruplexes, similar to that observed for other RGG motif bearing peptides. These data reveal that the hnRNP U RBD attenuates the RNA binding selectivity of its core RGG motifs to achieve an extensive RNA interactome. We propose that a critical role of RGG/RG motifs in RNA biology is to alter binding affinity or selectivity of adjacent RNA-binding domains.

biochemistry↗

bioRxiv2022

Context-dependence of T-loop mediated long-range RNA tertiary interactions

The architecture and folding of complex RNAs is governed by a limited set of highly recurrent structural motifs that form long-range tertiary interactions. One of these motifs is the T-loop, which was first identified in tRNA but is broadly distributed across biological RNAs. While the T-loop has been examined in detail in different biological contexts, the various receptors that it interacts with are not as well defined. In this study, we use a cell-based genetic screen in concert with bioinformatic analysis to examine three different, but related, T-loop receptor motifs found in the flavin mononucleotide (FMN) and cobalamin (Cbl) riboswitches. As a host for different T-loop receptors, we employed the env8 class-II Cbl riboswitch, an RNA that uses two T-loop motifs for both folding and supporting the ligand binding pocket. A set of libraries was created in which select nucleotides that participate in the T-loop/T-loop receptor (TL/TLR) interaction were fully randomized. Library members were screened for their ability to support Cbl-dependent expression of a reporter gene. While T-loops appear to be variable in sequence, we find that the functional sequence space is more restricted in the Cbl riboswitch, suggesting that TL/TLR interactions are context dependent. Our data reveal clear sequence signatures for the different types of receptor motifs that align with phylogenic analysis of these motifs in the FMN and Cbl riboswitches. Finally, our data suggest the functional contribution of various nucleobase-mediated long-range interactions within the riboswitch subclass of TL/TLR interactions that are distinct from those found in other RNAs. HighlightsO_LIThe T-loop motif frequently mediates tertiary interactions in RNA C_LIO_LIActivity-based screen used to explore T-loop mediate interactions in riboswitches C_LIO_LIResults from the screen were consistent with phylogenetic analysis C_LIO_LIT-loop/T-loop recepto{-}{-}{-}{-}r interactions are context dependent C_LI

biochemistry↗