Biology subjects

Aeschimann, F.

Publications and source records attributed to Aeschimann, F..

4 recordsLinked to original sources

bioRxiv2023

An optimised protocol for quality control of gene therapy vectors using Nanopore direct RNA sequencing

Despite recent advances made towards improving the efficacy of lentiviral gene therapies, a sizeable proportion of produced vector contains an incomplete and thus potentially nonfunctional RNA genome. This can undermine gene delivery by the lentivirus as well as increase manufacturing costs and must be improved to facilitate the widespread clinical implementation of lentiviral gene therapies. Here, we compare three long-read sequencing technologies for their ability to detect issues in vector design and determine Nanopore direct RNA sequencing to be the most powerful. We show how this approach identifies and quantifies incomplete RNA caused by cryptic splicing and polyadenylation sites, including a potential cryptic polyadenylation site in the widely used Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE). Using artificial polyadenylation of the lentiviral RNA, we also identify multiple hairpin-associated truncations in the analysed lentiviral vectors, which account for most of the detected RNA fragments. Finally, we show that these insights can be used for optimization of lentiviral vector design. In summary, Nanopore direct RNA sequencing is a powerful tool for the quality control and optimisation of lentiviral vectors, which may help to improve lentivirus manufacturing and thus the development of higher quality lentiviral gene therapies.

genomics↗

bioRxiv2019

Two parallel arms of the heterochronic pathway direct coordinated juvenile-to-adult transition through distinct LIN-29 isoforms

Robust organismal development relies on temporal coordination of disparate physiological processes. In Caenorhabditis elegans, the timely transition from juvenile to adult is controlled by the heterochronic pathway, a regulatory cascade of conserved proteins and small RNAs. The heterochronic pathway culminates in accumulation of the transcription factor LIN-29, which triggers coordinated execution of juvenile-to-adult (J/A) transition events. Here, we reveal that two LIN-29 isoforms fulfill distinct functions during the J/A transition. We show that the functional differences between the isoforms do not stem from differences in their sequences, but from their distinct spatiotemporal expression, and we propose that distinct LIN-29 dose sensitivities of the individual J/A transition events help to ensure their temporal ordering. We demonstrate that unique lin-29 isoform expression patterns are generated by the RNA-binding protein LIN-41 for lin-29a, and the transcription factor HBL-1 for lin-29b. By regulating both HBL-1 and LIN-41, the RNA-binding protein LIN-28 coordinates LIN-29 isoform activity. Thus, our findings reveal that a coordinated transition from juvenile to adult involves branching of a linear pathway to achieve timely control of multiple events.

developmental biology↗

bioRxiv2018

let-7 controls the transition to adulthood by releasing select transcriptional regulators from repression by LIN41

Development of multicellular organisms relies on faithful temporal control of cell fates. In Caenorhabditis elegans, the heterochronic pathway governs temporal patterning of somatic cells. This function may be phylogenetically conserved as several heterochronic genes have mammalian orthologues, and as the heterochronic let-7 miRNA and its regulator LIN28 appear to time the onset of puberty in mice and humans. Here, we have investigated how let-7 promotes the transition to adulthood in C. elegans. We find that let-7 controls each of three relevant processes, namely male and female sexual organ morphogenesis as well as changes in skin progenitor cell fates, through the same single target, lin-41. LIN41 in turn silences two pairs of targets post-transcriptionally, by binding and silencing their mRNAs. The EGR-type transcription factor LIN-29a and its co-factor, the NAB1/2 orthologous MAB-10, mediate control of progenitor cell fates and vulval integrity. By contrast, male tail development depends on regulation of the DM domain-containing transcription factors DMD-3 and MAB-3. Our results provide mechanistic insight into an exemplary temporal patterning pathway, demonstrate that let-7 - LIN41 function as a versatile regulatory module that can be connected to different outputs, and reveal how several levels of post-transcriptional regulation ultimately achieve effects through controlling transcriptional outputs.

developmental biology↗

bioRxiv2018

Timing mechanism of sexually dimorphic nervous system differentiation

In all animals, sexual differentiation of somatic tissue is precisely timed, yet the molecular mechanisms that control the timing of sexual differentiation, particularly in the brain, are poorly understood. We have used sexually dimorphic molecular, anatomical and behavioral features of the C. elegans nervous system to decipher a regulatory pathway that controls the precise timing of sexual differentiation. We find that the sexually dimorphic differentiation of postmitotic neurons in the male nervous system is abrogated in animals that carry a mutation in the miRNA let-7 and prematurely executed in animals either lacking the let-7 inhibitor lin-28, or the direct let-7 target lin-41, an RNA-binding, posttranscriptional regulator. We show that an isoform of a phylogenetically conserved transcription factor, lin-29a, is a critical target of LIN-41 in controlling sexual maturation of sex-shared neurons. lin-29a is expressed in a male-specific manner in a subset of sex-shared neurons at the onset of sexual maturation. lin-29a acts cell-autonomously in these neurons to control the expression of sexually dimorphic neurotransmitter switches, sensory receptor expression, neurite anatomy and connectivity, and locomotor behavior. lin-29a is not only required but also sufficient to impose male-specific features at earlier stages of development and in the opposite sex. The temporal, sexual and spatial specificity of lin-29a expression is controlled intersectionally through the lin-28/let-7/lin-41 heterochronic pathway, sex chromosome configuration and neuron type-specific terminal selector transcription factors. Two Doublesex-like transcription factors represent additional neuron-type specific targets of LIN-41 and are regulated in a similar intersectional manner, indicating the existence of modular outputs downstream of the heterochronic pathway. In conclusion, we have provided insights into the molecular logic of the timing of sexual differentiation in the C. elegans nervous system. Remarkably, the lin28/let7 axis also controls the timing of sexual differentiation in mice and humans thereby hinting toward a striking universality of the control mechanisms of sexual differentiation.

neuroscience↗