Optimisation de la production de cGAMP-VLP et développement de cGAMP-VLP ciblées

L'ADN double brin chez les mammifères est présent exclusivement dans le noyau et les mitochondries. Sa présence dans le cytosol est un marqueur d'infection virale ou de stress cellulaire. L'ADN double brin est détecté dans le cytosol par l'enzyme cGAS, qui produit après activation le dinucléotide cyclique 2'3'-cGAMP. Le 2'3'-cGAMP est un activateur de la protéine STING (STimulator of INterferon Genes), qui déclenche la production d'interférons de type 1 et l'activation de la voie NF-kB dans les cellules. Il a été montré que la voie cGAS-STING est activée lors d'infections virales par des virus à ADN, et que cette activation a un rôle anti-viral, et dans certains cas anti-tumoral. Le 2'3'-cGAMP produit dans les cellules durant la production de lentivirus est empaqueté dans les particules lentivirales nouvellement produites, et délivré dans les cellules avoisinantes, déclenchant une réponse anti-virale rapide. Ce mécanisme ouvre la possibilité de produire des pseudo-particules non-infectieuses contenant le cGAMP afin de le libérer dans des cellules d'intérêt. Ces particules pseudo virales ont été nommées cGAMP virus-like particles (cGAMP-VLP). Le cGAMP-VLP constitue un produit qui a été breveté et qui est en cours de développement pré-clinique. L'objectif de cette thèse était d'améliorer la production au laboratoire du cGAMP-VLP, d'étudier son mécanisme d'action et de développer une seconde génération. J'ai optimisé le protocole de production des cGAMP-VLP dans le but de le rentre compatible avec une éventuelle exploitation industrielle. Ainsi, j'ai optimisé les plasmides utilisés, le réactif de transfection et le format de production. J'ai ensuite caractérisé la réponse interféron de type I in vitro de différentes lignées cellulaires et de cellules primaires pertinentes dans le cadre d'une injection intra-tumorale de cGAMP-VLP. J'ai réalisé ces expériences en comparaison avec les di-nucléotides cycliques, ce qui m'a permis de mettre en évidence des spécificités de réponse pour le cGAMP-VLP. Enfin, j'ai analysé ex vivo la capture de cGAMP-VLP par des cellules immunitaires murines primaires, et j'ai pu montrer une capture préférentielle par les cellules dendritiques et les macrophages. Enfin, j'ai amorcé le développement de cGAMP-VLP ciblant spécifiquement des cellules d'intérêt. Ainsi, j'ai réalisé une preuve de concept avec des protéines virales qu'il était possible d'altérer le ciblage du cGAMP-VLP tout en conservant ses activités fusogènes. En conclusion, mes travaux de thèse ont permis d'améliorer nos capacités de production de cGAMP-VLP, ont apporté des éléments nouveaux sur son mécanisme d'action et ont ouvert la voie vers une seconde génération de cGAMP-VLP. L'ensemble de ces travaux contribuera à amener le cGAMP-VLP vers un développement clinique.Double stranded DNA in mammals is exclusively located inside the nucleus and in the mitochondria. Its presence in the cytosol is a marker of viral infection or cellular stress. Double stranded DNA is detected in the cytosol by the enzyme cGAS, that produces after activation the cyclic di-nucleotide 2'3'-cGAMP. 2'3'-cGAMP is an agonist of the STING protein (STimulator of INterferon Genes), that induces production of type I interferon and activates the NF-kB pathway in the cells. It has been shown that the cGAS-STING pathway is activated after infection by DNA viruses, and this activation has an anti-viral role, and in some cases also an anti-tumoral role. During lentivirus production, the 2'3'-cGAMP produced in the cells is packaged in newly produced lentiviral particles, and delivered to neighboring cells, inducing a rapid anti-viral response. This mecanism opens the possibility to produce non infectious viral like particles containing 2'3'-cGAMP for delivery in cells of interest. These viral like particles were named cGAMP virus like particles (cGAMP-VLP). The cGAMP-VLP is a product that has been patented and is in pre-clinical development. The goal of this thesis was to improve the production of cGAMP-VLPs in the laboratory, study its mecanism of action, and develop a second generation. I optimized the production protocol of the cGAMP-VLPs in order to make it compatible with a industrial production process. Thus, I optimized the transfection plasmids, the transfection reagent, and the production format. I then caracterized in vitro the type I interferon response of various cell lines and primary cells that are relevant in the case of an intra-tumoral injection of cGAMP-VLP. I compared these responses with the ones obtained with soluble cyclic di-nucleotides, and was able to show specificities of the reponses to cGAMP-VLPs. I then studied ex vivo the capture of cGAMP-VLPs by mouse primary immune cells, and I showed a preferential capture by dendritic cells and macrophages. Lastly, I started the development of cGAMP-VLPs able to target cells of interest. Using viral proteins, I made the proof of concept that it is possible to alter the cGAMP-VLP targets while keeping its fusogenic propreties intact. In conclusion, my PhD work improved our cGAMP-VLP production capacities, shed new light on its mecanism of action, and opened the way to a second generation cGAMP-VLPs. This work contributed to bringing the cGAMP-VLP closer to clinical developement

Transitioning from benchmarks to a real-world case of information-seeking in Scientific Publications

Although recent years have been marked by incredible advances in the whole development process of NLP systems, there are still blind spots in characterizing what is still hampering real-world adoption of models in knowledgeintensive settings. In this paper, we illustrate through a real-world zero-shot text search case for information seeking in scientific papers, the masked phenomena that the current process of measuring performance might not reflect, even when benchmarks are, in appearance, faithfully representative of the task at hand. In addition to experimenting with TREC-COVID and NFCorpus, we provide an industrial, expertcarried/annotated, case of studying vitamin B's impact on health. We thus discuss the misalignment between solely focusing on single-metric performance as a criterion for model choice and relevancy as a subjective measure for meeting a user's need

miR-128 Restriction of LINE-1 (L1) Retrotransposition Is Dependent on Targeting hnRNPA1 mRNA.

The majority of the human genome is made of transposable elements, giving rise to interspaced repeats, including Long INterspersed Element-1s (LINE-1s or L1s). L1s are active human transposable elements involved in genomic diversity and evolution; however, they can also contribute to genomic instability and diseases. L1s require host factors to complete their life cycles, whereas the host has evolved numerous mechanisms to restrict L1-induced mutagenesis. Restriction mechanisms in somatic cells include methylation of the L1 promoter, anti-viral factors and RNA-mediated processes such as small RNAs. microRNAs (miRNAs or miRs) are small non-coding RNAs that post-transcriptionally repress multiple target genes often found in the same cellular pathways. We have recently established that miR-128 functions as a novel restriction factor inhibiting L1 mobilization in somatic cells. We have further demonstrated that miR-128 functions through a dual mechanism; by directly targeting L1 RNA for degradation and indirectly by inhibiting a cellular co-factor which L1 is dependent on to transpose to new genomic locations (TNPO1). Here, we add another piece to the puzzle of the enigmatic L1 lifecycle. We show that miR-128 also inhibits another key cellular factor, hnRNPA1 (heterogeneous nuclear ribonucleoprotein A1), by significantly reducing mRNA and protein levels through direct interaction with the coding sequence (CDS) of hnRNPA1 mRNA. In addition, we demonstrate that repression of hnRNPA1 using hnRNPA1-shRNA significantly decreases de novo L1 retro-transposition and that induced hnRNPA1 expression enhances L1 mobilization. Furthermore, we establish that hnRNPA1 is a functional target of miR-128. Finally, we determine that induced hnRNPA1 expression in miR-128-overexpressing cells can partly rescue the miR-128-induced repression of L1's ability to transpose to different genomic locations. Thus, we have identified an additional mechanism by which miR-128 represses L1 retro-transposition and mediates genomic stability

Plants Encode a General siRNA Suppressor That Is Induced and Suppressed by Viruses

International audienceSmall RNAs play essential regulatory roles in genome stability, development, and responses to biotic and abiotic stresses in most eukaryotes. In plants, the RNaseIII enzyme DICER-LIKE1 (DCL1) produces miRNAs, whereas DCL2, DCL3, and DCL4 produce various size classes of siRNAs. Plants also encode RNASE THREE-LIKE (RTL) enzymes that lack DCL-specific domains and whose function is largely unknown. We found that virus infection induces RTL1 expression, suggesting that this enzyme could play a role in plant-virus interaction. To first investigate the biochemical activity of RTL1 independent of virus infection, small RNAs were sequenced from transgenic plants constitutively expressing RTL1. These plants lacked almost all DCL2-, DCL3-, and DCL4-dependent small RNAs, indicating that RTL1 is a general suppressor of plant siRNA pathways. In vivo and in vitro assays revealed that RTL1 prevents siRNA production by cleaving dsRNA prior to DCL2-, DCL3-, and DCL4-processing. The substrate of RTL1 cleavage is likely long-perfect (or near-perfect) dsRNA, consistent with the RTL1-insensitivity of miRNAs, which derive from DCL1-processing of short-imperfect dsRNA. Virus infection induces RTL1 mRNA accumulation, but viral proteins that suppress RNA silencing inhibit RTL1 activity, suggesting that RTL1 has evolved as an inducible antiviral defense that could target dsRNA intermediates of viral replication, but that a broad range of viruses counteract RTL1 using the same protein toolbox used to inhibit antiviral RNA silencing. Together, these results reveal yet another level of complexity in the evolutionary battle between viruses and plant defenses

miR-128 Restriction of LINE-1 (L1) Retrotransposition Is Dependent on Targeting hnRNPA1 mRNA

The majority of the human genome is made of transposable elements, giving rise to interspaced repeats, including Long INterspersed Element-1s (LINE-1s or L1s). L1s are active human transposable elements involved in genomic diversity and evolution; however, they can also contribute to genomic instability and diseases. L1s require host factors to complete their life cycles, whereas the host has evolved numerous mechanisms to restrict L1-induced mutagenesis. Restriction mechanisms in somatic cells include methylation of the L1 promoter, anti-viral factors and RNA-mediated processes such as small RNAs. microRNAs (miRNAs or miRs) are small non-coding RNAs that post-transcriptionally repress multiple target genes often found in the same cellular pathways. We have recently established that miR-128 functions as a novel restriction factor inhibiting L1 mobilization in somatic cells. We have further demonstrated that miR-128 functions through a dual mechanism; by directly targeting L1 RNA for degradation and indirectly by inhibiting a cellular co-factor which L1 is dependent on to transpose to new genomic locations (TNPO1). Here, we add another piece to the puzzle of the enigmatic L1 lifecycle. We show that miR-128 also inhibits another key cellular factor, hnRNPA1 (heterogeneous nuclear ribonucleoprotein A1), by significantly reducing mRNA and protein levels through direct interaction with the coding sequence (CDS) of hnRNPA1 mRNA. In addition, we demonstrate that repression of hnRNPA1 using hnRNPA1-shRNA significantly decreases de novo L1 retro-transposition and that induced hnRNPA1 expression enhances L1 mobilization. Furthermore, we establish that hnRNPA1 is a functional target of miR-128. Finally, we determine that induced hnRNPA1 expression in miR-128-overexpressing cells can partly rescue the miR-128-induced repression of L1′s ability to transpose to different genomic locations. Thus, we have identified an additional mechanism by which miR-128 represses L1 retro-transposition and mediates genomic stability

Normalized abundance of the three major classes of endogenous siRNAs.

<p><b>A</b>) ta-siRNAs. <b>B</b>) endoIR-siRNas. <b>C</b>) PolIV/PolV-siRNAs. Small RNAs from wild-type (Col), <i>35S</i>:<i>RTL1</i> (<i>RTL1</i>) transgenic plants and <i>dcl2dcl3dcl4 (dcl234)</i> mutants were classified as ta-siRNAs, endoIR-siRNas, or PolIV/PolV-siRNAs based on published annotation. Small RNA abundance was normalized to the total amount of conserved miRNAs. Each size of small RNA is indicated by a different color: 21 nt (blue), 22 nt (green), 23 nt (pink), and 24 nt (red).</p

RTL1 expression and localization.

<p><b>A)</b> RNA extracted from the total aerial part of wild-type plants (Col) three weeks after inoculation with water (mock), TCV, TVCV, CMV, or TYMV were subjected to oligo-dT reverse transcription followed by qPCR with primers specific to <i>RTL1</i>, <i>RTL2</i>, or <i>RTL3</i>. Analysis was done in triplicate. Results were normalized to <i>GAPDH</i>. <b>B)</b> RNA extracted from leaves and roots of 3 wk-old wild-type plants (Col) were subjected to oligo-dT reverse transcription followed by qPCR with RTL1 oligos. Analysis was done in triplicate. Results were normalized to <i>GAPDH</i>. <b>C)</b> RNA extracted from leaves of 3 wk-old wild-type plants (Col) and <i>35S</i>:<i>RTL1-Flag#2</i> (<i>RTL1-Flag</i> #2) plants were subjected to oligo-dT reverse transcription followed by qPCR with RTL1 oligos. Analysis was done in triplicate. Results were normalized to <i>GAPDH</i>. <b>D</b>) Onion epidermial cells transformed with a <i>35S</i>:<i>RTL1-GFP</i> construct were imaged using a Zeiss Axioskop 2 microscope and recorded using a Leica DC 300 FX digital camera (Leica). <b>E</b>) Proteins were extracted from 18-d-old seedlings of wild-type plants (Col) and <i>35S</i>:<i>RTL1-Flag</i> (<i>RTL1-Flag</i>) plants and hybridized with an anti-RTL1 antibody. Hybridization with an anti-RPL13 antibody serves as a loading control. <b>F)</b> Immunostaining of root cells from 8-d-old seedlings of wild-type plants (Col) and <i>35S</i>:<i>RTL1-Flag</i> (<i>RTL1-Flag</i>) plants was performed using an anti-RTL1 antibody and revealed with Alexa 488.</p

RTL1 cleaves dsRNA.

<p><b>A</b>) RNA gel blot detection of <i>IR71</i> precursor RNA in wild-type (Col), Col transformed with the <i>35S</i>:<i>RTL1</i> construct (<i>Col/RTL1</i>), <i>dcl234</i>, and <i>dcl234</i> transformed with the <i>35S</i>:<i>RTL1</i> construct (<i>dcl234/RTL1</i>). Transformants exhibiting a strong RTL1 developmental phenotype were analyzed. High molecular weight (HMW) RNAs extracted from flowers were hybridized with a probe complementary to the <i>IR71</i> RNA and with <i>25S</i> as loading control. <b>B</b>) RNAs extracted from wild-type seedlings were incubated or not with wild-type His-RTL1 and subjected to RT-PCR to detect IR71, IR2039, and At3g18145 (3’UTR) precursor RNAs. <b>C</b>) RNAs extracted from wild-type seedlings were incubated with wild-type or mutant His-RTL1 and subjected to RT-PCR reactions to detect At3g18145 (3’UTR) precursor RNAs. Comassie blue-stained gel shows approximately 200 ng of wild-type and mutant proteins. A schematic representation of RTL1 (residues 1–289) is shown at the top. Black and grey boxes correspond to RNaseIII and dsRBD (double stranded RNA Binding Domain) motifs, respectively. The conserved amino acids in the RNase III signature motif are highlighted in black and the residues E86, E89, E92, and D96 mutated in recombinant proteins indicated by an asterisk. <b>D</b>) RNAs extracted from wild-type seedlings were denaturated or not before incubation with His-RTL1 and subjected to RT-PCR reactions to detect IR71 precursor RNAs. RT-PCR amplification of U3 snoRNA sequences shows similar amount of RNA in each reaction.</p

The RTL1 RNaseIII domain is required for inhibition of transgene PTGS.

<p><b>A</b>) <i>N</i>. <i>benthamiana</i> leaves were infiltrated with a <i>35S</i>:<i>GU-UG</i> construct (<i>GU-UG</i>) together with either a wild-type <i>35S</i>:<i>RTL1</i> construct <i>(RTL1)</i>, a construct mutated in the RNaseIII domain (<i>RTL1mR3</i>) or a <i>35S</i>:<i>GFP</i> control <i>(GFP)</i>. Low molecular weight (LMW) RNAs were hybridized with a <i>GUS</i> probe and with <i>U6</i> as a loading control. <b>B</b>) <i>N</i>. <i>benthamiana</i> leaves were infiltrated with a <i>35S</i>:<i>GU-UG</i> construct (<i>GU-UG</i>) and a <i>35S</i>:<i>GUS</i> construct (<i>GUS)</i> together with either a wild-type <i>35S</i>:<i>RTL1</i> construct <i>(RTL1)</i>, a construct mutated in the RNaseIII domain (<i>RTL1mR3</i>), or a <i>35S</i>:<i>GFP</i> control <i>(GFP)</i>. LMW RNAs were hybridized with a <i>GUS</i> probe and with U6 as loading control. High molecular weight RNAs were hybridized with a <i>GUS</i> probe and with 25S as loading control. <b>C</b>) The <i>Arabidopsis</i> line <i>L1</i> carrying a <i>35S</i>:<i>GUS</i> transgene silenced by PTGS was transformed with a wild-type <i>35S</i>:<i>RTL1</i> construct <i>(RTL1)</i>. LMW <i>GUS</i> RNAs from two independent transformants were analyzed. Note that the images presented in this panel are internal to the images presented in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002326#pbio.1002326.s010" target="_blank">S9C Fig</a>. <b>D</b>) <i>N</i>. <i>benthamiana</i> leaves were infiltrated with a <i>35S</i>:<i>GU-UG</i> construct (<i>GU-UG</i>) together with either a wild-type <i>35S</i>:<i>RTL1</i> construct <i>(RTL1)</i>, tagged constructs (<i>RTL1-Myc</i>, <i>Myc-RTL1</i>, <i>RTL1-Flag</i>, <i>Flag-RTL1</i>), or a <i>35S</i>:<i>GFP</i> control <i>(GFP)</i>. LMW RNAs were hybridized with a <i>GUS</i> probe and with <i>U6</i> as a loading control. <b>E</b>) <i>N</i>. <i>benthamiana</i> leaves were infiltrated with a <i>35S</i>:<i>GU-UG</i> construct (<i>GU-UG</i>) together with either a wild-type tagged <i>35S</i>:<i>RTL1-Myc</i> construct <i>(RTL1-Myc)</i>, a construct mutated in the RNaseIII domain (<i>RTL1mR3-Myc</i>) or a <i>35S</i>:<i>GFP</i> control <i>(GFP)</i>. LMW RNAs were hybridized with a <i>GUS</i> probe and with <i>U6</i> as a loading control. Proteins were extracted and hybridized with an anti-Myc antibody. Ponceau staining serves as a loading control. <b>F</b>) The <i>Arabidopsis</i> line <i>L1</i> was transformed with either a wild-type-tagged <i>35S</i>:<i>RTL1</i> construct <i>(RTL1-Myc)</i> or a tagged construct mutated in the RNaseIII domain (<i>RTL1mR3-Myc</i>). Proteins were extracted from three independent <i>RTL1-Myc</i> transformants and eight independent <i>RTL1mR3-Myc</i> transformants and hybridized with an anti-Myc antibody. Ponceau staining serves as a loading control. <b>G</b>) LMW RNAs from <i>RTL1-Myc</i> and <i>RTL1mR3-Myc</i> transformants expressing comparable amount of proteins were hybridized with <i>GUS</i> and <i>TAS2</i> probes and with <i>U6</i> as a loading control.</p