301. Efficient Delivery of Nuclear and Cytoplasmic Proteins Fused to HIV-1 Gag Polypeptide By Means of Virus-Like Particles
Notice bibliographique
Résumé
The expression of the retroviral polypeptide Gag can induce the formation of virus-like particles (VLP). Upon expression, the polypeptide is targeted to the cell membrane and incorporated in the VLP during membrane budding. Chimeric proteins consisting of Gag polypeptide fused to different proteins were engineered and the VLP produced were used as vehicles for protein delivery. One major advantage of this approach is the low mutation risk due to the absence of DNA transfer and genomic integration. In this study, the C-Terminus of Gag from HIV-1 was fused to the green fluorescent protein (GFP), a chimeric transactivator (cTA) and a reprogamming factor (KLF4). VLP were produced by transfection using a stable cell line (293SF-pacLV) that expresses VSVg and Gag-pol. Analysis of the supernatants from producing cells by western blot confirmed the presence of Gag-GFP, -cTA and -KLF4. Confocal microscopy showed that the vast majority of the cells (> 90%) treated with VLP-GFP/polybrene complexes was successfully transduced. The cells also displayed a GFP signal almost exclusively localized inside the cytoplasm. Additional VSVg expression during the production facilitated the endosomal escape of VLP-GFP in transduced cells. The insertion of a nuclear localisation signal (NLS) shifted the localization of the GFP to the cell nucleus demonstrating that a nuclear protein could be successfully delivered by VLP. The experiment was thus repeated using two transcription factors. Lentiviral vectors were used to make two stable pools of HEK293 cells each containing a specific GFP reporter cassette. The GFP gene was regulated either by the CR5 promoter (specifically activated by the cTA) or by a minimal promoter fused to KLF4 transcription response elements (TRE). Transduction of the CR5-GFP pool with VLP-cTA/polybrene complexes showed a powerful activation of the reporter (365-fold compared to the negative control) as measured by flow cell cytometry two days post-transduction. Surprisingly, no activation was observed in the TRE-GFP pool three days after transduction by VLP-KLF4/polybrene complexes. Evidence obtained by transfection suggested that the Gag fusion inhibits KLF4 activity. To augment activity, the activation domain of VP16 was fused to KLF4. Transfection of a plasmid encoding Gag-VP16KLF4 strongly activated the reporter by a factor of 126-fold (6-fold higher than wild type KLF4). The ability of VLP produced with Gag-VP16KLF4 to activate transcription is currently under investigation. In summary, VLP based on HIV-1 Gag can deliver nuclear and cytoplasmic proteins directly into cells with a low mutation risk. Therefore, our platform for VLP production could be useful for several applications including cell reprogramming and genome editing oriented toward cell therapies of diseases like Duchenne muscular dystrophy.
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Comment cette classification a été obtenuedéplier
Prédiction distillée sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Apprise à partir de 10 348 étiquettes directes de Codex et de 10 348 étiquettes directes de Gemma. Le mode candidate est l'union des têtes enseignantes seuillées; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont ni des étiquettes humaines ni des étiquettes directes de modèles de pointe.
Scores Codex et Gemma par catégorie
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,000 | 0,000 |
| Méta-épidémiologie (sens strict) | 0,000 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,000 |
| Bibliométrie | 0,000 | 0,000 |
| Études des sciences et des technologies | 0,000 | 0,000 |
| Communication savante | 0,000 | 0,000 |
| Science ouverte | 0,000 | 0,000 |
| Intégrité de la recherche | 0,000 | 0,000 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,000 | 0,000 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule tête enseignante, pas un consensus.
Le détail, modèle par modèle et score par score, se trouve en fin de page sous « Comment cette classification a été obtenue ».