301. Efficient Delivery of Nuclear and Cytoplasmic Proteins Fused to HIV-1 Gag Polypeptide By Means of Virus-Like Particles
Bibliographic record
Abstract
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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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".