312 IL7 increases targeted lipid nanoparticle-mediated mRNA protein expression in T cells<i> in vitro</i> and<i> in situ</i> by enhancing T cell translation
Bibliographic record
Abstract
<h3>Background</h3> Chimeric Antigen Receptor T cells (CARTs) are a powerful anti-cancer therapy, demonstrating success in hematologic malignancies. The development of targeted lipid nanoparticle-mRNA (tLNP-mRNA) therapeutics has allowed for the generation of CARTs <i>in situ</i> and may resolve several challenges of conventional <i>ex vivo</i> viral-engineered CAR T cell products including scalability, access and mulitplexing. The <i>in situ</i> T cell transfection rate achieved by tLNP-mRNA varies from 4–20% of T cells expressing the protein of interest. As tLNP-mRNA platform efficacy may critically depend on the number, metabolic state, and localization of engineered T cells, we investigated whether cytokines could enhance protein expression. <h3>Methods</h3> We used tLNPs that target CD5, a marker expressed highly on mouse and human T cells. These tLNPs carried the mRNA for the reporter protein mCherry or encoded a fibroblast activated protein (FAP) targeted CAR. Mouse and human T cells were cultured with IL2, IL7, IL15 or activated using αCD3/CD28. CD5-mCherry-tLNPs or CD5-FAPCAR-tLNPs were added and protein expression was detected using flow cytometry. For <i>in vivo</i> studies, C57BL/6 mice were pretreated with IL7, injected with tLNPs and sacrificed 24 hours later. To analyze the T cell transcriptome, CD8<sup>+</sup> T cells were isolated from mouse spleens and cultured with IL2, IL7 or IL15 for 48 hours before being sequenced. <h3>Results</h3> We found that CD5-mCherry-tLNPs induced protein expression on 10% of resting T cells <i>in vivo</i> and ~15% of T cells <i>in vivo</i>. Culturing mouse and human T cells with IL7 significantly improved CD5-mCherry-tLNPs protein expression <i>in vitro</i>. This also occurred in the <i>in vivo</i> setting as pre-treating mice with IL7 elevated both the proportion and total number of mCherry expressing T cells. FAPCAR expression was also increased by combining CD5-FAPCAR-tLNPs with recombinant IL7. Transcriptomic analysis showed IL7 selectively increased pathways associated with protein translation. The significance of these transcriptomic changes was demonstrated by showing that after electroporation with mRNA, T cells cultured in IL7 produced more protein compared to IL2 or IL15. <h3>Conclusions</h3> T cells can be engineered <i>in situ</i> using CD5-targeted tLNPs and IL7 increases the protein expression induced by tLNPs. Our data suggests that the upregulation of translation-associated pathways in T cells by IL7 could be exploited to improve the expression of proteins <i>in situ</i> after tLNP administration. This provides a novel paradigm through which a T cell activating cytokine, instead of lymphodepletion, can potentiate in situ CAR T cell therapy. <h3>Ethics Approval</h3> This study was approved by The University of Pennsylvania’s Ethics Board; approval number 806099.
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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 | 0.001 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.000 |
| Bibliometrics | 0.000 | 0.001 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| 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".