Improving adeno-associated virus serotype 6 production and transduction for gene delivery and CAR T-cell therapy
Bibliographic record
Abstract
Chimeric antigen receptor (CAR)-T cell therapy is a promising immunotherapy that involves engineering T cells by adding a CAR to their surface to specifically target and "destroy" tumour cells.Adeno-associated viruses (AAVs) are well-suited for this application as they can deliver a DNA template for the knock-in of a transgene via homologous recombination with high efficiency.With the growing interest in immunotherapy, there is an increasing demand for AAV serotype 6 (AAV6), the serotype with the highest tropism for immune cells.However, the current limitations in AAV6 production and transduction pose significant challenges to effectively using this serotype in therapy.In this thesis, I aimed to improve the production and transduction capacity of AAV6 and to use this vector for site-specific integration of CAR into T cells.One of the significant limitations in AAV manufacturing is low-cell-density production.This is due to the Cell Density Effect, which limits the potential of suspension cell-based platforms to increase yields.We investigated the improvement of AAV6 production by transient transfection of suspension-adapted HEK293SF cells.We found that by providing plasmid DNA on a cell basis and at a medium cell density of 4 million cells per mL, we achieved titers above 10 10 viral genomes (VG)/mL, a significant increase compared to established methods.This medium-cell-density production method, validated in a 3-L bioreactor scale, lays the foundation for large-scale process operations, potentially solving the current vector shortage in AAV manufacturing.Another significant challenge in AAV transduction is the large number of AAV6 required for effective transduction, estimated at 10 6 viral genomes per cell.We evaluated the use of cellpenetrating peptides to enhance the transduction of AAV6 into T cells.We found that the peptides TAT-HA2 and LAH4 significantly improved the transduction efficiency, with LAH4 resulting in a 10-fold increase at the lowest multiplicity of infection tested.This approach could be further developed to improve the transduction of AAVs into other cell types.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.001 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.001 | 0.001 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.004 | 0.002 |
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 source (direct Gemma or distilled Codex), 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".