INDUCTION OF HUMAN HISTO-BLOOD GROUP A ANTIGEN EXPRESSION IN MOUSE CELLS BY GENE THERAPY USING LENTIVIRAL VECTORS HARBORING HUMAN ABH-RELATED GLYCOSYLTRANSFERASE GENES
Bibliographic record
Abstract
P966 Aims: We recently discovered that ABO-incompatibility is not a barrier in infant heart transplantation and that infant recipients of ABO-incompatible hearts develop B cell tolerance to specific donor-type ABH antigens. We hypothesize that induction of cell surface human histo-blood group A antigen expression in neonatal mice will induce specific B cell tolerance, allowing us to generate a murine model for mechanistic investigation. This study determined the feasibility of inducing human A antigen expression in murine cells, both in vitro and in vivo, using replication-deficient lentiviral vectors capable of stable long-term integration into the cellular genome. Methods: The human α-1,2-fucosyltransferase (FUT1; for H antigen expression) gene was cloned into a lentiviral vector, and the human A glycosyltransferase (A-trs; for A antigen expression) gene was cloned into a bicistronic lentiviral vector also containing the green fluorescent protein (GFP) gene. Infection-competent lentiviral particles were produced by co-transfection of 293T cells with FUT1 or A-trs vector and 2 helper plasmids. Viral supernatants were concentrated for titers up to 5x109 transducing units/ml. HeLa (human cervical carcinoma cells of histo-blood group O origin), LTA (mouse fibroblast) and SVEC-10 (mouse endothelial) cells were infected with FUT1 and/or A-trs lentiviral particles. GFP and cell surface H and A antigen expression were assessed by ‘Cellular ELISA’ (CELISA) and confocal microscopy. Neonatal BALB/c mice were injected with FUT1 and A-trs lentiviral particles; GFP was detected post-injection by flow cytometry. Results: Infection with FUT1 lentiviral particles enhanced H antigen expression by HeLa cells. Expression of A antigen was moderate in HeLa cells infected with only A-trs lentiviral particles and appreciably higher with FUT1 co-infection. LTA and SVEC-10 cells infected with FUT1 particles exhibited dose-dependent H antigen expression. Expression of A antigen by LTA and SVEC-10 cells occurred only when infected with both FUT1 and A-trs, suggesting the murine homologue of FUT1 does not provide H antigen expression sufficient for A antigen synthesis. Expression of A antigen was dose-dependent; doses of H and A lentivirus for optimal A antigen expression were determined. Fluorescent imaging of H and A antigen-positive cells was consistent with CELISA data. All cells exhibiting surface A antigen also showed GFP fluorescence. FACS analysis of peripheral mononuclear cells from mice injected as neonates with FUT1 and A-trs lentiviral particles revealed GFP expression in 18% of cells, 9 days post-injection. Conclusions: Our lentiviral vectors induced H and A antigen expression by human and mouse cells. Mouse cells required induction of H antigen for cell surface expression of A antigen. GFP expression in mice 9 days after neonatal injection with FUT1 and A-trs lentiviral particles suggests in vivo induction of H and A antigen expression.
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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".