327. Muscle Specific and Efficient Transgene Expression Using the Fast and Slow Troponin I Promoters in Cell Culture
Bibliographic record
Abstract
Gene replacement therapy for muscular diseases, such as Duchenne muscular dystrophy, using viral vectors requires strong promoters with the ability to confer muscle specific expression of the therapeutic gene product. Because skeletal muscle consists of a mixture of slow and fast muscle fibers, an ideal promoter for such application should direct efficient expression in both muscle fiber types. The genetic elements regulating expression of the fast and slow isoforms of troponin I (TnI), an abundant muscle protein, are relatively well characterized. Theoretically, a combination of vectors expressing transgenes regulated by TnIfast and TnIslow enhancers/promoters could be used to deliver strong and muscle specific transgene expression in every skeletal muscle fiber of the body. To evaluate the usefulness of the TnIfast and TnIslow enhancers/promoters for gene therapy, we have compared their strengths with the cytomegalovirus (CMV) and with the short (1.3-kb) murine muscle creatine kinase (MCK) promoters in cell culture. Plasmids expressing β-galactosidase (β-gal) regulated by MCK, CMV or TnIfast/TnIslow enhancers/promoters were constructed. The TnIfast construct includes the minimal promoter (200-bp) of the quail TnIfast gene linked to three copies (150-bp each) of that gene’s first-intron enhancer element (IRE). The TnIslow construct includes the minimal promoter (100-bp) of the human TnIslow gene linked to that gene’s upstream enhancer element of 160-bp (USE). Except for MCK, a small intron (100-bp) derived from SV40 was inserted between the promoter and β-bal. 293A cells and C2C12 myoblasts were transfected with the different constructs and β-gal expression was compared 48 hrs later. Analyses were also performed in culture of C2C12 myotubes 10 days after serum withdrawal. In 293 cells, MCK-, TnIfast-, and TnIslow-driven expression was at least 1300 folds weaker than CMV. In non-differentiated myoblasts, TnIslow-driven expression was 3.5 and 15 times better than TnIfast- and MCK-driven expression, respectively. Both TnIfast and TnIslow enhancers/promoters provided high-level β- gal expression in myotubes (35-40 % of CMV, and at least 10 times stronger than MCK). Our data confirmed the excellent muscle specificity and strength of TnIfast and TnIslow enhancer/promoter elements. Their relative small size makes them very attractive for usage in vectors with limited insert capacity such as AAV. These excellent properties warrant further in vivo gene transfer studies using viral vectors.
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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".