599. Deep Informatics Utilized to Design MiniPromoters for Driving PAX6-Like Retinal Expression with AAV
Bibliographic record
Abstract
Purpose: Gene-based therapies are making a comeback, as evidenced by the clinical success of Glybera, rAAV2. RPE65, and rAAV2. REP1. These therapies utilize ubiquitous promoters, however future gene therapies may benefit from promoters that can direct tissue- and cell-type specific expression. Capturing the expression of a human gene in a promoter small enough to use in AAV is challenging. Paired box six (PAX6) encodes a transcription factor with a complex and potentially therapeutically-useful retinal-expression pattern. Also, PAX6 has several previously published discrete regulatory regions (RRs), and therefore may contain modular regulation suitable for MiniPromoter design. Finally, mutations in PAX6 cause the vision-loss disorder aniridia, and a MiniPromoter capable of recapitulating the expression of PAX6 would be a useful tool for an aniridia gene therapy. Methods: PAX6 RRs were predicted using Hi-C, UCSC Genes, CAGE, 100 vertebrate phastCons, transcription factor binding site (TFBS), Segway and ChromHMM data. MiniPromoters were cloned into AAV genomes containing an emGFP reporter and WPRE, packaged into AAV2(Y272F, Y444F, Y500F, Y730F, T491V), and administered to postnatal day 14 mice by intravitreal injection. Ocular tissue was collected 30 days after injection, emGFP expression was evaluated by epifluorescent imaging of mouse retinas, and staining with antibodies against PAX6, Brn3a, syntaxin, and calbindin. MiniPromoters driving PAX6-like expression were examined for unique TFBSs using oPOSSUM 3. Results: A PAX6 containing highly interactive domain was revealed within which most previously published PAX6 RRs are located. Within this domain, 31 RRs were bioinformatically predicted, nine of which were selected to construct seven MiniPromoters. Of these MiniPromoters, two did not drive interesting emGFP expression in the retina, and five drove expression in the two retinal layers that express PAX6 (ganglion cell and inner nuclear). Twenty-one TFBSs were found to be unique to these MiniPromoters. Interestingly, one of the five (Ple255) not only drives expression in the same cell layers that express PAX6, but also in all the same cell types (ganglion, amacrine, and horizontal). Conclusions: Bioinformatic approaches can be used to design MiniPromoters capturing the endogenous expression pattern of a gene for a specific tissue. The resulting MiniPromoters may be important tools for future retinal gene therapy, with Ple255 being especially applicable to gene therapy for aniridia.
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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".