How do you design a cell-type-specific promoter for AAV gene therapy?

Annogen designs cell-type-restricted promoters by functionally measuring cis-regulatory activity across a target and its off-target cell panel, rather than inferring it from chromatin marks or sequence models. The hard part is not finding an element that is active; it is finding one whose activity is confined to the intended cell type at a therapeutically useful level, even where the capsid delivers off-target.

An element’s specificity only means something relative to the off-target contexts it was tested against. As such, we always make sure to include those contexts in the screen, either in cultured cells or in in vivo models, and counter-select against them, so specificity is a measured property rather than an annotation inherited from a name or a reference genome track. Often, our customers provide further input on the specific requirements of their therapy in such study designs.

SuRE™ fragments the human genome into millions of barcoded elements of roughly 200 to 2000 bp, each represented by dozens of barcodes, and quantifies activity by RNA barcode sequencing in the relevant cell type. The redundancy is what resolves the low-activity, high-restriction regime where sequence models and epigenomic prediction are least reliable. Increasingly, we rely on the data sets we have developed over the years to pre-select certain promoters and combinations to improve the pool from which we select. Several rounds of combining the best sequences, screening and selection yield patentable elements that have been validated directly in the relevant systems.

Specific elements are generally weaker than ubiquitous viral promoters; there is always a pay-off between such factors. We contrast candidates against spiked CMV, CAG, EF1a and your preferred references so you can directly compare and choose the characteristics your indication needs, rather than chasing an element that maximises both.

Where packaging is limiting, we condense elements to a few hundred base pairs and synthetic reassembly, or reduce the size of existing elements by sliding-window dissection, then read the compact variants functionally, since removing a binding site can shift strength and leakiness non-linearly. We have developed promoters as small as 270bp that still drive robust expression of therapeutic genes.

Talk to us. Send us your target cell type, the off-targets and vector, and we will outline a tailored screen.