How small can an AAV promoter be without losing expression?

Packaging capacity is one of the hardest constraints in adeno-associated virus (AAV) gene therapy, roughly 4.7 kb for single-stranded and 2.4 kb for self-complementary vectors, and the promoter competes with the transgene for that space. Making an element small that it matches your therapeutic gene is something SuRE™ can either take into account from the start, or something that we can optimize when starting from existing elements.

Designing new promoters with strict size limitations can be taken into account with SuRE™, whereby we either start with millions of small, random genomic fragments or rationally designed sequences. In our experience, transcriptional capacity makes a sheer drop when going below 150bp, so we typically use that as a minimum for single elements. With our hybrid screens we combine the most powerful elements, such that the resulting promoter will be a unique sequence of approximately 300bp that has the specificity and strength demanded by your therapy, while still leaving space for the transgene. We have developed promoters as small as 270bp that still drive robust expression of therapeutic genes.

Alternatively, when you want to start from your promoter of choice, Annogen can start from that sequence and perform a saturation mutagenesis profile, in which we create a synthetic SuRE™ library of every possibly single nucleotide variant, as well as sliding window deletions, and test their transcriptional strength to determine which regions can be deleted without losing activity or specificity. In a second iteration, unique, smaller elements will be functionally interrogated and the most optimal, validated elements will be provided to you. Such studies can be performed in as little as 3 months.

Changing an element’s length alters GC content and secondary structure, which can degrade packaging efficiency and worsen full-to-empty ratios, the numbers your manufacturing team lives by. Those constraints belong in the design filter, and we can report packaging-relevant properties alongside activity.

Annogen can screen very small elements, while we also want to be realistic that size, strength and specificity compete: a smaller element carries less regulatory information, and restricted elements are already weaker than ubiquitous viral promoters. Therefore, it is always a wager on how small you truly need to go and how much transcriptional activity you are willing to give up. Screening makes that frontier explicit so you choose the compromise deliberately rather than discover it too late, when it may impact your strategic decision beyond your control.

Finally, what is often not considered is that cryptic promoter activity in the inverted terminal repeat (ITR) and crosstalk across concatemers can perturb a compact cassette, so we advise confirming behaviour in the intended ITR context, not only in a bare reporter, something we can take along when screening a promoter for you.

Talk to us. If you are fitting an element into an AAV cassette, we can design and measure to your size budget.