ADAR RNA-editing switch enabled tunable AAV payload expression in vivo
A separate RNA dose activated a factor IX gene in mice that had previously been delivered by an AAV vector
In an August 5 preprint, researchers in Joseph Silverberg's group described a system in which AAV, a viral vector carrying genetic instructions, first delivers an inactive human factor IX gene to the liver. Lipid nanoparticles, which are fat-based particles that deliver RNA into cells, later provide the activation signal. A repeat dose of this RNA produced a new pulse of factor IX in mice.
After an AAV injection, its genetic instructions can remain in cells for a long time, allowing the cells to continue producing the encoded protein. Adjusting the amount of that protein later is difficult. The authors therefore separated the process into two steps. First, AAV deposits an inactive genetic instruction in the liver. A separate RNA drug then activates it.
The authors chose human factor IX, a protein involved in blood clotting, for the experiment. They replaced one of the gene's tryptophan codons with TAG. Cells interpret TAG as a stop signal and therefore terminate protein synthesis. Of the four tryptophan positions, the authors selected position 118. In cells, this variant produced no detectable factor IX, whereas variants at positions 240 and 261 still produced some protein.
To activate the gene, the researchers later administered a circular guide RNA to the mice. It recognizes factor IX RNA and recruits ADAR, a cellular enzyme that changes a single RNA base. ADAR converts adenosine into inosine, which the cell reads as guanosine. TAG therefore becomes TGG, a tryptophan codon, allowing the cell to produce factor IX again. In cells, the guide RNA restored protein production to approximately 38% of the level produced by the standard genetic instruction. Sequence analysis confirmed the change at codon 118.
The authors based their use of circular RNA on a 2022 study by Dhruva Katrekar and colleagues. In that study, a circular guide RNA persisted longer in cells than a linear guide and sustained editing for longer. Here, it serves as an external activation signal for an AAV payload that was delivered in an inactive state.
The mice first received AAV carrying the inactive instruction, followed two weeks later by the RNA trigger. After a 5 microgram dose, the mean factor IX concentration reached 48 ng/mL after one day and fell to 5 ng/mL by day seven. A repeat dose on day fifteen produced a second increase to 35 ng/mL. In a separate series, 3.75 micrograms of trigger RNA produced 31 ng/mL, whereas 15 micrograms produced 400 ng/mL.
In this mouse experiment, AAV deposited an inactive genetic instruction in the liver, while a later RNA dose controlled the timing and magnitude of the next factor IX pulse.