AK-OTOF-101: How a gene therapy that restores hearing works
Exciting news for AAV-mediated gene therapies: in a first ever, the gene therapy AK-OTOF-101 by Akouos (wholly owned subsidiary of Eli Lilly and Company) has restored hearing in an 11 yo with profound deafness since birth.
As a gene therapy guy, here is a brief explanation of how AK-OTOF-101 works.
The deafness treated (DFNB9) is caused by loss-of-function mutations in the gene otoferlin (OTOF). Otoferlin is essential to transmit electrical signals from the ear to the brain.
AK-OTOF-101 aims at restoring hearing by supplementing a functional copy of otoferlin. The transgene by itself (~6 kb) exceeds the packaging capacity of AAVs (~4.4 kb). And this is before considering that the transgene needs a promoter and a poly-adenylation (poly-A) site.
Akous has circumvented this limitation by adopting a dual vector strategy. In dual vector strategies, transgenes are split in two fragments (5' and 3'), equipped with elements to stich up the whole gene (or mRNA, or protein), and the two halves are packaged in separate rAAVs (split rAAVs). When both split rAAVs co-transduce the same cell, the therapeutic product can be reconstituted, like a jigsaw puzzle.
For AK-OTOF-101, Akous uses a “hybrid” dual vector strategy. In short,
- split rAAV1 genome harbours (in this order): a promoter, the 5' transgene, a donor splice site, and a highly recombinogenic sequence (HRS),
- split rAAV2 genome harbours (in this order): an HRS, an acceptor splice acceptor, the 3' transgene and a poly-A site.
In cells, AAVs single strand genomes are converted to double strand ones. In co-transduced cells, double strand genomes of the split rAAV1 and rAV2 recombine via the HRS, a sequence with a high propensity to recombine (Figure 1). In AK-OTOF-1, this is a 77bp sequence from the F1 phage referred to as AK.
In a minority of cases, the split rAAVs recombine via the ITRs, and not the AK (Figure 2).
In both instances, the recombination reconstitutes the whole transgene, with an upstream promoter and a downstream poly-A (if the two AAVs are joined in the right order and orientation).
When the transgene is transcribed, the mRNA contains one HRS (recombination via HRS) or two HRS and an ITR (recombination via ITR) between transgene fragments. At this point, the presence of a splice donor downstream of the 5' transgene fragment and of a splice acceptor upstream of the 3' transgene fragment is instrumental for the success of the gene therapy approach. Together, the splicing sites ensure that the spliceosome rejoins the two transgene fragments (as if they were exons of an endogenous gene) and excise the accessory elements in between (as if an intron).
A fully spliced mRNA for a functional protein can now be exported to the cytoplasm.
And this is how, in short, a full otoferlin can be expressed in auditory cells transduced by AK-OTOF-101 to restore hearing.
