Novel Gene Therapies to Treat Usher Syndrome Type 1F: Gene Editing

In a third strategy to circumvent the size limitation of PCDH15, we focused on the common R245X mutation in PCDH15 that causes Usher 1F. This mutation is a single C>T transition, which converts the arginine at position 245 to a stop codon (R245X). We reasoned that we could use an adenine base editor (ABE) to reverse the mutation and to restore expression of normal PCDH15. We first developed a Pcdh15-R245X mouse model, by swapping about 60 bases of the mutant human sequence into the corresponding mouse exon.

Mutant human sequence swapped into the mouse gene to introduce the R245X mutation
Mutant human sequence swapped into the mouse gene to introduce the R245X mutation. There are five nucleotide differences in the human sequence, but four are silent so the expressed protein is unchanged except for the R>X mutation. Editing is designed to change the T back to a C, by editing A to G on the antisense strand.

We included the flanking human sequence so that ABEs and guide RNAs (gRNAs) optimized in this model could be directly used in human without re-optimizing. Mice homozygous for the R245X allele were completely deaf and exhibited profound balance deficits, while heterozygous mice were unaffected. With in vitro assays, we tested different ABEs and gRNAs to optimize editing efficiency. We found that an adenine base editor fused to Cas9 is capable of reverting the complementing mutant adenine (A>G on the antisense strand produces T>C on the sense strand), which restores the wild-type PCDH15 coding sequence. We then packaged split-intein versions of the best ABEs and gRNAs into AAV vectors and injected them into the cochleas of the R245X mice. For these experiments we used a modified R245X model in which one Pcdh15 allele carried the mutation and the other allele was floxed. A late-onset Myo15-Cre also in the model deleted the second allele starting about P0, so that the hair bundles could develop normally before deletion of Pcdh15. We observed moderate hearing rescue after delivery of the split-intein ABE. This method is promising and has potential for further optimization.

This project is a collaboration with David Liu and Ben Kleinstiver.