Despite their clinical importance, the pathogenic mechanisms underlying SLC26A4 variants remain largely unexplored, leading to a lack of effective treatments. Potential therapeutic solutions for SLC26A4 variants include gene replacement (or augmentation) and genome editing to correct specific variants. In 2019, Kim et al. (37) demonstrated the potential of gene replacement therapy for pendrin-related hearing loss by injecting an adeno-associated virus containing Slc26a4 cDNA into the inner ear of Slc26a4 knockout mice. Later, Takeda et al. (38) demonstrated that transuterine gene transfer of Slc26a4 cDNA into the otocysts of Slc26a4 deficient mice could restore hearing and vestibular functions. However, these replacement approaches, by providing exogenous coding sequences, may result in complications arising from overexpression or ectopic expression of the wild-type gene in treated cells (18–20). In addition, gene replacement therapies have limited durability and may require repeated administration, increasing the likelihood of side effects such as immunogenicity (21).
In contrast, genome editing with the CRISPR/Cas9 system offers a potential one-time treatment strategy to efficiently and permanently correct pathogenic variants. Recently, several derivative techniques have been developed to improve the performance of the CRISPR/Cas9 system, such as base editing and prime editing (39). Base editing can directly modify a single nucleotide without a template, while prime editing can provide more possible sites of action to increase the editing efficiency (40). Despite these advances, the long-term efficacy of these genome editing approaches in vivo is controversial (41). In addition, most somatic cells are non-dividing, which limits the development of therapeutic strategies using the aforementioned CRISPR/Cas9 techniques that require a precise HDR pathway (42). This may be particularly relevant for the treatment of HHI, as most cells in the inner ear are in the terminal stage of differentiation and are non-dividing. In our unpublished study, we tried HDR and base editing approaches to correct the SLC26A4 c.919-2A > G site in cells. However, the HDR approach failed with a low correction rate and the base editing showed not only low editing efficiency but also various types of unwanted off-target editing (data not shown). It is unclear whether the low correction rates were due to the genomic sequence, location, or the CRISPR/Cas9 strategy itself.
Recently, a unique CRISPR/Cas9-based HITI strategy was developed to enable targeted gene insertion in non-dividing cells both in vitro and in vivo, thus showing great translational potential for inner ear therapeutics (28). In this study, we investigated the editing efficiency of HITI in human cells harboring SLC26A4 c.919-2A > G, the most common SLC26A4 variant in East Asian populations. The aim of this study was to correct the SLC26A4 transcript missplicing caused by c.919-2A > G variant (chr7-107 323 898-A-G) by introducing the designed HITI donor sequence with the entire exon 8 and its flanking intronic segments (chr7:107 323 848–107 325 735, 1 888bp, see Fig. 2) into the target region with the aim of restoring normal mRNA spliced transcript with non-skipped exon 8. To validate the efficiency of the HITI strategy, we used NGS to determine whether the donor sequence was successfully inserted into the expected locus. Although the HEK293T cells suggested a better sgRNA targeting efficiency than iPSCs, only 0.15% of the sequencing reads showed correct integration in the final performance of HITI.
To our knowledge, this study was among the first in the literature to apply HITI to correct pathogenic HHI variants. Despite the low efficiency, the presence of the QueryCI sequence (Fig. 5B) indicated that HITI-mediated genome editing had occurred, providing proof of concept. Several factors likely contributed to the low editing efficiency. First, based on our experience with other CRISPR/Cas9 assays (data not shown), the T-rich sequence near the c.919-2 locus may affect the sgRNA binding efficiency. Second, HITI-mediated genome editing requires the donor sequence to be in a specific forward/reverse orientation, which may require multiple CRISPR cutting steps and reduce efficiency. Third, to be compatible with the adeno-associated virus system, our CRISPR/Cas9 system was cloned separately into the two-plasmid system; therefore, only double-transfected cells had a chance to be edited, which reduced the yield of successful transfection.
The emergence of the HITI strategy provides a novel approach for genome editing in non-dividing inner ear cells during terminal differentiation. However, current CRISPR/Cas9 techniques, including HITI, fail to effectively correct the misspliced mRNA transcript of the SLC26A4 c.919-2 variant, indicating its unsuitability for CRISPR/Cas9-mediated treatments. To improve efficacy, it may be beneficial to explore additional sgRNAs that target the downstream region, particularly the intronic region near the 3' end of exon 8. The longer intron 8 provides more potential sgRNA targets for future HITI strategies. Despite promising in vitro results in HEK293T cells, correction efficiency remained low, highlighting the challenges of applying CRISPR/Cas9 approaches to this variant. Our experience in this genomic region provides valuable insights to guide future research and avoid trial-and-error approaches. Given the genetic diversity of HHI, further investigation of the translational potential of the HITI strategy in inner ear therapeutics is warranted.