Cabp2-Gene Therapy Restores Inner Hair Cell Calcium Currents and Improves Hearing in a DFNB93 Mouse Model

Abstract

Clinical management of auditory synaptopathies like other genetic hearing disorders is currently limited to the use of hearing aids or cochlear implants. However, future gene therapy promises restoration of hearing in selected forms of monogenic hearing impairment, in which cochlear morphology is preserved over a time window that enables intervention. This includes non-syndromic autosomal recessive hearing impairment DFNB93, caused by defects in the CABP2 gene. Calcium-binding protein 2 (CaBP2) is a potent modulator of inner hair cell (IHC) voltage-gated calcium channels CaV1.3. Based on disease modeling in Cabp2–/– mice, DFNB93 hearing impairment has been ascribed to enhanced steady-state inactivation of IHC CaV1.3 channels, effectively limiting their availability to trigger synaptic transmission. This, however, does not seem to interfere with cochlear development and does not cause early degeneration of hair cells or their synapses. Here, we studied the potential of a gene therapeutic approach for the treatment of DFNB93. We used AAV2/1 and AAV-PHP.eB viral vectors to deliver the Cabp2 coding sequence into IHCs of early postnatal Cabp2–/– mice and assessed the level of restoration of hair cell function and hearing. Combining in vitro and in vivo approaches, we observed high transduction efficiency, and restoration of IHC CaV1.3 function resulting in improved hearing of Cabp2–/– mice. These preclinical results prove the feasibility of DFNB93 gene therapy.

CaBP1 and 2 enable sustained CaV1.3 calcium currents and synaptic transmission in inner hair cells

“…1-4), we thus next asked, how this may influence the hearing of the animals. As previously observed 20 , unilateral injection of the PHP.eB- Cabp2 - eGFP resulted in strong eGFP immunofluorescence signal in the vast majority of hair cells and elsewhere (Fig. S4A-B).…”

“…This was significantly stronger when compared to the inactivation observed in the IHCs of Cabp1 or Cabp2 single KOs in similar conditions (Fig. 1C) 11,12,20 , and even when comparing to data from 5-week-old Cabp2 KO mice obtained at physiological temperature (Fig. S1D), further substantiating the evidence of severity and early onset of the DKO phenotype.…”

“…Among the three putative splice isoforms of CaBP2 differing in the N-terminal aminoacid sequence, the alternative long isoform, termed CaBP2-alt, may be the most abundant splice variant in the IHCs, as suggested by in situ hybridization 19 , while SGNs seem to express none. So far our gene-rescue experiments were conducted with the conventional L-isoform only, which however shows strong inhibition of Ca V 1.3 inactivation in the HEK cells as well as IHCs 12,20 . Alternative splicing also generates three CaBP1 variants 19 .…”

Gene mutations as a non-invasive measure of adult cochlear implant performance: Variable outcomes in patients with select TMPRSS3 mutations

“…Future high powered genetic cochlear implant outcome studies will help drive this field forward [22]. Patients with genetic mutations that lack benefit from a cochlear implant would warrant high priority for future gene therapy and auditory brainstem implantation consideration [23,24].…”

Diversity matters — extending sound intensity coding by inner hair cells via heterogeneous synapses

“…Interestingly, bassoon (Frank et al , 2010), but not RIM2a (Jung et al , 2015) or RIM‐BP2 (Krinner et al , 2017), seems required for establishing the normal variance of maximal synaptic Ca 2+ influx of IHC AZs. Differences in the Ca 2+ channel complexes (Fig 5D) between IHC AZs might originate from their subunit composition as well as alternative splicing of Ca V 1.3α1 (Shen et al , 2006; Scharinger et al , 2015; Ohn et al , 2016; Vincent et al , 2017), which may influence binding of EF‐hand Ca 2+ binding proteins (CaBPs, calmodulin; Grant & Fuchs, 2008; Schrauwen et al , 2012; Picher et al , 2017a; Oestreicher et al , 2021), multidomain proteins of the AZ (e.g., RIM‐BPs and RIM), and adapters (e.g., Gipc3, unpublished). For example, a genetic manipulation of the differentially spliced Ca V 1.3α1 C‐terminus that is expected to abolish the long Ca V 1.3α1 splice variant (Scharinger et al , 2015) indeed resulted in a mild alteration of Ca 2+ influx at IHCs, but the functional relevance for sound encoding remains to be clarified (Ohn et al , 2016).…”