AAV-mediated transgene delivery targeting spiral ganglion nonsensory cells

Abstract

In-situ neuronal reprogramming in the cochlea through gene therapy offers an avenue to restore hearing loss caused by neuronal damage. One possible source of neuronal conversion is the nonspiral ganglion cells (NSGCs), which include satellite cells, Schwann cells, and otic mesenchyme cells. A major obstacle for this approach is the vector-mediated transgene delivery toward NSGCs. Herein, we sought to assess the transduction profile of adeno-associated virus (AAV) serotypes with peripheral glial cell tropism in the murine inner ear. AAV-1, AAV-DJ, and AAV-PHP.eB with a cytomegalovirus promoter-driven enhanced green flourescent protein (eGFP) reporter were injected into CBA/CaJ neonatal mice via the posterior semicircular canal. One week postinjection, the cochlear tissue was collected for immunohistochemistry in whole-mount and mid-modiolar sections to assess the colocalization of eGFP within the NSGCs in the osseous spiral lamina and Rosenthal's canal. The contralateral ear served as an internal control. Auditory brain responses (ABRs) were recorded 30 days postinjection to assess for hearing loss. AAV-1 and AAV-DJ demonstrated 30-32% transduction efficacy of Pou3f4 immunopositive otic mesenchyme cells, whereas transduction efficacy of Sox2 or Sox10 positive Schwann cells and satellite cells was 0.8-1.82% for all serotypes. At 30 days postinjection, ABR thresholds in the injected mice were comparable to those of the noninjected control. We were able to transduce otic mesenchyme cells among SGNCs in the spiral ganglion region, whereas transduction of Schwann cells and satellite cells continues to pose challenges with AAV-1, AAV-DJ, and AAV-PHP.eB serotypes.

Keywords: AAV; Schwann cells; gene delivery; otic mesenchyme cells; satellite cells.

Fig. 1

AAV-1, AAV-DJ, and AAV-PHP.eB demonstrate GFP distribution in the OSL, RC, and the OC regions in cochlear whole mount. (a–d) Control cochlear whole mount specimen with Sox10+ (red) which highlights the cochlear supporting cells in the OC and glial cells in OSL and OC area. Phalloidin+ (orange) cells delineate the cytoskeletal structure of the cochlear supporting cells and hair cells in the OC region. White dashed lines delineate the division among OC, OSL, and RC regions. (b–d) Neonatal cochleae injected with AAV-1, AAV-DJ, and AAV-PHP.eB displayed robust GFP+ signals in the OC, OSL, and RC regions at varying intensities. AAV, adeno-associated virus; OC, organ of Corti; OSL, osseous spiral lamina; RC, Rosenthal’s canal.

Fig. 2

Pou3f4+ mesenchymal cells are preferentially transfected by AAV-1 and AAV-DJ. (a) AAV serotypes mediated GFP expression colocalized with Pou3f4+ (red) cells. (b) AAV serotypes also demonstrated transfected-GFP expression that colocalized with a few Sox2+ (magenta) cells. (c) AAV-DJ and AAV-PHP.eB demonstrated AAV-mediated GFP expression colocalizing with a few Sox10+ (red) cells. The non-Sox2/10 positive cells in the RC represent SGN cells. (d) Transfection efficacy of the selected AAV vectors regarding Sox10+, Sox2+, and Pou3f4+ cells. AAV-1, AAV-DJ and AAV-PHP.eB transfects 1.20–1.82% of the Sox10+ cells within the RC. White arrowheads depict areas with colocalized GFP and Sox2/10 or Pou3f4 signals. The Sox2+ cells were 0.88–1.39%, respectively. AAV-1 and AAV-DJ transfection ratio of Pou3f4 was 30.1 and 32.5%, whereas AAV-PHP.eB was 10.1%. ** Denotes significant level P less than 0.0 and *** denotes significance level P less than 0.001. AAV, adeno-associated virus; RC, Rosenthal’s canal; SGN, spiral ganglion neuron.

Fig. 3

Threshold shifts are not significantly elevated in mice injected with AAV-1, AAV-DJ, and AAV-PHP.eB via posterior semicircular canal. The left ears of mice injected with AAV-1, AAV-DJ, and AAV-PHP.eB were averaged and compared to those of uninjected contralateral ears (n = 11) at 30 dpi. Injected ears (red) have thresholds comparable to those of uninjected control mice (black), with no statistical difference (P = 0.09). This indicates that PSCC is well-tolerated in neonatal mice. AAV, adeno-associated virus; dpi, days postinjection; PSCC, posterior semicircular canal.