Journal Club: Targetted expression of genes in cochlear supporting cells using Lfng-enhancer.

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Today's journal article

Seist R, Copeland JS, Tao L, Groves AK. Rational design of a Lfng-enhancer AAV construct drives specific and efficient gene expression in inner ear supporting cells. Hear Res. 2025 Mar;458:109203. 

Why I picked this article

In the previous journal club, the research was about the role of supporting cells in the cochlea, related to ototoxic damage. This research is also about supporting cells; the research focuses on how to deliver DNA material specifically to supporting cells, in the context of gene therapy. 

Supporting cells are not sensory cells or neurons; however, they play very important roles in maintaining the cochlear environment under normal homeostasis and under pathological conditions. When supporting cell functions are disrupted, it can lead to sensorineural hearing loss. For example, the most common gene affected in genetic sensorineural hearing loss is Gjp2, a gene for protein connexin 26, found in supporting cells and fibrocytes. 

Targeting supporting cells is central to many regeneration strategies. The current AAV-based vectors for gene therapy favour targeting of hair cells or are too broad across multiple cell types. There is a need to develop a targeted gene delivery strategy for supporting cells. This research project designs an AAV-based gene delivery cassette with an enhancer DNA sequence that switches on genes almost exclusively in supporting cells, offering a cleaner handle for future therapies and lineage‑tracing.

Some of the research findings

Strategy for designing: 
  • Capsid: A pair of inner-ear tropic capsids 
  • Enhancer discovery: with enhancer DNA from Lunatic fringe (Lfng) was sought to restrict expression to supporting cells. 
  • Lfng is a gene expressed broadly during early development, but then becomes specific to supporting cells only, and continues to be expressed in supporting cells of both the cochlea and the vestibular system.
  • After re-analysis of published data (Tao 2021), three candidate Lfng enhancers (En1, En2 and En3) each ~1kb long, were selected based on the chromatin accessibility and active histone marks at the Lfng locus in cochlear supporting cells.
Gene delivery vectors: 
  • AAV‑ie capsid packaging either Lfng‑En1/En2/En3 enhancers driving expression of enhanced green fluorescent protein (EGF), or a broad CBh promoter (control).
    • Lfng-En1-EGFP
    • Lfng-En2-EGFP
    • Lfng-En3-EGFP
    • CBh-EGFP (control)
  • A Cre‑inducible version (AAV‑Lfng‑En3‑CreER) was also built for recombination experiments.
Effectiveness of gene delivery using these vectors - testing: 
  • Mouse strain and ages: outbred ICR mice (both sexes) for EGFP reporter experiments; Ai3 Cre reporter mice for CreER testing.
  • Neonatal P1 animals received an injection of viral vectors via the posterior semicircular canal
  • Cochlear tissues were collected and analyzed at P7 and P21.
  • For CreER, tamoxifen at P18–P19 before P21 harvest.
Cell‑type specific gene delivery:
  • Lfng‑En3 drove robust EGFP in border cells, inner phalangeal cells, inner and outer pillar cells, and all three rows of Deiters’ cells, from apex to base.
  • Vestibular supporting cells were also labelled; hair‑cell labelling was minimal (a weak signal noted in a Myo7a+/Sox2+ type II hair cell).
  • The CBh control was expressed broadly in sensory and non‑sensory inner‑ear cells and showed expression in the brain; in contrast, Lfng‑En3 constructs did not label CNS cells in the examined regions.
  • Testing at later age: AAV‑Lfng‑En3‑CreER plus tamoxifen at P18–P19 drove strong recombination across inner‑ear supporting cells in reporter mice, enabling inducible, supporting‑cell‑specific genetics.
Their construct, based on Lfng-E3 enhancer‑driven AAV, can achieve supporting‑cell‑specific expression with minimal CNS off‑target after neonatal delivery.
From part of Figure 2C. Supporting cells that successfully received genes and expressed EGFP (green) vs sensory cells (red). Seist et al. 2025

Haruna's takeaway

Since the demonstration of successful hearing restoration by OTOF-gene therapy, the race is on to develop gene therapies for other genetic sensorineural hearing loss. This research on the identification and development of targeted gene expression in supporting cells is a progress towards such therapy targeting genes that impact supporting cell health and cause sensorineural hearing loss.  By the time sensorineural hearing loss develops, in many cases, hair cells may be lost or supporting cells may have changed their properties as part of their injury response. With enhancer-driven expression in the target cell, the next step will need to show that this remains effective even in an unhealthy, pathologically changed cochlear environment.

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This is Haruna's 70/100 of the 100-day challenge to post a science blog article every day! I love inner ear biology & cochlear physiology.