corrected common TGFBI corneal-dystrophy mutations in human corneal epithelial cell models.
Evidence
This preclinical cell and donor-tissue study tested ABE8e-NG RNA editing in R124C and R555W HCE models and AAV serotypes in HCE cells and healthy human corneal donor tissue.
Caveat
The work is limited to cell models and healthy donor tissue, so in vivo efficacy and durability remain untested.
Simplified
PURPOSE: Lattice and granular corneal dystrophy comprise two common TGFBI-associated autosomal dominant corneal disorders. Existing therapies are only temporizing and carry significant morbidity. Here, we develop a novel therapeutic approach using an adenine base editor (ABE) to correct common .
METHOD: We generated two human corneal epithelial (HCE) cell models harboring a copy of the most common disease-causing TGBFI mutations, R124C or R555W. These lines were electroporated with an ABE8e-NG-encoding mRNA and guide RNAs targeting the mutations. The resulting A•T-to-G•C editing efficiencies and off-target (OT) effects were assessed by amplicon sequencing. GFP-expressing adeno-associated viruses (AAVs) with different capsid types were transduced into HCE cells and healthy human corneal donor tissues, and GFP fluorescence was evaluated.
RESULTS: Using all-RNA delivery for ABE8e-NG, we achieved 91% and 62% correction of the pathogenic adenines in HCE TGFBIR124C/WT and TGFBIR555W/WT cells, without editing the wild-type allele. Indel formation was negligible (<0.2%), bystander adenine editing was minimal (<0.7%), and editing at top computationally predicted OT sites was modest (<1.2% at all but 1 of the 20 OT sites analyzed), suggesting minimal safety concerns. Correction of TGFBIR124C/WT in HCEs rescued the aberrant lysosomal localization of TGFBI. We further identified AAV1 as the most effective serotype for gene delivery into both human corneal donor tissue and HCE cells.
CONCLUSIONS: Our study demonstrates the feasibility and safety of CRISPR as a new therapeutic strategy for correcting common TGFBI mutations in corneal dystrophies, paving the way for further preclinical testing.
Key numbers
91%
Correction Efficiency R124C
Percentage of pathogenic adenines corrected in TGFBI R124C cells.
62%
Correction Efficiency R555W
Percentage of pathogenic adenines corrected in TGFBI R555W cells.
99.7%
AAV1 Transduction Efficiency
Percentage of GFP-positive cells after AAV1 transduction.
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CRISPR Gene Editing