In vivo Adenine Base Editing rescues adrenoleukodystrophy in a humanized mouse model

X-linked adrenoleukodystrophy (ALD) is an inherited neurometabolic disorder caused by mutations in the ABCD1 gene encoding the peroxisomal ABC transporter and ALD is a progressive disorder that affects mainly the brain, adrenal glands, spinal cord and testis with accumulations of toxic metabolites of very long-chain fatty acids (VLCFA), that fail to enter peroxisome and subsequent beta-oxidation. However, the targeted gene therapy for ALD is currently not yet well-established. Here, we describe the application of dual adeno-associated viral vectors (AAVs) for the delivery of split adenine base editors (ABE) that are then reconstituted by trans-splicing inteins in ALD patient-derived fibroblasts and the in vivo mutational gene correction with dual AAV-PHP.B split-NG-ABE8e(V106W) in novel humanized ALD (hALD) mouse model. We found that our hALD mouse model showed a significant increase of mutated human ABCD1 mRNA, and Docosanoic acid (C22:0), Tetracosanoic acid (C24:0), and Hexacosanoic acid (C26:0) levels were similarly affected. Furthermore, we also demonstrated that adenine base editing enables the therapeutically relevant mutational correction in patient-derived fibroblasts with ~ 2.25% and in vivo mouse brain tissues (~5.5% (DNA) or ~10% (cDNA), spinal cord (~ 5.1%), liver (~4.5%) and adrenal gland (~2%). Finally, using isolated tissues with LC-MS/MS analysis, we showed a significant reduction of plasma levels of C26:0/C22:0, a crucial diagnostic markers for ALD. All these results and the established mouse model with human genomic sequence allow in vivo assessment of clinically relevant gene correction strategies with split-ABEs and serve as a significant step towards treating human ALD disease.