In vivo adenine base editing rescues adrenoleukodystrophy in a humanized mouse model

Abstract

X-linked adrenoleukodystrophy (ALD), an inherited neurometabolic disorder caused by mutations in ABCD1 , which encodes the peroxisomal ABC transporter, mainly affects the brain, spinal cord, adrenal glands, and testes. In ALD patients, verylong-chain fatty acids (VLCFAs) fail to enter the peroxisome and undergo subsequent b-oxidation, resulting in their accumulation in the body. It has not been tested whether in vivo base editing or prime editing can be harnessed to ameliorate ALD. We developed a humanized mouse model of ALD by inserting a human cDNA containing the pathogenic variant into the mouse Abcd1 locus. The humanized ALD model showed increased levels of VLCFAs. To correct the mutation, we tested both base editing and prime editing and found that base editing using ABE8e(V106W) could correct the mutation in patient-derived fi broblasts at an ef fi ciency of 7.4%. Adenoassociated virus (AAV)-mediated systemic delivery of NGABE8e(V106W) enabled robust correction of the pathogenic variant in the mouse brain (correction ef fi ciency: ,,, 5.5%), spinal cord ( ,,, 5.1%), and adrenal gland ( ,,, 2%), leading to a significant reduction in the plasma levels of C26:0/C22:0. This established humanized mouse model and the successful correction of the pathogenic variant using a base editor serve as a signi fi- cant step toward treating human ALD disease. Pathogenic mutations in ABCD1 result in the accumulation of C24:0 and C26:0 VLCFAs in several tissues. 2 - 4 Accumulated VLCFAs can cause pathologic changes, such as cerebral in fl ammation or demyelination in the central nervous system (CNS). 5 Based on symptom presentation, ALD can be classi fi ed into six subtypes: childhood cerebral, adolescent cerebral, adult cerebral, adrenomyeloneuropathy, Addison-only, and asymptomatic. 4 The diagnosis of ALD requires the identi fi cation of ABCD1 mutations along with elevation of plasma VLCFAs (mainly C26:0/C22:0 and C26:0). 6,7 The absence of a genotype-phenotype correlation and the possible existence of secondary modi fi er genes or environmental factors make it challenging to manage ALD. 8,9 Most therapeutic approaches have focused on the control of VLCFA accumulation via Lorenzo ' s oil, lovastatin, hematopoietic stem cell transplantation (HPSCT), or conventional gene therapy. 10 - 14 However, Lorenzo ' s oil and lovastatin do not provide permanent treatment. 12,13 HPSCT requires bone marrow suppression in patients, which makes them vulnerable to infection and several toxicities. 15 - 17 Lastly, gene therapy does not fundamentally correct the pathogenic mutation, and if a lentivirus is used for such therapy, viral genomes can be randomly integrated into the genome, potentially causing side effects such as tumorigenesis. 18 Thus, the precise correction of the causal mutation via an appropriate vector is required.