Cozmescu, Andrei Claudiu; (2023) Liver directed lentiviral gene therapy for ARC syndrome. Doctoral thesis (Ph.D), UCL (University College London).

Text CC_Liver directed lentiviral gene therapy for ARC syndrome_For Submission_June 2023.pdf - Other Access restricted to UCL open access staff until 1 July 2024. Download (5MB)

Abstract

Arthrogryposis-renal dysfunction-cholestasis (ARC) syndrome is an inherited disorder typically arising from mutations in the vacuolar protein sorting 33 homolog B (VPS33B) gene. This incurable autosomal recessive disease is characterised by congenital joint contractures, renal tubular acidosis, and neonatal cholestatic jaundice. The liver cholestasis is caused by the hepatocytes’ inability to secrete several bile components, leading to liver fibrosis, cirrhosis, and terminal-stage liver disease. Following breakthrough ex vivo treatments of numerous inherited monogenic disorders, studies on animal models suggest that lentiviral vectors might be safe for in vivo use. Thus, this project aimed to develop and evaluate the efficacy and safety of lentiviral gene therapy for the liver phenotype of ARC syndrome. For this, a liver-specific Lenti.LP1.cohVPS33B vector and a constitutive Lenti.EF1α.cohVPS33B vector have been produced. In vitro tests revealed that both vectors restored VPS33B expression and function in a liver cell model for ARC syndrome, but none of the vectors transduced sufficient hepatocytes to achieve phenotypic improvement in the in vivo studies in liver-specific Vps33bfl/fl-Alfp-Cre knock-out mice. In vivo studies in 9-month-old Vps33bfl/+-Alfp-Cre mice treated with the two lentiviral vectors have raised concerns over the safety of the Lenti.EF1α.cohVPS33B treatment. Injections with clodronate-encapsulated liposomes temporarily deplete the liver-resident Kupffer cells, leading to improved lentiviral vector hepatocyte transduction. Thus, the potential therapeutic benefits of clodronate pre-treatment prior to Lenti.LP1.cohVPS33B administration was further investigated in neonatal Vps33bfl/fl-Alfp-Cre mice. To accelerate phenotype development these mice were fed a 0.5% cholic acid diet at 12 weeks and were sacrificed for analysis a week thereafter. Compared to mice treated solely with the Lenti.LP1.cohVPS33B vector, the clodronate-primed mice displayed reduced weight loss and improved blood parameters on the cholic acid diet. These results hold promise for ARC patients and underscore the significance of optimal hepatocyte transduction for lentiviral gene therapy.

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