Liver-targeted therapies in Zellweger spectrum disorders

Our clinical center has pioneered the organ and cell transplantation approaches to treat Zellweger spectrum disorders (ZSD). The present work aimed to develop innovative tools to evaluate peroxisome biogenesis (PB) in vitro and in a pre-clinical model, in order to assess the potential benefit of hepatocyte transplantation in ZSD. ZSD are inborn errors of metabolism (IEM) caused by genetic alterations in PEX genes leading to peroxisome biogenesis disorder (PBD). The phenotype is broad and extends between neonatal death by liver failure, in severe ZSD, to developmental delay and/or deafness and/or blindness, in milder forms of the disease (Chapter 1). Diagnosis is supported by peroxisomal marker quantification in blood and urine, and is further confirmed by sequencing the 13 PEX genes associated with ZSD. Supportive care is standard since no curative treatment have been validated in clinical practice. Drug screening on ZSD patient skin fibroblasts uncovered several compounds able to restore PB in vitro. Some of these molecules are currently under clinical trials (NCT01838941, NCT03856866). In addition, we and other reported favorable bio-clinical short-term outcomes after hepatocyte transplantation (HT) or liver transplantation (LT) in mild ZSD patients. Methods to evaluate PBD in vitro are time-consuming and exhibit low sensitivity on cells from mild ZSD patients. In Chapter 2, we developed a stable expression system for an enhanced green fluorescent protein fused with a peroxisome targeting signal 1 (eGFP-PTS1). eGFP-PTS1 is a fluorescent peroxisomal reporter used to evaluate PB. The transduction efficiency ranged from 45% to 85% and eGFP-PTS1 expression was maintained more than 8 passages. The eGFP-PTS1 signal was then digitally analyzed and validated to evaluate PB in human skin fibroblasts and mouse embryonic fibroblasts isolated from our optimized mild ZSD mouse model (see Chapter 3). Live cell imaging of transduced cells allowed peroxisome motility measurement. In the future, this tool could be used to screen for molecules able to restore PB in vitro. Available ZSD mouse models are fragile and difficult to breed. It makes them unsuitable to evaluate rigorously HT as a therapeutic approach in ZSD. Pex1 p.G844D (equivalent to the human PEX1 p.G843D mutation associated with a mild ZSD phenotype) is an hypomorphic allele which has been developped in a C57BL/6 mouse background to create a mild ZSD mouse model. We backcrossed the murine allele in an NMRI mouse strain, known for delivering large litters and for its robustness. In Chapter 3, we report the longitudinal evaluation of this new mild ZSD mouse model. Hepatomegaly and liver glycogen metabolism impairment are further deciphered. Our mouse model exhibited classical peroxisomal marker alterations including the urine organic acid profile. We used this model to evaluate HT impact on disease markers (see Chapter 4). LT is indicated in the management of several IEM. Yet, organ shortage limits LT availability, especially for IEM patients who do usually not exhibit classical LT indication criteria (e.g. decompensated cirrhosis). HT has been developed to overcome some of the LT limitations. Our clinical transplant program initially treated one mils ZSD patient using HT and showed positive biochemical outcomes, 18 months post-HT, highlighted by the marked reduction of some peroxisomal metabolites (i.e., the C27 bile acids intermediates and the very long-chain fatty acids). To confirm the benefit of HT in mild ZSD, we evaluated two doses of syngeneic HT in our ZSD mouse model. In Chapter 4, we relate that HT was feasible and safe in our mouse model. Unfortunately, none of the HT protocols could achieve persistent liver engraftment more than 24 h. In accordance with that, no impact on peroxisomal markers could be detected. We and a Japanese pediatric liver transplant team reported biochemical and clinical improvement up to 2 years after living-donor LT in two mild ZSD patients. Long term clinical outcomes post-LT for ZSD are lacking. In Chapter 5, the biochemical and clinical results 17 years post-LT for the first mild ZSD patient are described along with two others mild ZSD patients who benefited from living-donor LT. In Chapter 6, a general discussion and perspectives are presented along with a comparison between LT and a potential gene therapy for ZSD. In Chapter 7, publications from international collaborations are reported.(MED - Sciences médicales) -- UCL, 202