Crigler-Najjar type I (CNI) syndrome is a recessively inherited disorder characterized by severe unconjugated hyperbilirubinemia caused by uridine diphosphoglucuronosyltransferase 1A1 (UGT1A1) deficiency. The disease is lethal due to bilirubin-induced neurological damage unless phototherapy is applied from birth. However, treatment becomes less effective during growth, and liver transplantation is required. To investigate the pathophysiology of the disease and therapeutic approaches in mice, we generated a mouse model by introducing a premature stop codon in the UGT1a1 gene, which results in an inactive enzyme. Homozygous mutant mice developed severe jaundice soon after birth and died within 11 d, showing significant cerebellar alterations. To rescue neonatal lethality, newborns were injected with a single dose of adeno-associated viral vector 9 (AAV9) expressing the human UGT1A1. Gene therapy treatment completely rescued all AAV-treated mutant mice, accompanied by lower plasma bilirubin levels and normal brain histology and motor coordination. Our mouse model of CNI reproduces genetic and phenotypic features of the human disease. We have shown, for the first time, the full recovery of the lethal effects of neonatal hyperbilirubinemia. We believe that, besides gene-addition-based therapies, our mice could represent a very useful model to develop and test novel technologies based on gene correction by homologous recombination.—Bortolussi, G., Zentilin, L., Baj, G., Giraudi, P., Bellarosa, C., Giacca, M., Tiribelli, C., Muro, A. F. Rescue of bilirubin-induced neonatal lethality in a mouse model of Crigler-Najjar syndrome type I by AAV9-mediated gene transfer.

The feline leukemia virus subgroup C receptor (FLVCR) is a heme export protein that is required for proerythroblast survival and facilitates macrophage heme iron recycling. However, its mechanism of heme export and substrate specificity are uncharacterized. Using [55 Fe]heme and the fluorescent heme analog zinc mesoporphyrin, we investigated whether export by FLVCR depends on the availability and avidity of extracellular heme-binding proteins. Export was 100-fold more efficient when the medium contained hemopexin (K d < 1 pM) compared with albumin (K d ‫؍‬ 5 nM) at the same concentration and was not detectable when the medium lacked heme-binding proteins. Besides heme, FLVCR could export other cyclic planar porphyrins, such as protoporphyrin IX and coproporphyrin. However, FLVCR has a narrow substrate range because unconjugated bilirubin, the primary breakdown product of heme, was not transported. As neither protoporphyrin IX nor coproporphyrin export improved with extracellular hemopexin (versus albumin), our observations further suggest that hemopexin, an abundant protein with a serum concentration (6.7-25 M) equivalent to that of the iron transport protein transferrin (22-31 M), by accepting heme from FLVCR and targeting it to the liver, might regulate macrophage heme export and heme iron recycling in vivo. Final studies show that hemopexin directly interacts with FLVCR, which also helps explain why FLVCR, in contrast to some major facilitator superfamily members, does not function as a bidirectional gradient-dependent transporter. Together, these data argue that hemopexin has a role in assuring systemic iron balance during homeostasis in addition to its established role as a scavenger during internal bleeding or hemolysis.The biological roles of heme are diverse and encompass most areas of cell metabolism and gene regulation. Heme serves as a prosthetic group in the hemeproteins, which are involved in crucial biological functions, including oxygen binding (hemoglobin and myoglobin), oxygen metabolism (oxidases, peroxidases, catalases, and hydroxylases), electron transfer (cytochromes) (4), and signal transduction (nitric-oxide synthases) (5). In addition, heme serves as a signaling molecule in erythropoiesis and other physiological systems. Because heme generally signals by binding and inactivating a transcriptional repressor (e.g. Bach1) or translational inhibitor (e.g. heme-regulated inhibitor and thus eIF␣2 kinase), its impact is immediate (6 -8). Perhaps for this reason, heme is utilized in time-sensitive processes, such as the regulation of circadian rhythm (9), N-end rule pathway/protein ubiquitination (10), and globin synthesis. Heme is also required for microRNA processing (11). However, excess free or non-protein-bound heme damages lipid, protein, and DNA through the generation of reactive oxygen species, resulting in cellular injury and death (12). Therefore, free cellular heme levels must be tightly regulated to provide an adequate supply yet avoid heme toxicities (4,(13)(14)(15).Most studies of h...

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