1. MURINE HISTIDINEMIA: AMELIORATION USING RETROVIRAL GENE TRANSFER

Introduction: Histidinaemia is an autosomal recessive disorder in man affecting the hepatic enzyme histidase resulting in elevated plasma and urinary histidine. A mouse model with a similar phenotype lends itself to investigating methods of gene therapy.

Aim: To revert the biochemical phenotype in by in vivo retroviral gene transfer.

Methods: A cDNA encoding normal histidase was cloned into the LNCX retroviral construct under the control of the CMV promoter. Virus was concentrated and administered to histidinaemic mice primed for hepatocyte transfection by partial hepatectomy. HPLC flow scintillation was used to assess in vivo catalysis of¹⁴C-histidine. Hepatic cytosol was examined for histidase expression by Western analysis and enzyme activity by conversion of histidine to urocanic acid.

Results: Infected animals showed a 33%±15% enhancement of metabolism as shown by in vivo histidine breakdown at 7 days (p<0.05 c.f. controls). Phenotype amelioration continued until 14 days but returned to pre-operative levels at 21 days post infection. Histidase activity from liver tissue from transduced animals ranged from 46.1±13.3 to 81.8±22 pmols of urocanic acid formation/min/mg total protein compared with 8.8±3.7 in histidinaemic controls and 1315±47.4 observed in wild type controls.

Conclusion: Murine histidinaemia provides a model for testing gene therapy for hepatocyte enzyme defects. Therapeutic retroviral gene transfer was sufficient to ameliorate the enzyme deficiency in this model and restored enzyme function in excess of 14 days. Immune reaction to expressed normal protein in this model is unlikely from previous hepatocellular transplantation studies and down regulation of the therapeutic gene by CMV promoter silencing is a likely explanation of early loss of enzyme function.

2. THE PROGNOSTIC IMPLICATIONS OF HYPERAMMONEMIA IN ACUTE LIVER FAILURE

Background: Extensive animal research has suggested that ammonia (NH₄) has a pivotal role in …