Current strategies for hepatic gene therapy are either quantitatively inefficient or suffer from lack of permanent gene expression. We have utilized an animal model of hereditary tyrosinaemia type I (HT1), a recessive liver disease caused by deficiency of fumarylacetoacetate hydrolase (FAH), to determine whether in vivo selection of corrected hepatocytes could improve the efficiency of liver gene transfer. As few as 1,000 transplanted wild-type hepatocytes were able to repopulate mutant liver, demonstrating their strong competitive growth advantage. Mutant hepatocytes corrected in situ by retroviral gene transfer were also positively selected. In mutant animals treated by multiple retrovirus injections >90% of hepatocytes became FAH positive and liver function was restored to normal. Our results demonstrate that in vivo selection is a useful strategy for hepatic gene therapy and may lead to effective treatment of human HT1 by retroviral gene transfer.

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Liver regeneration after partial hepatectomy and most chemical injuries occurs by the division of fully differentiated hepatocytes and does not require stem or progenitor cells.

We have cloned and characterized the expression of a Kv1.5 K+ channel (cKv1.5) from canine colonic smooth muscle. The amino acid sequence displayed a high level of identity to other K+ channels of the Kv1.5 class in the core region between transmembrane segments S1-S6; however, identity decreased to between 74 and 82% in the NH2 and COOH terminal segments, suggesting that cKv1.5 is a distinct isoform of the Kv1.5 class. Functional expression of cKv1.5 in oocytes demonstrated a channel highly selective for K+, which activates in a voltage-dependent manner on depolarization to membrane potentials positive to -40 mV. At room temperature the channel showed fast activation (time to half of peak current, 5.5 ms) and slow inactivation that was incomplete after 20-s depolarizations. Single channel analysis of the channel expressed in oocytes displayed a linear current-voltage curve and had a slope conductance of 9.8 +/- 1.1 pS. Northern blot analysis demonstrated differential expression of cKv1.5 in smooth muscles of the gastrointestinal tract and abundant expression in several vascular smooth muscles. We propose that cKv1.5 represents a component of the delayed rectifier current in both vascular and visceral smooth muscles.

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