The liver represents an excellent organ for gene therapy since many genetic disorders result from the deficiency of liver-specific gene products. We have previously demonstrated that transgenic mouse hepatocytes can be heterologously transplanted into congenic recipients where they survived indefinitely and continued to function as hepatocytes.Here we demonstrate the autologous transplantation of retrovirally transduced canine hepatocytes. At least 1 x 109 hepatocytes or 5% of the liver mass can be transplanted by the portal vasculature. In two animals we have transplanted hepatocytes transduced with a retroviral vector containing the human a,-antitrypsin cDNA under transcriptional control of the cytomegalovirus promoter. Both animals had sificant human a1-antitrypsin in the serum for 1 month. Although the serum levels of human a1-antitrypsin eventually fell due to inactivation of the cytomegalovirus promoter, PCR analysis demonstrated that a significant fraction of transduced hepatocytes migrated to the liver and continued to survive in vivo.The results suggest that gene therapy of hepatic deficiencies may be achieved by hepatocellular transplantation after genetic reconstitution with the use of promoters of cellular genes that are active in the normal liver.Many laboratories have developed methods for the transduction of genes into cultured primary hepatocytes (1-5). To use this method for gene therapy, one must be able to introduce the genetically altered cells back into the animal, preferably to the liver. Using transgenic mouse hepatocytes as a model, we (6) and others (7) have recently demonstrated that freshly isolated mouse hepatocytes can be transplanted into the liver of congenic recipients by direct splenic or portal vein injection. The grafted cells migrated to the liver and functioned indefinitely as hepatocytes after transplantation.The next critical step is to determine whether hepatocytes that undergo in vitro manipulation and retroviral transduction can be transplanted back into the animal and function in a manner similar to the transgenic mouse models. Wilson et al. (8) transplanted heterologous hepatocytes after retroviral transduction with the human low density lipoprotein receptor into low density lipoprotein-deficient rabbits but found limited short-term expression. We have, however, selected the canine model to develop methodologies for autologous transplantation of virally transduced hepatocytes because (i) the size of a young dog parallels that of a human infant and (ii) there are canine hepatic-deficiency disorders that can be used as models for gene therapy. We selected the human a1-antitrypsin (hAAT) cDNA as a marker gene since it encodes a secretable protein that can be easily detected in the serum of recipients with an RIA that utilizes a species-specific hAAT antibody. We show here that at least 1 x 109 hepatocytes can be transplanted into the animals and, after retroviral gene transduction, autologous hepatocytes transplanted by the portal circulation remain in the liver...

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