Somatic gene therapy has been proposed as a means to achieve systemic delivery of therapeutic proteins. However, there is limited evidence that current methods of gene delivery can practically achieve this goal. In this study, we demonstrate that, following a single intramuscular administration of a recombinant adeno-associated virus (rAAV) vector containing the ␤-galactosidase (AAV-lacZ) gene into adult BALB͞c mice, protein expression was detected in myofibers for at least 32 weeks. A single intramuscular administration of an AAV vector containing a gene for human erythropoietin (AAV-Epo) into mice resulted in dosedependent secretion of erythropoietin and corresponding increases in red blood cell production that persisted for up to 40 weeks. Primary human myotubes transduced in vitro with the AAV-Epo vector also showed dose-dependent production of Epo. These results demonstrate that rAAV vectors are able to transduce skeletal muscle and are capable of achieving sustained expression and systemic delivery of a therapeutic protein following a single intramuscular administration. Gene therapy using AAV vectors may provide a practical strategy for the treatment of inherited and acquired protein deficiencies.

CCAAT/enhancer-binding protein (C/EBP) isoforms are thought to be important regulators of the hepatocyte phenotype. However, the specific physiological roles of different isoforms are poorly understood because hepatocytes express multiple C/EBPs, and various isoforms have overlapping functions. To identify the functions of C/EBP␣ in mature hepatocytes, replicationdefective adenovirus vectors were used to efficiently and homogeneously overexpress the mouse C/EBP␣ gene in a SV40 virus-conditionally transformed rat hepatocyte line that can be induced to express C/EBP␤ and C/EBP␦ but that has little endogenous C/EBP␣ expression. Hepatocytes were infected with a recombinant adenovirus vector carrying the cDNA for C/EBP␣ driven by Rous sarcoma virus promoter elements (AdCEBP␣) or a similar vector carrying the Escherichia coli lacZ gene (Ad␤gal). Staining for ␤-galactosidase demonstrated an infection efficiency of 100% at a multiplicity of infection of 25 plaque-forming units/cell and persistence of foreign gene expression for at least 9 days. Cultures infected with AdCEBP␣ had 50-fold higher levels of C/EBP␣ mRNA and protein than those infected with Ad␤gal, but similar expression of C/EBP␤. Infection with AdCEBP␣ inhibited proliferation in cells expressing little C/EBP␤, even when proliferation was driven by the SV40 transforming antigen, and also blunted mitogenic induction of the c-myc proto-oncogene in nontransformed cells with high levels of C/EBP␤. Although overexpression of C/EBP␣ consistently increased C/EBP␣ DNA binding activity, it was not sufficient for albumin expression. Infection with AdCEBP␣ only increased albumin mRNA levels in nontransformed cells that also expressed relatively high levels of C/EBP␤. Thus, in hepatocytes, C/EBP␣ has a dominant antiproliferative function, but must interact with other factors to regulate hepatocyte-specific gene expression.

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