Gene Therapy (2002) 9, 127–134  2002 Nature Publishing Group All rights reserved 0969-7128/02 $25.00 www.nature.com/gt RESEARCH ARTICLE Cirrhotic rat livers with extensive fibrosis can be safely transduced with clinical-grade adenoviral vectors. Evidence of cirrhosis reversion J Garcia-Ban ˜ uelos 1 , F Siller-Lopez 1 , A Miranda, LK Aguilar 2 , E Aguilar-Cordova 3 and J Armendariz-Borunda 1 1 Institute of Molecular Biology in Medicine and Gene Therapy, CUCS, University of Guadalajara, Guadalajara, Mexico; 2 Advantagene, Inc., San Diego, CA, USA; and 3 Harvard Gene Therapy Initiative, Boston, MA, USA Adenoviral vectors efficiently target normal liver cells; how- ever, a clear-cut description of the safety boundaries for using adenovectors in hepatic cirrhosis has not been settled. With this in mind, we used a first-generation, replication- deficient adenoviral vector carrying the E. coli lacZ gene (Ad5βGal) to monitor therapeutic range, biodistribution, tox- icity and transduction efficiency in Wistar rats made cirrhotic by two different experimental approaches resembling alcoholic cirrhosis and biliary cirrhosis in humans. Further, we show proof of concept on fibrosis reversion by a ‘thera- peutic’ Ad-vector (AdMMP8) carrying a gene coding for a collagen-degrading enzyme. Dose–response experiments with Ad5βGal ranging from 1 × 10 8 –3 × 10 12 viral particles (vp) per rat (250 g), demonstrated that adenovirus-mediated gene transfer via iliac vein at 3 × 10 11 vp/rat, resulted in an approximately 40% transduction in livers of rats made cir- rhotic by chronic intoxication with carbon tetrachloride, com- Keywords: cirrhosis; gene transfer; safety; fibrosis reversion Introduction Liver cirrhosis is a worldwide health problem. Cirrhosis is a major liver disease for which there are no completely satisfactory therapies. Cirrhotic livers are characterized by extensive fibrosis throughout the entire hepatic paren- chyma, especially around central and portal veins. The deposition of excessive fibrous or collagenous proteins in the subendothelial space or Space of Disse results in decreased free exchange flow between hepatocytes and sinusoidal blood. The cellular effects of these collagenous materials and other non-collagenous components, especially on hepatocytes, cause synthetic and metabolic dysfunction characteristic of advanced liver disease. 1,2 Removing the fibrous septa might result in benefit for subjects undergoing liver fibrosis due to the functional re-establishment of the hepatocyte–sinusoid flow exchange. Thus, delivery of genes coding for collagen- Correspondence: J Armendariz-Borunda, Institute of Molecular Biology in Medicine and Gene Therapy, CUCS, University of Guadalajara, Apdo Postal 2-123, Guadalajara, Jal, Mexico 44281 Received 1 May 2001; accepted 20 November 2001 pared with approximately 80% in control non-cirrhotic livers. In rats made cirrhotic by bile-duct obstruction only, 10% efficiency of transduction was observed. Biodistribution analyses showed that vector expression was detected prim- arily in liver and at a low level in spleen and kidney. Although there was an important increase in liver enzymes between the first 48 h after adenovirus injection in cirrhotic animals compared to non-transduced cirrhotic rats, this hepatic dam- age was resolved after 72–96 h. Then, the cDNA for neutro- phil collagenase, also known as Matrix Metalloproteinase 8 (MMP8), was cloned in an Ad-vector and delivered to cir- rhotic rat livers being able to reverse fibrosis in 44%. This study demonstrates the potential use of adenoviral vectors in safe transient gene therapy strategies for human liver cirrhosis. Gene Therapy (2002) 9, 127–134. DOI: 10.1038/sj/gt/3301647 degrading enzymes may represent a novel therapy strategy for liver cirrhosis. Various vector types have been shown to be efficient at delivering genes to normal livers. 3–7 Similarly, the use of viral and nonviral vectors for gene delivery to func- tionally compromised livers has been instrumental to establish ‘proof of concept’ in several experimental mod- els. Various strategies involving adenoviral vectors have been used. 8,9 However, a key issue concerning toxicology, biodistribution and safety when using adenoviral vectors in cirrhotic animals, remains unresolved. Part of this study was designed to evaluate biodistribution, liver tox- icity and efficiency of transduction of a reporter gene in hepatic cells of cirrhotic Wistar rats. We also wanted to delimit the ‘therapeutic window’ for potential and safe use of these vectors in a given clinical setting. Adenoviral vectors were chosen because they have been shown to be efficient vehicles for delivering genes to the liver and their distribution has been extensively analyzed in heal- thy animals. However, the potential toxicity of these vec- tors, especially to the liver, was a major concern. Animals with decreased liver function may have increased sensi- tivity to hepatotoxic effects of adenoviral vector adminis- tration. Therefore, we analyzed liver enzyme profiles of