Gene Therapy (2001) 8, 1343–1346  2001 Nature Publishing Group All rights reserved 0969-7128/01 $15.00 www.nature.com/gt BRIEF COMMUNICATION Observed incidence of tumorigenesis in long-term rodent studies of rAAV vectors A Donsante 1 , C Vogler 2 , N Muzyczka 3 , JM Crawford 4 , J Barker 5 , T Flotte 3 , M Campbell-Thompson 4 , T Daly 1,6 and MS Sands 1 1 Department of Internal Medicine, and 2 Department of Pathology, St Louis University School of Medicine, St Louis, MO; 3 Powell Gene Therapy Center, 4 Department of Pathology, University of Florida, Gainesville, FL; and 5 Jackson Laboratory, Bar Harbor, ME, USA Gene therapy using recombinant adeno-associated virus vectors (rAAV) is generally considered safe. During the course of a study designed to determine the long-term effi- cacy of rAAV-mediated gene therapy initiated in newborn mice with the lysosomal storage disease, mucopolysacchar- idosis type VII (MPSVII), a significant incidence of hepato- cellular carcinomas and angiosarcomas was discovered. A hepatocellular carcinoma was first detected in a 35-week- old mouse and by 72 weeks of age, three out of five rAAV- treated MPSVII mice had similar lesions. These types of tumors had not been seen previously in long-term studies of MPSVII mice using recombinant enzyme or bone marrow transplantation. In an attempt to ascertain whether mouse strain or GUSB expression confers susceptibility to tumor formation, we histopathologically examined untreated nor- mal mice of the same strain, untreated MPSVII mice, and Keywords: adeno-associated virus; MPSVII; gene therapy; lysosomal storage disease; tumorigenesis Recombinant adeno-associated virus (rAAV) vectors are generally considered safe. No side-effects have been reported in the many in vivo studies designed to assess the efficacy of rAAV vectors. 1 Wild-type AAV (wtAAV) is not associated with any disease and the virus integrates with high frequency into a single site on human chromo- some 19. However, the site specificity is lost in rAAV vec- tors and they have been shown to either persist as epi- somes or to integrate randomly into the host genome. 2–5 Therefore, the potential for rAAV to have adverse side- effects due to insertional mutagenesis may exist. In this issue of Gene Therapy, Daly et al 6 report on the long-term (1 year) efficacy of intravenous neonatal rAAV treatment for murine mucopolysaccharidosis type VII (MPSVII). Following submission of the paper, three of five rAAV-treated MPSVII mice that were part of a lon- gevity study were killed at approximately 18 months of age for histochemical, biochemical, and histopathological analyses. All of the animals appeared healthy and the three animals killed were chosen at random. Interest- Correspondence: MS Sands, Box 8007, 660 S Euclid Ave, Washington University School of Medicine, St Louis, MO 63110, USA 6 Current address: Department of Pathology, University of Alabama, Birmingham, AL, USA Received 29 March 2001; accepted 5 July 2001 normal mice overexpressing human GUSB for the presence of tumors and increased hepatocyte replication. The results of these studies do not indicate that MPSVII mice or mice overexpressing human GUSB are susceptible to tumor for- mation; however, the number of animals examined is too small to draw definitive conclusions. Results from quantitat- ive PCR performed on the tumor samples suggest that the tumors are probably not caused by an insertional mutagenesis event followed by the clonal expansion of a transformed cell. In a separate study, a relatively large group of mice injected with varying doses and types of rAAV vec- tors had no evidence of hepatic or vascular tumors. Although the mechanism of tumor formation is currently unknown, the tumorigenic potential of rAAV vectors must be rigorously determined in long-term in vivo studies. Gene Therapy (2001) 8, 1343–1346. ingly, the average GUSB-specific activities in the liver, spleen, kidney, heart, lung, brain, and serum were vir- tually identical to those measured in other treated MPSVII animals analyzed at 1 year. 6 At necropsy, two of the three animals had obvious circumscribed 1 to 2 cm in diameter tan-brown tumor nodules beneath the capsular surface of the liver. Following this unexpected discovery, the remaining two rAAV-treated MPSVII mice and eight age-matched normal controls were killed and examined. In addition, tissue from animals from the same study that had died or been killed earlier in the experiment were retrospectively examined for abnormalities. In all, six treated MPSVII animals with tumors were discovered (see Table 1). Five animals had hepatocellular carcinomas (HCC) (Figure 1a–c), one had a metastatic angiosarcoma (AS) (Figure 2a–c), and one of the five that had HCC also had an angiosarcoma. None of the eight age-matched controls had gross lesions at 18 months of age. Several hypotheses could explain the formation of the tumors. The first hypothesis is that MPSVII mice are pre- disposed to tumors. However, since untreated MPSVII mice have a reduced lifespan (6 to 10 months), 6–8 they may not live long enough to develop neoplasms. To address the possibility that the tumors might be strain- specific or specific to older MPSVII animals that have sur- vived because of treatment, four groups of mice were