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Observed Incidence of Tumorigenesis in Long-Term Rodent Studies of Raav Vectors

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Observed Incidence of Tumorigenesis in Long-Term Rodent Studies of Raav Vectors 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 Donsante1, C Vogler2, N Muzyczka3, JM Crawford4, J Barker5, T Flotte3, M Campbell-Thompson4, T Daly1,6 and MS Sands1 1Department of Internal Medicine, and 2Department of Pathology, St Louis University School of Medicine, St Louis, MO; 3Powell Gene Therapy Center, 4Department of Pathology, University of Florida, Gainesville, FL; and 5Jackson Laboratory, Bar Harbor, ME, USA Gene therapy using recombinant adeno-associated virus normal mice overexpressing human GUSB for the presence vectors (rAAV) is generally considered safe. During the of tumors and increased hepatocyte replication. The results course of a study designed to determine the long-term effi- of these studies do not indicate that MPSVII mice or mice cacy of rAAV-mediated gene therapy initiated in newborn overexpressing human GUSB are susceptible to tumor for- mice with the lysosomal storage disease, mucopolysacchar- mation; however, the number of animals examined is too idosis type VII (MPSVII), a significant incidence of hepato- small to draw definitive conclusions. Results from quantitat- cellular carcinomas and angiosarcomas was discovered. A ive PCR performed on the tumor samples suggest that the hepatocellular carcinoma was first detected in a 35-week- tumors are probably not caused by an insertional old mouse and by 72 weeks of age, three out of five rAAV- mutagenesis event followed by the clonal expansion of a treated MPSVII mice had similar lesions. These types of transformed cell. In a separate study, a relatively large group tumors had not been seen previously in long-term studies of of mice injected with varying doses and types of rAAV vec- MPSVII mice using recombinant enzyme or bone marrow tors had no evidence of hepatic or vascular tumors. Although transplantation. In an attempt to ascertain whether mouse the mechanism of tumor formation is currently unknown, the strain or GUSB expression confers susceptibility to tumor tumorigenic potential of rAAV vectors must be rigorously formation, we histopathologically examined untreated nor- determined in long-term in vivo studies. Gene Therapy mal mice of the same strain, untreated MPSVII mice, and (2001) 8, 1343–1346. Keywords: adeno-associated virus; MPSVII; gene therapy; lysosomal storage disease; tumorigenesis Recombinant adeno-associated virus (rAAV) vectors are ingly, the average GUSB-specific activities in the liver, generally considered safe. No side-effects have been spleen, kidney, heart, lung, brain, and serum were vir- reported in the many in vivo studies designed to assess tually identical to those measured in other treated the efficacy of rAAV vectors.1 Wild-type AAV (wtAAV) MPSVII animals analyzed at 1 year.6 At necropsy, two of is not associated with any disease and the virus integrates the three animals had obvious circumscribed 1 to 2 cm in with high frequency into a single site on human chromo- diameter tan-brown tumor nodules beneath the capsular some 19. However, the site specificity is lost in rAAV vec- surface of the liver. Following this unexpected discovery, tors and they have been shown to either persist as epi- the remaining two rAAV-treated MPSVII mice and eight somes or to integrate randomly into the host genome.2–5 age-matched normal controls were killed and examined. Therefore, the potential for rAAV to have adverse side- In addition, tissue from animals from the same study that effects due to insertional mutagenesis may exist. had died or been killed earlier in the experiment were In this issue of Gene Therapy, Daly et al6 report on the retrospectively examined for abnormalities. In all, six long-term (1 year) efficacy of intravenous neonatal rAAV treated MPSVII animals with tumors were discovered treatment for murine mucopolysaccharidosis type VII (see Table 1). Five animals had hepatocellular carcinomas (MPSVII). Following submission of the paper, three of (HCC) (Figure 1a–c), one had a metastatic angiosarcoma five rAAV-treated MPSVII mice that were part of a lon- (AS) (Figure 2a–c), and one of the five that had HCC also gevity study were killed at approximately 18 months of had an angiosarcoma. None of the eight age-matched age for histochemical, biochemical, and histopathological controls had gross lesions at 18 months of age. analyses. All of the animals appeared healthy and the Several hypotheses could explain the formation of the three animals killed were chosen at random. Interest- 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 Correspondence: MS Sands, Box 8007, 660 S Euclid Ave, Washington may not live long enough to develop neoplasms. To University School of Medicine, St Louis, MO 63110, USA 6Current address: Department of Pathology, University of Alabama, address the possibility that the tumors might be strain- Birmingham, AL, USA specific or specific to older MPSVII animals that have sur- Received 29 March 2001; accepted 5 July 2001 vived because of treatment, four groups of mice were Recombinant AAV toxicity A Donsante et al 1344 Table 1 Tumor identification in AAV-injected newborn mutants that received non-ablative bone marrow trans- mps/mps mice plantation at birth. We also examined transgenic mice that harbor the same expression cassette encoded by the Animal Noa Age Cause of death Diagnosisb Metastasesc rAAV vector9,10 to evaluate the possibility that over- (weeks) expression of human GUSB is oncogenic. All of the experiments described above were performed in mice on 8374 35 killed HCC NE the same genetic background (C57Bl/6), except for a 6669 52 spontaneous HCC NE 7648 52 spontaneous AS (ut) spl, bm mutation in the minor histocompatibility locus bm1 in the 6654 78 killed HCC colon animals not receiving bone marrow transplantation. Only 7645 78 killed HCC, AS NE the rAAV-treated MPSVII mice exhibited HCC or angio- (br) sarcomas, though one of the bone marrow transplanted 6477 78 killed HCC NE animals that received radiation had a pulmonary adeno- carcinoma. These data are consistent with previous bone a41 treated animals killed between one and 35 weeks of age had marrow transplantation and enzyme replacement studies no visible lesions, but have not been examined microscopically. where the tumor incidence is low.8,11–14 The only example Twelve treated mice either died spontaneously or were killed between 35 and 78 weeks of age and had no gross lesions. of HCC described in these studies occurred in a 494-day- bHCC and AS indicate hepatocellular carcinoma and angiosarcoma, old normal animal receiving irradiation and bone mar- respectively. The primary angiosarcomas were in the uterus (ut) row transplantation.8 None of the treated MPSVII mice and at the base of the brain (br). developed HCC. These results suggest that the tumors cThe metastatic lesions were visible on gross examination and con- found in the current study are unique to animals receiv- firmed histologically. Mice without visible lesions in other organs ing rAAV. However, we have not evaluated the tumori- were not thoroughly examined (NE) for metastases. spl and bm indicate spleen and bone marrow, respectively. genic potential of rAAV in normal mice. Studies to evalu- ate the toxicity of rAAV in nomal animals is currently underway. examined histologically for tumors (Table 2). They It is also possible that MPSVII mice could be at include: (1) the rAAV-treated MPSVII mice; (2) the nor- increased risk for liver tumors if they have an increased mal untreated controls from the efficacy study; (3) hepatocyte replication rate. To determine the fraction of mutants that received bone marrow transplantation at hepatocytes undergoing replication, young adult animals birth following 100 rads of gamma radiation; and (4) (MPSVII mice, normal mice, and transgenic mice overex- Figure 1 Morphology of hepatocellular carcinomas observed in rAAV-treated MPSVII mice. (a) A normal mouse liver has hepatocytes aligned in regular cords and portal spaces (arrowhead). Kupffer cells line the normal sinusoids and there are occasional Ito cells. Hepatocytes are regular in size and have small nuclei with inconspicuous nucleoli. Mitotic figures are not seen in the normal liver (toluidine blue, 1 cm = 31 microns). (b) The interface (arrowhead) between a tumor nodule (bottom) and normal liver (top) is distinct, although there is no tumor capsule. The neoplastic tumor cells in the nodule have marked pleomorphism with variation in cell size and large bizarre nuclei (arrow) in contrast to the regularly sized hepatocytes in the adjacent normal liver (toluidine blue, 1 cm = 31 microns). (c) Although there are areas of preserved liver cell cords, the neoplastic cells in the grossly identified tumor nodules are larger and more pleomorphic than normal hepatocytes. The nuclei are variable in size and have prominent nucleoli. There were occasional multinucleated cells and occasional large bizarre mitotic figures were seen, as in this figure with a tripolar mitosis (arrow) (toluidine blue, 1 cm = 31 microns). Figure 2 Morphology of angiosarcomas and metastases observed in rAAV-treated MPSVII mice. (a) One animal had an angiosarcoma that involved the uterus, spleen and bone marrow. In the spleen, the lesions were distinct from the adjacent spleen, multifocal and had large blood-filled vascular spaces along with more solid areas (hematoxylin and eosin, 1 cm = 322 microns). (b) The bone marrow from the same animal had foci of similar vascular neoplasia (hematoxylin and eosin, 1 cm = 322 microns). (c) The more solid areas consisted of variably sized vessels with erythrocytes admixed with small, closely packed endothelial cells (hematoxylin and eosin, 1 cm = 50 microns). Gene Therapy Recombinant AAV toxicity A Donsante et al 1345 Table 2 Clinical findings in aged treated and untreated mice Mouse genotypea Treatment No.
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