Vector-related Tumorigenesis Not Found in Ornithine Transcarbamylase-deficient Mice
Authors: Dwight D. Koeberl
Journal: Molecular Therapy (2006)

The possibility that AAV vector-mediated gene therapy is linked to the development of hepatic tumors in ornithine transcarbamylase (OTC)-deficient mice is an evolving story that has held the attention of researchers at the junction of the fields of biochemical genetics and gene therapy. Moscioni and colleagues previously presented an abstract reporting effective AAV-mediated gene therapy in two strains (spf and spf-ash) of OTC-deficient mice [1Morizono H. et al.Enhanced rescue of OTC deficiency using novel adeno-associated virus vectors.Mol. Genet. Metab. 2005; 84: 230-231Google Scholar]. The discovery of hepatic tumors in some treated mice at the end of this long-term study was an important caveat however. In this issue of Molecular Therapy, two new articles from this group describe the results of efficient AAV-mediated gene therapy in these OTC-deficient mice and provide further details of the incidence of hepatic tumors in these strains of mice [2Bell P. et al.Analysis of Tumors Arising in Male B6C3F1 Mice with and without AAV Vector Delivery to Liver.Mol. Ther. 2006; 14: 34-45Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 3Moscioni D. et al.Long-Term Correction of Ammonia Metabolism and Prolonged Survival in Ornithine Transcarbamylase-Deficient Mice Following Liver-Directed Treatment with Novel Adeno-associated Viral Vectors.Mol. Ther. 2006; 14: 25-33Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar]. Improved therapeutic options are clearly needed for the X-linked human metabolic disorders known as OTC deficiency. OTC deficiency can lead to early lethality due to neonatal-onset hyperammonemia in severely affected males despite the availability of dietary and drug therapy. A subset of females carrying an inactivating mutation of the OTC gene also exhibit symptoms because of skewed inactivation of the X chromosome. However, females can also present with life-threatening hyperammonemia. Mutation of the OTC gene in humans has not been linked to an increased risk of hepatic tumors in surviving patients, such as mildly affected males, or in female carriers. Although liver transplantation can be effective, many affected males die prior to transplantation, leading some metabolic specialists to forego treatment of severely affected patients [4Saudubray J.M. et al.Liver transplantation in urea cycle disorders.Eur. J. Pediatr. 1999; 158: S55-S59Crossref PubMed Google Scholar]. The previously reported evaluation of adenovirus vector-mediated gene therapy for OTC was complicated by transient transgene expression and marked toxicity [5Raper S.E. et al.Developing adenoviral-mediated in vivo gene therapy for ornithine transcarbamylase deficiency.J. Inherit. Metab. Dis. 1998; 21: 119-137Crossref PubMed Scopus (32) Google Scholar, 6Raper S.E. et al.Fatal systemic inflammatory response syndrome in a ornithine transcarbamylase deficient patient following adenoviral gene transfer.Mol. Genet. Metab. 2003; 80: 148-158Abstract Full Text Full Text PDF PubMed Scopus (1132) Google Scholar]. The isolation of additional AAV serotypes from primates has vastly improved the prospects for liver-targeted therapy in disorders of metabolism such as OTC deficiency [7Gao G.P. et al.Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy.Proc. Natl. Acad. Sci. USA. 2002; 99: 11854-11859Crossref PubMed Scopus (1193) Google Scholar]. In the first of the companion studies reported in this issue, Moscioni and colleagues evaluated delivery of the mouse OTC gene by AAV vectors pseudotyped with AAV2, AAV7, AAV8, or AAV9 capsids in adult OTC-deficient mice of the spf and spf-ash strains [3Moscioni D. et al.Long-Term Correction of Ammonia Metabolism and Prolonged Survival in Ornithine Transcarbamylase-Deficient Mice Following Liver-Directed Treatment with Novel Adeno-associated Viral Vectors.Mol. Ther. 2006; 14: 25-33Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar]. Vectors pseudotyped with AAV7, 8 or 9 capsids were effective, as evidenced by normalization of a disease-related biomarker, urinary orotate, fifteen days after administration of the therapy. Furthermore, functional testing consisting of a hyperammonemic challenge confirmed the benefit of vector treatment from 21 to over 250 days post-administration. Interestingly, prior to 21 days, the pseudotyped AAV vectors did not protect against symptoms of hyperammonemia, underlining the potential importance of early stabilization of disease progression in patients prior to the onset of therapeutic OTC expression during a future clinical trial. Detectable transgene was expressed in the mice by the vector for up to one year. However, the development of hepatic tumors in mice monitored for longer periods of time raised the spectre that AAV vectors could be tumorigenic, as had been suggested by a previous long-term study involving gene therapy in a mouse model of Sly disease, a lysosomal storage disorder [8Donsante A. et al.Observed incidence of tumorigenesis in long-term rodent studies of rAAV vectors.Gene Ther. 2001; 8: 1343-1346Crossref PubMed Scopus (212) Google Scholar]. In addition, while the new paper reports compelling data regarding the efficacy of pseudotyped AAV vectors in OTC deficiency, important questions remain regarding the feasibility of AAV vector-mediated gene therapy in this disorder, given the evidence for the need for early treatment of this disorder and the potential for reduced efficacy of AAV vectors in very young animals [9Ghosh A. et al.Long-term correction of murine glycogen storage disease type Ia by recombinant adeno-associated virus-1-mediated gene transfer.Gene Ther. 2006; 13: 321-329Crossref PubMed Scopus (56) Google Scholar, 10Koeberl D.D. et al.Early, sustained efficacy of adeno-associated virus vector-mediated gene therapy in glycogen storage disease type Ia.Gene Ther. 2006; (Epub ahead of press.)Google Scholar]. The importance of long-term follow-up and post mortem evaluation of vector-treated animals in preclinical studies was re-emphasized by the authors, because the incidence of tumors could easily have been missed if all experimental animals had not been fully analyzed. In the accompanying report, Bell and colleagues analyze the incidence of hepatic tumors in AAV vector-treated and untreated OTC-deficient mice in some detail [2Bell P. et al.Analysis of Tumors Arising in Male B6C3F1 Mice with and without AAV Vector Delivery to Liver.Mol. Ther. 2006; 14: 34-45Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar]. Initially, grave concerns were raised because the incidence of hepatic tumors was higher following AAV vector administration into spf-ash and wild-type mice than in untreated, control littermates. Further analysis seemed to alleviate these concerns somewhat, because the increased incidence of hepatic tumors was associated with AAV vectors encoding the control LacZ gene, but not the OTC gene. Furthermore, isolated tumor tissue generally contained lower numbers of vector copies than the surrounding liver tissue and did not express the transgene. Only one of four tumors evaluated by Southern blot analysis contained an integrated copy of the AAV vector, rendering it unlikely that integrative damage was linked to tumor development. None of the tumors occurred in younger, three month-old mice, consistent with age-related onset of the tumors at a frequency consistent with previous reports for the B6C341 hybrid mouse background from which the OTC-deficient mice were derived [11Buchmann A. et al.Mutational activation of the c-Ha-ras gene in liver tumors of different rodent strains: correlation with susceptibility to hepatocarcinogenesis.Proc. Natl. Acad. Sci. USA. 1991; 88: 911-915Crossref PubMed Scopus (175) Google Scholar]. The limitations of this study stem mainly from the fact that it was not designed a priori to detect increased tumor risk in response to AAV vector delivery. The number of animals analyzed was relatively small and none of the mice received an AAV vector not carrying a transgene ("empty vector"). In addition, the investigators were not able to score all of the mice in the study for the presence of tumors and/or hepatocellular carcinoma (HCC). However, even if we assume that all of the treated spf-ash mice that were unscored for HCC were affected, the risk for HCC was still not significantly increased by administration of the therapeutic vector. The possible link between LacZ expression and tumorigenesis observed in the study cannot be explained at this time, and these findings contrast with the results of a previous large-scale study that failed to detect any increase in tumor incidence related to administration of a LacZ-containing vector [12Bell P. et al.No evidence for tumorigenesis of AAV vectors in a large-scale study in mice.Mol. Ther. 2005; 12: 299-306Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar]. Cumulative data indicates that the risk of development of liver tumors, especially HCC, remains low in the initial year following AAV vector administration [2Bell P. et al.Analysis of Tumors Arising in Male B6C3F1 Mice with and without AAV Vector Delivery to Liver.Mol. Ther. 2006; 14: 34-45Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 8Donsante A. et al.Observed incidence of tumorigenesis in long-term rodent studies of rAAV vectors.Gene Ther. 2001; 8: 1343-1346Crossref PubMed Scopus (212) Google Scholar, 12Bell P. et al.No evidence for tumorigenesis of AAV vectors in a large-scale study in mice.Mol. Ther. 2005; 12: 299-306Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar]. Long-term, prospective studies in normal mice and other species using vectors carrying different transgenes, and including a vector carrying no transgene, would be required to determine definitively whether there is any AAV vector-related risk of tumorigenicity. However, even such studies could overlook disease- and model-specific risks. What conclusion can we draw regarding the likelihood that a therapeutic AAV vector increases the risk of hepatic tumors? Probably not.