AAV Joins the Rank of Genotoxic Vectors
Authors: Utpal P. Davé, Kenneth Cornetta
Journal: Molecular Therapy (2021)

Cancer is one of the most feared diagnoses, and any treatment that increases the chance of a secondary malignancy requires careful consideration before offering it to patients. Integrating vectors based on retroviruses and lentiviruses carry the risk of malignancy related to insertional mutagenesis (IM), a process where vector insertion alters expression of critical genes involved with cell growth and differentiation. Adeno-associated viral (AAV) vectors have been touted as safer, given that these vectors generally persist episomally with infrequent integration. Unfortunately, mounting evidence supports the notion that AAV vectors can be genotoxic. In 2001, Donsante et al.1Donsante A. Vogler C. Muzyczka N. Crawford J.M. Barker J. Flotte T. Campbell-Thompson M. Daly T. Sands M.S. Observed incidence of tumorigenesis in long-term rodent studies of rAAV vectors.Gene Ther. 2001; 8: 1343-1346Crossref PubMed Scopus (207) Google Scholar reported the development of hepatocellular carcinoma (HCC) after AAV gene transfer in a murine model of mucopolysaccharidosis type VII (MPSVII). Subsequent reports identified risk factors associated with HCC development, including dose, strong promoter/enhancer elements, and exposure in the neonatal period.2Chandler R.J. Sands M.S. Venditti C.P. Recombinant Adeno-Associated Viral Integration and Genotoxicity: Insights from Animal Models.Hum. Gene Ther. 2017; 28: 314-322Crossref PubMed Scopus (87) Google Scholar The majority of malignant cells had integrated AAV sequences resulting from recombination, which often deleted the vector transgene. The most common site associated with murine HCC is the Rian gene. A liver-specific enhancer-promoter region in wild-type AAVs and AAV vectors has also been implicated in HCC risk.3Logan G.J. Dane A.P. Hallwirth C.V. Smyth C.M. Wilkie E.E. Amaya A.K. Zhu E. Khandekar N. Ginn S.L. Liao S.H.Y. et al.Identification of liver-specific enhancer-promoter activity in the 3′ untranslated region of the wild-type AAV2 genome.Nat. Genet. 2017; 49: 1267-1273Crossref PubMed Scopus (49) Google Scholar Whether the observation in mice was applicable to the human use of AAV vectors has been debated, but recent reports indicate that the risk of cancer in patients needs further consideration. A report by Nguyen et al.4Nguyen G.N. Everett J.K. Kafle S. Roche A.M. Raymond H.E. Leiby J. Wood C. Assenmacher C.A. Merricks E.P. Long C.T. et al.A long-term study of AAV gene therapy in dogs with hemophilia A identifies clonal expansions of transduced liver cells.Nat. Biotechnol. 2020; (Published online November 16, 2020)https://doi.org/10.1038/s41587-020-0741-7Crossref Scopus (54) Google Scholar noted clonal expansion of liver cells in a canine model of factor VIII. The animals were followed for 10 years, and two dogs had stable factor VIII levels for the first 4 years after treatment, but the levels increased 4-fold over subsequent years. The animals were treated with different doses, promoter elements, and delivery systems (single-chain versus two cF8 chain vectors). Liver biopsies from six of the dogs, including the two with elevated factor VIII, had evidence of clonal cell expansion based on the frequency of vector integrations. For the most common clones, nearly half of the integrations were near growth regulatory genes. Now, two studies reported in this issue of Molecular Therapy provide additional findings of concern. Li et al.5Li Y. Miller C.A. Shea L.K. Jiang X. Guzman M.A. Chandler R.J. Ramakrishnan S.M. Smith S.N. Venditti C.P. Vogler C.A. et al.Enhanced efficacy and increased long-term toxicity of CNS-directed, AAV-based combination therapy for Krabbe disease.Mol. Ther. 2020; 29 (this issue): 691-701Abstract Full Text Full Text PDF Scopus (8) Google Scholar describe a high incidence of HCC in a murine model of Krabbe disease (Twitcher mice). Both wild-type and Twitcher neonatal mice received treatment directed at the hematopoietic system (by transplantation of syngeneic marrow after total body irradiation) and CNS-directed therapy (intrathecally and intracranially administered AAV9 vector expressing galactosylceramidase). Animals were further divided into subgroups, treated with or without L-cycloserine (which reduces psychosine accumulation). While the vector was not administered intravenously, significant liver uptake was documented. Twitcher mice had a marked increase in life span. While a small percentage of animals treated with the double therapy developed HCC, 14/16 Twitcher mice treated with transplant, AAV9, and L-cycloserine developed HCC. Moreover, 19/19 wild-type mice treated with the three interventions also developed HCC. Ten of ten tumors evaluated had integration within the Rian locus, and additional integrations near genes associated with HCC were found. The authors hypothesize that both the radiation and L-cycloserine (which decreases pro-apoptotic ceramide) contributed to the development of HCC after AAV gene therapy. A report by Dalwadi et al.6Dalwadi D.A. Torrens L. Abril-Fornaguera J. Pinyol R. Willoughby C. Posey J. Llovet J.M. Christian Lanciault C. Russell D.W. Markus Grompe M. Naugler W.E. Liver Injury Increases the Incidence of HCC following AAV Gene Therapy in Mice.Mol. Ther. 2020; 29 (this issue): 680-690Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar evaluated the hypothesis that adult mice would have an increased risk of AAV-associated HCC if there was increased hepatocyte turnover. They evaluated two interventions, partial hepatectomy, and inflammation related to non-alcoholic fatty liver disease (NAFLD). They compared a control vector (tdTomato transgene) and an editing AAV vector targeting the Rian gene and sacrificed animals 6 months after AAV exposure. The editing vector produced HCC in all male neonatal mice and approximately 30% of female mice, regardless of diet. In adult mice treated with the editing vector, 5% on a regular diet developed HCC, while 100% of mice treated with a NAFLD-inducing diet or a partial hepatectomy developed HCC. Those animals treated with the control vector and NAFLD-inducing diet also developed HCC, but at half the frequency. Given the high incidence of liver inflammation in the human population, these findings raise concern for late-onset HCC after AAV therapy. Whereas IM is a new development in AAV therapy, the concept has an extensive history in retrovirology. Studies on replication-competent murine leukemia viruses (MLVs) and the initial description of frequent insertional mutation of proto-oncogenes in human gene therapy of severe combined immunodeficiency (SCID)-X1 have underscored the importance of clonal expansion and retroviral integration site mapping in retroviral gene therapy. The earliest complications can be attributable to several factors, such as the vector copy number, the vector's potential as a transcriptional enhancer and/or strong promoter, the unique susceptibility of certain target cells such as HSC, the underlying disease biology, and the potential transforming effects of the transgene. Among these, the shift from gammaretroviruses to lentiviruses seems to have alleviated the risks of enhancer and promoter insertions by the potent MLV long terminal repeat (LTR) elements. Even so, recent cases of clonal expansion from lentiviral gene therapy have been disconcerting. In the treatment of beta thalassemia patients, insertion into HMGA2 probably accounted for clonal expansion. Furthermore, in a trial of chimeric antigen receptor gene transduction, one patient's transduced T cells showed persistence of a clone with insertional inactivation of a TET2 allele; this resulted in complete loss of TET2 since the patient had a loss of function missense mutation in the other allele. The AAV studies cited here underscore the importance of underlying disease biology. For example, SCID-X1 due to loss of function of IL2RG is much more susceptible to leukemic complications than SCID due to adenosine deaminase deficiency (ADA). However, in November 2020, an ADA-SCID patient was diagnosed with leukemia 4 years out from gammaretroviral gene therapy. Whether this leukemia was a sporadic chance event or due to insertional mutation from the retroviral vector remains to be seen. The US Food and Drug Administration (FDA) has required monitoring of patients treated with retroviral and lentiviral vectors for clonal dominance and cancer (https://www.fda.gov/regulatory-information/search-fda-guidance-documents/long-term-follow-after-administration-human-gene-therapy-products). While the current guidance does not require laboratory evaluations for patients treated with AAV, the accumulating data raise the question as to whether the current recommendations should be revisited. Monitoring integrations after AAV gene transfer will be more challenging given that integration of extra-chromosomal DNA can occur over long periods of time. Moreover, many AAV applications target organs, such as the liver and the CNS, where sampling poses serious risks to the patient. These issues appear more pressing given the recent clinical hold placed on the uniQue's Hope B Trial for hemophilia (http://www.uniqure.com/investors-newsroom/press-releases.php). A patient was diagnosed with HCC 1 year after treatment, and we do not yet know if the cancer was related to the AAV vector. Finally, does cancer risk negate development of genetic modification? We would argue the answer is no. Secondary cancers remain a significant risk for our patients treated with radiation, chemotherapy, and many cytotoxic drugs that treat non-malignant disease. These adverse events are part of our discussions with patients as we embark on these therapies. It is the risk:benefit ratio that will decide whether a therapy will be utilized. A challenge for gene therapy is the potentially long latency before adverse events are observed. Research aimed at understanding the relationship between vector design, patient genotype, and prior genotoxic treatments is needed to help assess and mitigate risks. Liver Injury Increases the Incidence of HCC following AAV Gene Therapy in MiceDalwadi et al.Molecular TherapyOctober 22, 2020In BriefThis study demonstrates that adult mice exposed to rAAV gene therapy in the context of chronic liver disease developed HCC at high frequency. Further, female mice are less susceptible to rAAV-induced HCC compared to males, part of which is due to a more favorable immune milieu related to estrogen. Full-Text PDF Enhanced Efficacy and Increased Long-Term Toxicity of CNS-Directed, AAV-Based Combination Therapy for Krabbe DiseaseLi et al.Molecular TherapyDecember 31, 2020In BriefBrain-directed, adeno-associated-virus-mediated gene therapy synergizes with bone-marrow transplantation and substrate reduction therapy to dramatically increase treatment efficacy in mice with Krabbe disease. However, nearly 95% of the treated mice developed liver cancer. DNA sequence analysis revealed AAV integrations within or near the Rian locus, tumor suppressors, and proto-oncogenes. Full-Text PDF