AAV Vectors, Insertional Mutagenesis, and Cancer
Authors: David W Russell
Journal: Molecular Therapy (2007)

By now we have all heard a lot about retroviral vectors and cancer, but other types of vectors have typically been left out of these discussions. However, results presented at the recent American Society of Gene Therapy meeting and just published in Science1Donsante A Miller DG Li Y Vogler C Brunt EM Russell DW et al.AAV vector integration sites in mouse hepatocellular carcinoma.Science. 2007; 317: 477Crossref PubMed Scopus (461) Google Scholar show that this is a subject that goes beyond retroviruses, hematopoietic cells, and gamma C transgenes. In this study, mice developed hepatocellular carcinomas (HCCs) that contained adeno-associated virus (AAV) vector proviruses at a specific chromosomal locus, raising important questions about the safety of in vivo gene therapy. The experiments were a collaboration among three laboratories: Mark Sands's group, which generated the mice; the lab of Dan Miller, who isolated the vector:chromosome junctions; and my own (let's just say I made sure Dan got the samples from Mark).To summarize the findings, normal newborn mice injected intravenously with an AAV vector containing a cytomegalovirus (CMV) enhancer and human β-glucuronidase gene had an increased incidence of HCC (33–56% vs. 8.6% for untreated controls). Four integrated vector proviruses were identified by inverse PCR methods in the tumors from four mice, and all four mapped to a 6-kilobase region on mouse chromosome 12 (Figure 1), referred to here as the AAV-HCC locus. These specific proviruses were not detected in adjacent normal tissue, and the tumors had upregulated expression of neighboring chromosomal transcripts, including some that encode multiple small nucleolar RNAs (snoRNAs) and microRNAs. Given that AAV vectors typically integrate throughout the genome,2Nakai H Wu X Fuess S Storm TA Munroe D Montini E et al.Large-scale molecular characterization of adeno-associated virus vector integration in mouse liver.J Virol. 2005; 79: 3606-3614Crossref PubMed Scopus (142) Google Scholar,3Miller DG Trobridge GD Petek LM Jacobs MA Kaul R Russell DW Large-scale analysis of adeno-associated virus vector integration sites in normal human cells.J Virol. 2005; 79: 11434-11442Crossref PubMed Scopus (122) Google Scholar the consistent identification of proviruses in the same locus suggests that insertional mutagenesis by the AAV vector led to the malignant transformation of the hepatocytes.Unfortunately, the provirus structures could not be completely established in these experiments, despite multiple attempts to do so. Only one junction was recovered from each provirus, so the other ends were not localized. Thus, it is formally possible that large rearrangements or even translocations occurred, as is sometimes observed with AAV vectors.2Nakai H Wu X Fuess S Storm TA Munroe D Montini E et al.Large-scale molecular characterization of adeno-associated virus vector integration in mouse liver.J Virol. 2005; 79: 3606-3614Crossref PubMed Scopus (142) Google Scholar,3Miller DG Trobridge GD Petek LM Jacobs MA Kaul R Russell DW Large-scale analysis of adeno-associated virus vector integration sites in normal human cells.J Virol. 2005; 79: 11434-11442Crossref PubMed Scopus (122) Google Scholar However, this seems unlikely, because it would have separated the unidentified junctions from adjacent, upregulated chromosomal transcripts. Second, we do not know which parts of the vector genome were present in the proviruses, other than a portion of the CMV enhancer and one terminal repeat. Because AAV vector proviruses typically contain terminal deletions, a functional β-glucuronidase gene might not even have been present.How much integration occurred?The copy numbers of the integrated proviruses ranged from 0.03 to 0.27 per diploid mouse genome, which, although lower than one might expect, is still consistent with clonal expansion of a single transformed hepatocyte in each tumor. That is because hepatocytes are polyploid (often 8N or even 16N)4Epstein CJ Gatens EA Nuclear ploidy in mammalian parenchymal liver cells.Nature. 1967; 214: 1050-1051Crossref PubMed Scopus (47) Google Scholar, and tumors can contain significant numbers of nonhepatocyte cells, both of which would dilute vector genomes. The low copy numbers and unpredictable nature of AAV provirus structures represent technical problems that can prevent the isolation of AAV vector–chromosome junctions. This can make the identification of a specific provirus impossible by Southern blots or PCR, when probes or primers require the presence of a deleted portion of the vector. In fact, initial attempts by more than one researcher failed to isolate junctions from these same tumor samples.These findings should be considered in light of what we know about AAV vector integration frequencies, and a few back-of-the-napkin calculations can be informative. In other experiments, adult mice that received an intravenous injection of approximately 1010Seitz H Royo H Bortolin ML Lin SP Ferguson-Smith AC Cavaille J A large imprinted microRNA gene cluster at the mouse Dlk1-Gtl2 domain.Genome Res. 2004; 14: 1741-1748Crossref PubMed Scopus (428) Google Scholar vector particles/g contained integrated AAV vector genomes in the liver at roughly 0.05 copies per diploid cell genome.5Nakai H Yant SR Storm TA Fuess S Meuse L Kay MA Extrachromosomal recombinant adeno-associated virus vector genomes are primarily responsible for stable liver transduction in vivo.J Virol. 2001; 75: 6969-6976Crossref PubMed Scopus (365) Google Scholar Given the ploidy of hepatocytes and the presence of other cell types in the liver, this can conservatively be rounded up to 0.1 integrated vector genomes per hepatocyte. Because there are approximately 6 × 107 hepatocytes in an adult mouse liver,6Rhim JA Sandgren EP Degen JL Palmiter RD Brinster RL Replacement of diseased mouse liver by hepatic cell transplantation.Science. 1994; 263: 1149-1152Crossref PubMed Scopus (504) Google Scholar a random distribution of integration sites would produce an integration every 483 base pairs on average (2.9 × 109 base pairs in the mouse genome divided by 6 × 106 integrations), so about 12 hepatocytes would be expected to contain an integrant within the 6-kilobase window found to be associated with HCC. Although every experiment is different, and the assumptions are inherently inaccurate, the bottom line from this sort of analysis is that mice treated with AAV vectors in a typical gene therapy experiment (about 1011 serotype 2 vector particles injected intravenously into an adult) are likely to contain multiple hepatocytes with integrants at the AAV-HCC locus and every other locus as well. So, even though AAV is often thought of as an episomal vector, integration is frequent enough to saturate the genome and result in a provirus in every gene. Because AAV vectors depend entirely on cellular factors for integration, and they integrate at existing, chromosomal breaks7Miller DG Petek LM Russell DW Adeno-associated virus vectors integrate at chromosome breakage sites.Nat Genet. 2004; 36: 767-773Crossref PubMed Scopus (176) Google Scholar just as naked DNA can,8Lin Y Waldman AS Capture of DNA sequences at double-strand breaks in mammalian chromosomes.Genetics. 2001; 158: 1665-1674Crossref PubMed Google Scholar this sobering thought may also apply to other "nonintegrating" vectors, in which rare integration frequencies combined with large vector doses could still produce substantial insertional mutagenesis.In the experiments that produced proviral insertions at AAV-HCC, the mice were injected as neonates, so in comparison with adults the total number of hepatocytes was smaller, but the dose was higher when adjusted for body weight (1.5 × 1011 vector genomes per pup). A separate study9Miller DG Wang PR Petek LM Hirata RK Sands MS Russell DW Gene targeting in vivo by adeno-associated virus vectors.Nat Biotechnol. 2006; 24: 1022-1026Crossref PubMed Scopus (93) Google Scholar with similar neonatal mouse dosing produced total vector copy numbers (episomal and integrated) equivalent to those of the adult mouse experiments described above. Thus, it seems likely that each mouse injected as a newborn also had a provirus in every gene, and it is not surprising that integration sites were found so close together in four different mice.How many oncogenic hits were required?In the case of hematopoietic cells, a single cell containing a specific integrated provirus can replicate and expand to vast cell numbers, thereby undergoing spontaneous mutations that could result in additional oncogenic hits. It is possible that all leukemias linked to retroviral vector integrations contain these types of secondary, cooperating mutations. This is much harder to envision in the case of HCC, because normal, individual hepatocytes do not undergo a massive, multi-log expansion during which random mutations could occur in cooperating oncogenes. Even if we assume that multiple hepatocytes contained proviruses at the AAV-HCC locus, the number is still too small for a second spontaneous mutation to consistently occur during subsequent proliferation.Another possibility is that the transformed hepatocytes contained multiple AAV integrants. With an average integration frequency of 0.1 proviruses per hepatocyte, the Poisson distribution predicts that 0.45% of hepatocytes will contain two proviruses and 0.015% will contain three proviruses. In a mouse with 6 × 107 hepatocytes, that equals 2.7 × 105 or 9.1 × 103 hepatocytes, respectively. Although at first glance this seems like a lot of room for combinatorial complexity, these odds also predict that only a small percentage of the few hepatocytes with proviruses at the AAV-HCC locus will contain additional proviruses, ruling out an essential second provirus insertion in tumor formation. It should also be noted that no additional proviruses were found in the tumor samples despite several attempts to isolate them. This suggests that a single AAV vector provirus is enough to transform a mouse hepatocyte under appropriate conditions.Why did it cause cancer?The AAV-HCC locus on chromosome 12 contains several maternally and paternally imprinted genes (including Dlk1, Gtl2, Rtl1, Rian, Mirg, and Dio3),10Seitz H Royo H Bortolin ML Lin SP Ferguson-Smith AC Cavaille J A large imprinted microRNA gene cluster at the mouse Dlk1-Gtl2 domain.Genome Res. 2004; 14: 1741-1748Crossref PubMed Scopus (428) Google Scholar,11Lin SP Youngson N Takada S Seitz H Reik W Paulsen M et al.Asymmetric regulation of imprinting on the maternal and paternal chromosomes at the Dlk1-Gtl2 imprinted cluster on mouse chromosome 12.Nat Genet. 2003; 35: 97-102Crossref PubMed Scopus (386) Google Scholar,12Shimoda M Morita S Obata Y Sotomaru Y Kono T Hatada I Imprinting of a small nucleolar RNA gene on mouse chromosome 12.Genomics. 2002; 79: 483-486Crossref PubMed Scopus (12) Google Scholar and uniparental disomy for chromosome 12 causes multiple developmental defects,13Georgiades P Watkins M Surani MA Ferguson-Smith AC Parental origin-specific developmental defects in mice with uniparental disomy for chromosome 12.Development. 2000; 127: 4719-4728PubMed Google Scholar confirming a functional role for imprinting. The locus is similar in many ways to the imprinted Igf2 gene cluster associated with abnormal growth and cancer.14Maher ER Reik W Beckwith-Wiedemann syndrome: imprinting in clusters revisited.J Clin Invest. 2000; 105: 247-252Crossref PubMed Google Scholar,15Feinberg AP Cui H Ohlsson R DNA methylation and genomic imprinting: insights from cancer into epigenetic mechanisms.Semin Cancer Biol. 2002; 12: 389-398Crossref PubMed Scopus (169) Google Scholar At present, it is not known if the AAV vector proviruses were located in the paternal or maternal alleles, or if methylation and imprinting were affected. The callipyge, or "beautiful buttocks," mutation responsible for muscular hypertrophy maps to the syntenic locus in sheep and causes overexpression of Dlk1 and Gtl2.16Charlier C Segers K Karim L Shay T Gyapay G Cockett N et al.The callipyge mutation enhances the expression of coregulated imprinted genes in cis without affecting their imprinting status.Nat Genet. 2001; 27: 367-369Crossref PubMed Scopus (143) Google Scholar However, these genes were not overexpressed in the tumors containing proviruses. The human DLK1 gene has been reported to be a tumor suppressor in renal cell carcinoma,17Kawakami T Chano T Minami K Okabe H Okada Y Okamoto K Imprinted DLK1 is a putative tumor suppressor gene and inactivated by epimutation at the region upstream of GTL2 in human renal cell carcinoma.Hum Mol Genet. 2006; 15: 821-830Crossref PubMed Scopus (84) Google Scholar and decreased GTL2 expression was observed in some neuroblastomas,18Astuti D Latif F Wagner K Gentle D Cooper WN Catchpoole D et al.Epigenetic alteration at the DLK1-GTL2 imprinted domain in human neoplasia: analysis of neuroblastoma, phaeochromocytoma and Wilms' tumour.Br J Cancer. 2005; 92: 1574-1580Crossref PubMed Scopus (110) Google Scholar but insertional mutagenesis should not decrease expression of a possible tumor suppressor gene by direct, cis-acting effects, given the polyploidy of hepatocytes and the presence of many alleles without proviruses. So even though these genes have been implicated in cell growth and cancer, it is difficult to relate these prior observations to AAV-induced tumorigenesis.A more likely basis for the oncogenic effects of vector integration was the overexpression of adjacent transcripts telomeric to the proviruses. The Rian gene contains several snoRNAs and was overexpressed 9- to 539-fold in tumors that contained only 0.1–0.27 provirus copies per diploid genome, so per integrant there was an even more dramatic increase in expression. snoRNAs direct sequence-specific methylation and pseudouridylation of ribosomal RNAs, spliceosomal snRNAs, and possibly messenger RNAs.19Bachellerie JP Cavaille J Huttenhofer A The expanding snoRNA world.Biochimie. 2002; 84: 775-790Crossref PubMed Scopus (507) Google Scholar The targets of the multiple snoRNAs present at the provirus insertion locus are not known, but sequence analysis suggests that at least some do not target ribosomal RNA or snRNAs,20Xiao Y Zhou H Qu LH Characterization of three novel imprinted snoRNAs from mouse Irm gene.Biochem Biophys Res Commun. 2006; 340: 1217-1223Crossref PubMed Scopus (8) Google Scholar thus specific messenger RNAs may have been affected. A link between snoRNA expression and cancer has not previously been established, so these findings may represent a novel mechanism of tumor formation. Similarly, the Mirg gene containing several microRNAs was overexpressed 3.1- to 73-fold in tumors, and microRNAs have recently been shown to play important roles in cancer.21Calin GA Croce CM MicroRNA signatures in human cancers.Nat Rev Cancer. 2006; 6: 857-866Crossref PubMed Scopus (6526) Google Scholar Thirty-two of 386 known mouse microRNAs are present here,22Griffiths-Jones S Grocock RJ van Dongen S Bateman A Enright AJ miRBase: microRNA sequences, targets and gene nomenclature.Nucleic Acids Res. 2006; 34: D140-D144Crossref PubMed Scopus (3572) Google Scholar with thousands of potential targets that could have global influences on gene expression. Because of the complexity of this locus, it may take many years to establish how provirus insertions in it can cause cancer. However, given the worldwide importance of HCC to human health, and the small number of patients receiving AAV-mediated gene therapy, the real significance of these findings may ultimately be a better understanding of liver cancer.What about other animal models?The first report of AAV vector-induced liver tumors was in mice with mucopolysaccharidosis type VII (MPS VII) that received the same β-glucuronidase vector referred to in this Commentary.23Donsante A Vogler C Muzyczka N Crawford JM Barker J Flotte T et al.Observed incidence of tumorigenesis in long-term rodent studies of rAAV vectors.Gene Ther. 2001; 8: 1343-1346Crossref PubMed Scopus (216) Google Scholar Because integrated proviruses were not isolated from these tumors, an early hypothesis was that the MPS VII mouse strain was tumor-prone, and the longer life span of treated mice just provided the necessary time for tumor formation. However, follow-up studies showed that MPS VII mice treated by bone marrow transplantation did not have an increased incidence of HCC, and normal mice that received the same vector did.1Donsante A Miller DG Li Y Vogler C Brunt EM Russell DW et al.AAV vector integration sites in mouse hepatocellular carcinoma.Science. 2007; 317: 477Crossref PubMed Scopus (461) Google Scholar In retrospect, the tumors in the original MPS VII mice may have contained integrated proviruses that were technically difficult to isolate.There are other reports of liver tumors in mice that received AAV vectors. In some cases this may be due to known effects of the vector-encoded transgenes such as the HBx protein expressed from the woodchuck hepatitis virus post-transcriptional regulatory element,24Embury JE Charron CC Poirier AE Zori A Carmichael R Flotte TR et al.Long term portal vein administration of AAV-WPRE vector results in increased incidence of neoplastic disease and hepatic pathology.Mol Ther. 2006; 13: S83Abstract Full Text Full Text PDF Google Scholar or short hairpin RNA-related toxicity leading to increased hepatocyte division.25Grimm D Beer S Komatsubara K Lee JS Koh S Wang L et al.Adverse shRNA cytotoxicity can accelerate tumorigenesis in genetically predisposed mice.Mol Ther. 2007; 15: S266Abstract Full Text Full Text PDF Scopus (116) Google Scholar Of greater concern is a retrospective study describing an increased incidence of hepatic tumors in mice treated with vectors containing a lacZ transgene (but not an ornithine transcarbamylase transgene) driven by the human thyroid hormone binding globulin promoter.26Bell P Moscioni AD McCarter RJ Wu D Gao G Hoang A et al.Analysis of tumors arising in male B6C3F1 mice with and without AAV vector delivery to liver.Mol Ther. 2006; 14: 34-44Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar It is hard to imagine lacZ as an oncogene, so insertional mutagenesis could have played a role in these mice. The tumors contained adequate amounts of vector genomes based on real-time PCR, but no integration junctions were described. These isolated examples must be considered in light of the many studies done in rodents, dogs, and primates wherein no increased incidence of tumors was noted (references too numerous to include).What experimental differences might explain the appearance of tumors in the study described here? First, these mice were injected as neonates, when hepatocytes are more actively dividing, potentially increasing the frequency of integration, the amount of cell proliferation after integration, and the length of time for tumor development, which occurred preferentially in older mice. Although these are probably important factors, other studies did not report increased tumor formation after neonatal injection (discussed in ref. 23), and the animals with a higher tumor incidence after lacZ vector injection were injected as young adults.26Bell P Moscioni AD McCarter RJ Wu D Gao G Hoang A et al.Analysis of tumors arising in male B6C3F1 mice with and without AAV vector delivery to liver.Mol Ther. 2006; 14: 34-44Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar Second, there may be vector-specific effects, if the CMV enhancer is prone to activating neighboring genes, especially at that particular locus, or if the β-glucuronidase gene promoted cell proliferation in some manner. While these are real possibilities, the CMV enhancer has been widely used in gene therapy without being linked to tumor formation, and transgenic mice with massive overexpression of β-glucuronidase do not seem to be tumor-prone27Vogler C Galvin N Levy B Grubb J Jiang J Zhou XY et al.Transgene produces massive overexpression of human β-glucuronidase in mice, lysosomal storage of enzyme, and strain-dependent tumors.Proc Natl Acad Sci USA. 2003; 100: 2669-2673Crossref PubMed Scopus (38) Google Scholar (although, interestingly, one strain was believed to be tumor-prone as a result of integration at a specific, unidentified site). Third, the particular strain of mice may be relevant, because there can be large differences in the incidence of liver cancer in different mouse strains (see http://tumor.informatics.jax.org/mtbwi/tumorFrequencyGrid.do), and mice may be more tumor-prone than other species. Finally, some tumors may have gone undetected in negative studies, if histological analysis was incomplete, the tumors were asymptomatic, or the follow-up was too short. Given the long latency observed in mouse experiments, similar tumors could take several years to develop in large animals. At this point, it is not clear what factors are critical for tumor formation, and until these factors are identified, it is important to remember that the bulk of experimental evidence suggests that tumors are not routinely observed after AAV vector administration. Sorting out the problem will require several experiments, including comparisons of different transgenes, promoters, delivery routes, and animal ages, all with long-term follow-up and careful analysis for the presence of tumors. It will be especially important to study large animals injected as newborns.Will it happen in humans?The short answer is that we do not know yet, and it is impossible to say what the chances of tumor formation will be in a clinical trial. Still, some points are worth considering. First, there is a syntenic locus on human chromosome 14 with similar regulation to the AAV-HCC locus in mice.28Cavaille J Seitz H Paulsen M Ferguson-Smith AC Bachellerie JP Identification of tandemly-repeated C/D snoRNA genes at the imprinted human 14q32 domain reminiscent of those at the Prader-Willi/Angelman syndrome region.Hum Mol Genet. 2002; 11: 1527-1538Crossref PubMed Scopus (181) Google Scholar Second, the number of hepatocytes is much larger in humans (approximately 1.5 × 1011 hepatocytes/liver),29Wilson ZE Rostami-Hodjegan A Burn JL Tooley A Boyle J Ellis SW et al.Inter-individual variability in levels of human microsomal protein and hepatocellularity per gram of liver.Br J Clin Pharmacol. 2003; 56: 433-440Crossref PubMed Scopus (137) Google Scholar so if integration occurs at a frequency similar to that in mice, there would be about 3 × 104 hepatocytes with proviruses at the AAV-HCC locus. This number is high enough that a 100- or even 1,000-fold lower dose per kilogram as compared with that used in mice would not eliminate the risk of integration at AAV-HCC. In addition, it raises the possibility of multi-hit oncogenesis by AAV, because over 108 and 107 hepatocytes would have two or three proviruses, respectively. Thus, we should assume that intravenous delivery of AAV vectors in clinical trials will result in hepatocytes with proviruses at AAV-HCC. Whether those proviruses will cause cancer is not known, and humans could be very different from mice in that regard. In the clinical trial of liver-directed gene therapy for hemophilia B, an immune response against capsid proteins ultimately led to the elimination of transduced hepatocytes,30Manno CS Pierce GF Arruda VR Glader B Ragni M Rasko JJ et al.Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response.Nat Med. 2006; 12: 342-347Crossref PubMed Scopus (1537) Google Scholar so these patients may not reflect what will happen when long-term gene delivery becomes more effective. Finally, in the case of mice, a very large number of animals used by many investigators in over a decade of research has only now led to the first documented cases of oncogenesis due to an integrated AAV vector provirus. So even if humans do behave like mice, tumors may be observed only in some clinical situations and in a small minority of individuals receiving gene therapy.Should clinical trials continue?When I have asked gene therapists what these findings mean for clinical trials with AAV, the answer is almost always that they should continue. And, although this is a bit like asking a surgeon if we need to operate, I agree with this sentiment for the most part (the recently reported death that occurred after administration of an AAV vector for the treatment of arthritis is presumably unrelated and not considered here). We need to accept that there is an unquantified possibility of tumor formation in clinical trials using AAV, and do the best we can to consider this when estimating risk-to-benefit ratios, which is where investigators will almost certainly disagree. From my own perspective, some trials using AAV seem too risky even with the limited data in hand, such as gene therapy for MPS VII using a β-glucuronidase vector. In utero gene therapy may also be risky, if human, fetal hepatocyte biology is similar to that of newborn mice. In other cases, the potential risks are less concerning, especially untreatable, fatal diseases in which preclinical studies suggest that AAV may provide some benefit. Hemophilia lies between these extremes, because there is a wealth of data in multiple animal models demonstrating therapeutic clotting factor levels after AAV-mediated gene therapy, and no reported increase in tumors. However, the potential benefits of hemophilia gene therapy as compared with protein infusions are difficult to quantify. At a minimum, when trials are done with AAV, consent forms should mention the risk of developing tumors in a balanced way, and follow-up screening for liver tumors should be considered. In the meantime, we can all agree on the need for further research, and some questions will only be answered with human trials. By now we have all heard a lot about retroviral vectors and cancer, but other types of vectors have typically been left out of these discussions. However, results presented at the recent American Society of Gene Therapy meeting and just published in Science1Donsante A Miller DG Li Y Vogler C Brunt EM Russell DW et al.AAV vector integration sites in mouse hepatocellular carcinoma.Science. 2007; 317: 477Crossref PubMed Scopus (461) Google Scholar show that this is a subject that goes beyond retroviruses, hematopoietic cells, and gamma C transgenes. In this study, mice developed hepatocellular carcinomas (HCCs) that contained adeno-associated virus (AAV) vector proviruses at a specific chromosomal locus, raising important questions about the safety of in vivo gene therapy. The experiments were a collaboration among three laboratories: Mark Sands's group, which generated the mice; the lab of Dan Miller, who isolated the vector:chromosome junctions; and my own (let's just say I made sure Dan got the samples from Mark). To summarize the findings, normal newborn mice injected intravenously with an AAV vector containing a cytomegalovirus (CMV) enhancer and human β-glucuronidase gene had an increased incidence of HCC (33–56% vs. 8.6% for untreated controls). Four integrated vector proviruses were identified by inverse PCR methods in the tumors from four mice, and all four mapped to a 6-kilobase region on mouse chromosome 12 (Figure 1), referred to here as the AAV-HCC locus. These specific proviruses were not detected in adjacent normal tissue, and the tumors had upregulated expression of neighboring chromosomal transcripts, including some that encode multiple small nucleolar RNAs (snoRNAs) and microRNAs. Given that AAV vectors typically integrate throughout the genome,2Nakai H Wu X Fuess S Storm TA Munroe D Montini E et al.Large-scale molecular characterization of adeno-associated virus vector integration in mouse liver.J Virol. 2005; 79: 3606-3614Crossref PubMed Scopus (142) Google Scholar,3Miller DG Trobridge GD Petek LM Jacobs MA Kaul R Russell DW Large-scale analysis of adeno-associated virus vector integration sites in normal human cells.J Virol. 2005; 79: 11434-11442Crossref PubMed Scopus (122) Google Scholar the consistent identification of proviruses in the same locus suggests that insertional mutagenesis by the AAV vector led to the malignant transformation of the hepatocytes. Unfortunately, the provirus structures could not be completely established in these experiments, despite multiple attempts to do so. Only one junction was recovered from each provirus, so the other ends were not localized. Thus, it is formally possible that large rearrangements or even translocations occurred, as is sometimes observed with AAV vectors.2Nakai H Wu X Fuess S Storm TA Munroe D Montini E et al.Large-scale molecular characterization of adeno-associated virus vector integration in mouse liver.J Virol. 2005; 79: 3606-3614Crossref PubMed Scopus (142) Google Scholar,3Miller DG Trobridge GD Petek LM Jacobs MA Kaul R Russell DW Large-scale analysis of adeno-associated virus vector integration sites in normal human cells.J Virol. 2005; 79: 11434-11442Crossref PubMed Scopus (122) Google Scholar However, this seems unlikely, because it would have separated the unidentified junctions from adjacent, upregulated chromosomal transcripts. Second, we do not know which parts of the vector genome were present in the proviruses, other than a portion of the CMV enhancer and one terminal repeat. Because AAV vector proviruses typically contain terminal deletions, a functional β-glucuronidase gene might not even have been present. How much integration occurred?The copy numbers of the integrated proviruses ranged from 0.03 to 0.27 per diploid mouse genome, which, although lower than one might expect, is still consistent with clonal expansion of a single transformed hepatocyte in each tumor. That is because hepatocytes are polyploid (often 8N or even 16N)4Epstein CJ Gatens EA Nuclear ploidy in mammalian parenchymal liver cells.Na