Gene Therapy (2003) 10, 1791–1799 & 2003 Nature Publishing Group All rights reserved 0969-7128/03 $25.00 www.nature.com/gt REVIEW Illegitimate DNA integration in mammalian cells HWu¨ rtele1, KCE Little2 and P Chartrand2,3 1Programme de Biologie Mole´culaire, Universite´ de Montre´al, Montre´al, Canada; 2Department of Medicine, Division of Experimental Medicine, McGill University, Montre´al, Que´bec, Canada; and 3Centre Hospitalier de l’Universite´ de Montre´al and De´partement de Pathologie et de Biologie Cellulaire, Universite´ de Montre´al, Montre´al, Que´bec, Canada Foreign DNA integration is one of the most widely exploited therapy procedures can result in illegitimate integration of cellular processes in molecular biology. Its technical use introduced sequences and thus pose a risk of unforeseeable permits us to alter a cellular genome by incorporating a genomic alterations. The choice of insertion site, the degree fragment of foreign DNA into the chromosomal DNA. This to which the foreign DNA and endogenous locus are modified process employs the cell’s own endogenous DNA modifica- before or during integration, and the resulting impact on tion and repair machinery. Two main classes of integration structure, expression, and stability of the genome are all mechanisms exist: those that draw on sequence similarity factors of illegitimate DNA integration that must be con- between the foreign and genomic sequences to carry out sidered, in particular when designing genetic therapies. homology-directed modifications, and the nonhomologous or Gene Therapy (2003) 10, 1791–1799. doi:10.1038/ ‘illegitimate’ insertion of foreign DNA into the genome. Gene sj.gt.3302074 Keywords: illegitimate DNA integration; DNA repair; transgenesis; recombination; mutagenesis Introduction timate integration. Homology-dependent genome mod- ifications rely on mechanisms that make use of sequence The development of methods to manipulate and modify similarities between the incoming DNA and the targeted the genome is one key achievement of molecular biology. locus to induce homologous recombination. These Foreign DNA integration is one of those widely used processes usually give rise to predictable results, such techniques, allowing us to modify a cell’s genetic that the configurations of the modified loci, and of the content. The integration process begins with the delivery integrated DNA, can be predefined by the investigators. of the DNA to the cell. Many transfection procedures Techniques employing these mechanisms have numer- have been developed that enable DNA to enter into the ous applications.11 Among others, small fragment homo- cytoplasm, but its passage into the nucleus is mainly logous recombination (SFHR) employs short (400– mediated by cellular processes.1,2 After its entry into the 800 bp) DNA fragments to modify any locus and can nucleus, a large proportion of the foreign DNA is rapidly be used to correct punctual mutations.12 Other techni- degraded or diluted among subsequent cell divisions.3 ques, such as classic gene targeting, are routinely used to However, molecules containing an origin of replication introduce DNA to a specific site or to knock-out genes.13 (typically derived from viruses) can persist for long However, while these methods hold great promise for periods as extrachromosomally replicating episomes the field of gene therapy, and for the development of under certain conditions (such as expression of viral experimental tools to investigate genome dynamics, we tumour antigens or association with the nuclear matrix).4 must take into account the fact that a significant Alternatively, in approximately one cell per thousand, proportion of cells integrate DNA into their genomes the introduced foreign DNA will integrate into chromo- via homology-independent means, collectively referred somal DNA (although this figure may vary somewhat to as illegitimate integration. Illegitimate integration depending on cell type).5,6 This phenomenon is not events are usually more frequent than homology- limited to laboratory transfections as many natural directed integration: the ratio of homology versus non- cellular processes, such as integration of DNA from homology-dependent integration can be anywhere from apoptotic bodies,7 repair of chromosomal lesions by 4:1 to 1:1 000 000, depending on experimental conditions insertion of mitochondrial DNA,8 or retrotransposition and cell types, but illegitimate integration is typically events, give rise to de novo integration of DNA.9,10 Thus 1000–10 000 times more frequent than targeted integra- DNA integration may be seen as an ongoing natural tion (see Smith14 and references therein). These mechan- process, which can be harnessed to artificially introduce isms give rise to much less predictable integrated modifications to a cell’s genetic content. structures, as one usually cannot preselect the genomic DNA can become integrated into chromosomes by two site of integration, nor the resulting foreign DNA– main processes: homology-dependent means and illegi- chromosome structure. The conditions governing how the cell chooses which mode of integration to employ are Correspondence: Dr P Chartrand, 1560 Sherbrooke Est, Montreal, Que´bec, unclear, but may be dictated by cell cycle-dependent Canada H2L 4M1 availability of DNA-modifying machinery: homology- Received 10 December 2002; accepted 28 March 2003 dependent machinery is available mainly in G2/S, while Illegitimate DNA integration HWu¨rtele et al 1792 homology-independent means are present during most suggesting that viral factors can also be involved in the of the cell cycle (see below).15 integration.18–20 Thus, some viral DNA integration events It must therefore be stressed that DNA often integrates appear to be identical in nature to any type of foreign into the genome in ways that we do not fully understand, DNA integration (for example SV40 DNA), while those nor totally control. In the context of gene therapy, it is depending on viral-encoded machinery are clearly likely that local or systemic delivery of DNA to cells will different. These types of virus-specific events will not eventually result in illegitimate integration of DNA be considered here, but it should be noted that many of molecules. This appears to happen at a low fre- the mechanisms involved in the illegitimate integration quency.16,17 However, we will see in this review that of foreign DNA could also be involved, at one point or the resultant integrated structure can be surprisingly another, in viral DNA integration. complex and unpredictable compared to the expected structure of targeted integration, making it difficult to evaluate the risks inherent in these events (see Figure 1). Extrachromosomal DNA is often modified A brief note regarding the source of integrating DNA before integration into chromosomal DNA discussed in this review, since much of the early work done on illegitimate integration described the integration The first part of this article addresses the status of foreign of viral DNA. Viral DNA per se appears to integrate into DNA before, during or shortly after integration (see the genome in the same way as any foreign DNA. Figure 2a). As mentioned earlier, it has been shown that However, available data suggest that viral-encoded most transfected DNA is rapidly degraded upon integrases (such as with retroviruses) may target incom- introduction into cells. Molecules that are not degraded ing DNA to particular genomic loci, and influence (at can be modified extrachromosomally in many different least in part) the mechanisms of integration. Indeed, ways. Homologous recombination can modify extra- characterization of retroviral integration processes has chromosomal DNA very efficiently using either inter- or shown only a partial dependence on host cell factors, intramolecular sequence homology and can result in multiple products depending on the organization of the sequences sharing identity.21–26 The end products of extrachromosomal homologous recombination are simi- lar to those obtained during chromosomal homologous recombination, suggesting that at least some mechanisms are shared by the two processes.27–31 Transfected DNA can also be mutated at high frequency (on the order of 1%) by homology-indepen- dent means including point mutations, deletions, and more complex rearrangements such as insertion of genomic DNA.32–34 (see also our unpublished work). Linear extrachromosomal DNA can also be efficiently circularized in the nucleus, and different molecules can be joined together to form concatamers35 by processes referred to as nonhomologous end joining (NHEJ). NHEJ often involves short sequence homology between the joined ends, and additions or deletions of approximately 25 nucleotides or less at the junctions.34,36,37 Similar processes have been shown to occur during chromoso- mal double-strand break (DSB) repair,27,29,38 which again suggests that extrachromosomal and chromosomal re- pair are mediated by similar cellular machinery. Integrated DNA is modified in the same ways as extrachromosomal DNA Studies aimed at characterizing integrated foreign DNA reveal modifications very similar to those observed in nonintegrating extrachromosomal DNA. However, it must be kept in mind that it is not known whether the integrated DNA was modified before or during integra- Figure 1 Difference between homology-directed genome modification and tion, since the observations are typically made after the illegitimate DNA integration. (a) The chromosome structure resulting integration has taken place.