A new analysis of vector integration has shown how integration site varies among three different groups of retroviruses1
Integration sites of retroviruses
Even before we knew what retroviruses were we knew they could induce malignancy.2 We now know that a number of them are directly associated with cell transformation through mechanisms such as insertional mutagenesis.3, 4 Nonetheless, the utility of retroviruses as gene vectors has led to their widespread use in vivo and in vitro. So the relatively recent appearance of leukaemia in two child recipients of retroviral vectors was perhaps less of a surprise to retrovirologists than it was to gene therapists. However, the effect of these sad occurrences on the field has been profound: in particular, it has installed the goal of the ability to control the sites of vector integration as a holy grail for gene therapy researchers.
Early studies that attempted to identify integration sites used laborious techniques such as positional cloning,5 isochore analysis6 and FISH.7 However, powerful PCR methodologies and the completion of the human genome sequence have revolutionized the identification of vector integration sites. Several large studies have shown that retroviral integration (and hence retroviral vector delivery) is far from random: factors such as base composition6, 8 and the presence of alphoid9 or Alu repeats10 as well as transcriptional activity all influence the integration site.
Now Rick Mitchell and co-workers have extended these previous findings in a large and well-controlled analysis of new and previously identified integration sites, which compares site preference of murine (MLV), avian (ASLV) and human (HIV) retroviruses. Chromosomal distribution, GC content, transcriptional activity and distance from cellular gene transcription start sites were compared using appropriate controls.
The authors confirmed that HIV has a tendency to integrate in regions rich in expressed genes and they also showed that it does not favour integration in the intervening CpG islands. These results were cell type independent and it is likely that other viral vectors will demonstrate similar patterns. They also confirmed previous findings that MLV favours transcriptional start sites. By contrast, their new data showed that ASLV has only a slight predilection for transcriptionally active regions and no start site preference. Moreover, many ASLV integrations occur in intergenic regions.
It would be interesting to extend these studies to cells with different cycling properties than those that these authors studied. Vector integration is complete within 24 h of infection and often considerably sooner in rapidly cycling cells,11 so the authors' analysis at 48 h might have marginally over-represented integrations in such cells. It would also be interesting to compare integration sites in growth-arrested cells to those in otherwise identical cycling cells. This would be of particular relevance to those that use lentiviruses (including HIV as studied here) to deliver genes to nondividing cells.
What guides the integration event?
The clear inference from these new data is that different retroviral preintegration complexes (PICs) recognize different features of genomic nucleoproteins, but what is the crucial link in this process? Integrase itself has only a weak nucleotide sequence preference12 so it is much more likely that it is the DNA associated proteins.
Nucleosome structure is important for both ASLV integration, where DNA ligation occurs more efficiently in compact chromatin, and for HIV, where the reverse is true.13 Addition of the transcription factor HNF3 inhibited ASLV end joining at the HNF3 sites.13 This together with previous data that showed that ASLV integration does not favour transcriptionally active sites14 would have predicted the Mitchell findings and it is nice to see this so comprehensively confirmed.
Could covalent modifications of Histone proteins be important for target site selection? Lysine 4 and 9 of H3 are methylated at transcriptional start sites15 and both H3 and H4 have varying acetylation patterns depending on whether they are in open reading frames or intergenic regions16 Other chromosomal proteins also redistribute with transcription and at least one, the INI/SNF5 complex, has been shown to interact with HIV-1 integrase.17 Any or all of these factors (and others) might affect integration site choice.
What are the implications for gene therapy?
Mitchell and co-workers suggest that the large number of intergenic integrations seen with ASLV might make it a good gene vector candidate since it could avoid the risk of insertional mutagenesis. The assumption that noncoding regions are ‘safe’ and do not play a significant role in cellular regulation is however not completely secure and there is good published data on their importance in influencing heterochromatin formation (for reviews see Henikoff,18 Fischle et al and 19 Maison and Almouzni20). Retrotransposons, endogenous relatives of retroviruses, certainly do affect heterochromatin formation.21, 22 Thus noncoding regions might play important regulatory roles in the genome.
There are also doubts about the subsequent transcriptional activity of an ASLV inserted in an intergenic region. Spreading chromatinization might silence the provirus. So multiple integrations might be needed to achieve at least one that is regularly transcribed, which in turn make it more likely that one of these would affect a critical site.
Overall, Mitchell and co-workers have completed a well-executed and comprehensive study that adds to our knowledge of retroviral integration and the factors that affect it. Bushman's group is one of the major players in this field and the work takes us one step further towards understanding and ultimately controlling retroviral integration. However, there is more to be discovered before we can make definitive decisions about our choice of retroviral gene vectors. It is perhaps significant that despite the apparent predilection of HIV for transcriptionally active sites, and its abundance, only one case of probable insertional mutagenesis has been documented reasonably securely23 and a second with another lentivirus, Feline immunodeficiency virus.24 Many of the target cells for HIV are lymphocytes with a short lifespan, but there are also many infected longer lived cells of the monocyte macrophage series in every carrier. For this reason, if the virus had some potential for oncogenicity one might have expected this to appear in at least some of the 40 million infected individuals who are each producing millions of integrating viruses each day. Arguably the lytic nature of HIV has concealed such a tendency that might be revealed as they are used as vectors. However, there is no room for complacency. If evidence of insertional mutagenesis from the use of any specific retro or lentiviral vector comes to light subsequently, we will have to think seriously about our choices and indeed whether with our current state of knowledge we can risk any integrating vectors in human studies.
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We thank Professor J Neil for helpful comments. â–ª
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Mok, H., Lever, A. Vector integration: Location, location, location. Gene Ther 12, 1–2 (2005). https://doi.org/10.1038/sj.gt.3302415
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DOI: https://doi.org/10.1038/sj.gt.3302415