1. Introduction
Retroviral vectors have gained an increasing value in gene therapy because they stably deliver therapeutic genes to the host cell genome. These therapeutic genes are supposed to rectify consequences of inherited and acquired mutated genes in the host cell genome, or alter host cell function to cure diseases. In the following section we will discuss the biology and life cycle of retroviruses which starts with viral entry into the host cell, reverse transcription of viral RNA, nuclear import of the provirus, and finally integration of viral DNA into the cell host genome (Flint, Racaniello et al. 2004). Integration involves viral and host cellular proteins. Their role is discussed in the third and fourth sections of this chapter. Recently, the process of integration site selection (which is where the viral DNA integrates with the host cell DNA) has been quite understood throughout many
1.1. Retrovirus structure and life cycle
Viruses are obligate parasites which depend on living cells to multiply. Their ability to deliver stable RNA and DNA into cells has determined their use in gene therapy. In 1983 Mann et al. developed one of the first retroviral gene therapy vectors for delivery
Retroviruses belong to the
The life cycle of the retrovirus consist of several steps. It begins with the binding of the viral envelope to cellular receptors, which enables fusion of the viral envelope with the cellular membrane. Consequently, the viral particle is uncoated, liberating the viral core into the cell cytoplasm. The viral DNA is reverse transcribed to DNA. Then, the viral DNA is transported to the nucleus where it is integrated into the host cell’s genome. From there, viral DNA is transcribed to RNA, some of which is translated to proteins. The viral RNA is packed in a viral particle along with viral proteins. Then, virion is produced when viral particles bud from the hosting cells (Escors and Breckpot).
1.2. Integration
The retroviral enzyme integrase (IN) plays a vital role in integration. It exists as a tetramer (dimer-of-dimers) inside the virion or the preintegration complex. IN facilitate viral DNA integration
In summary, retroviral DNA integration is catalyzed by the viral protein integrase, but host cell proteins play a significant role in enhancing the efficiency of the reaction, and preventing autointegration.
2. Integration site preferences of retroviruses and retroviral vectors
While Integration of viral DNA can take place anywhere in the host cell genome and there is no strict host sequence for site selection, many studies showed that site selection is not a haphazard process (Schroder, Shinn et al. 2002; Wu, Li et al. 2003; Mitchell, Beitzel et al. 2004). In vitro studies demonstrated that some DNA-binding proteins can prevent contact of IN to target DNA and subsequently block the integration reaction at their binding sites (Pryciak and Varmus 1992; Bushman 1994). On the contrary, bending or distortion of DNA seems to enhance integration (Pryciak, Muller et al. 1992; Pryciak and Varmus 1992; Katz and Skalka 1994; Pruss, Bushman et al. 1994; Pruss, Reeves et al. 1994). Furthermore, studies showed that DNA wrapping around nucleosomes promotes distortion of DNA and thus promotes integration in the nucleosomes-bound DNA (Pryciak, Sil et al. 1992; Pryciak and Varmus 1992; Pruss, Bushman et al. 1994). All of the previous studies show that there are certain integration site preferences in DNA substrate in
Avian retroviruses and vectors show only a weak preference for integration around genes (about 40%) and no MLV-like preference for 5’ ends of transcription units (Mitchell, Beitzel et al. 2004; Narezkina, Taganov et al. 2004). Interestingly, high levels of transcription may even inhibit ASV integration in genes (Weidhaas, Angelichio et al. 2000; Maxfield, Fraize et al. 2005). These preferences are consistent with the above-described data from the in vitro system, which used nucleosomal arrays (Taganov, Cuesta et al. 2004). Interestingly, the human T-leukemia virus type 1 (HTLV-1) and mouse mammary tumor virus (MMTV), like avian retroviruses, do not specifically target genes and transcription start sites (Derse, Crise et al. 2007; Faschinger, Rouault et al. 2008).
Lastly, it appears that there is a symmetric base preferences surrounding integration sites for integration of HIV-1, SIV, MLV, and avian sarcoma-leukosis viruses (Crise, Li et al. 2005; Holman and Coffin 2005). These weak consensus sequences are virus specific and possibly reflect the influence of IN on integration site selection (Holman and Coffin 2005). This proposal is supported by the symmetry of the target site sequence, because IN likely functions as a tetramer (Coffin et al., 1997; Flint et al., 2004; Wu et al., 2005; and see above).
In summary, the integration preferences described in this section are distinct for different group of retroviruses. The first group including HIV-1, HIV-2, SIV, and FIV, show a preferential integration into genes (Daniel and Smith 2008). While the second group, consisting of MLV and FV, integrate in 5' ends of transcription units and CpG islands. The last group consists of AVLS, HTLV-1, and MMTV (Daniel and Smith 2008). This group shows weak or even no preferences for gene or transcription start sites. Also, it appears that DNA sequence has a role in integration site selection. However, other factors (cellular cofactors and cellular structures) are likely to be the principal controllers of integration site selection.
3. Mechanism of integration site selection
As mentioned before, IN has a low specificity for binding to host cell DNA. So, it seems that host cell proteins participate in the integration process. Using the yeast two-hybrid system, Debyser and coworkers have identified a new HIV-1 IN-binding protein, termed LEDGF/p75 (Cherepanov, Maertens et al. 2003). LEDGF/p75 is required for efficient integration of HIV-1 DNA. Also, LEDGF/p75 is a transcription factor and has a C-terminal IN-binding domain and N-terminal chromatin-binding domain (Cherepanov, Maertens et al. 2003; Cherepanov, Devroe et al. 2004; Vanegas, Llano et al. 2005; Llano, Vanegas et al. 2006; Turlure, Maertens et al. 2006). Chromatin binding is mediated by PWWP and AT-hook motifs in the N-termianl domain of LEDGF/p75 (Llano, Vanegas et al. 2006; Turlure, Maertens et al. 2006). In addition, LEDGF/p75 was detected in association with preintegration complexes of HIV-1 and FIV in cultured cells (Llano, Vanegas et al. 2006). Moreover, LEDGF/p75 halts proteasomal degradation of ectopically expressed HIV-1 IN, therefore it might assist to the stability of preintegration complexes during infection (Maertens, Cherepanov et al. 2003; Llano, Vanegas et al. 2006). Also, LEDGF/p75 null cells showed that the residual integration sites in these cells no longer take place in active genes (Shun, Raghavendra et al. 2007). However, integration occurred preferentially near promoters and CpG islands (Shun, Raghavendra et al. 2007). The symmetric base preferences surrounding the integration site remained preserved (Holman and Coffin 2005). As a result, in the absence of LEDGF/p75, HIV-1 integration site preferences resemble those of MLV (Shun, Raghavendra et al. 2007). All these results strongly support the hypothesis that LEDGF/p75 targets HIV-1 (and other lentiviral) integration into active genes by tethering the IN protein to chromatin.
Although LEDGF/p75 appears to be a major HIV-1 IN-binding cellular protein, other factors are likely involved in integration site selection by HIV-1 and HIV-1-based vectors. Analysis of robust number of integration sites demonstrated that preferred integration sites are found in the vicinity of certain computer-predicted epigenetic marks, such as histone H3 K4 methylation, H4 acetylation, or H3 aceytlation (Kalpana, Marmon et al. 1994). These results may suggest that the chromatin structure, including the histone code, may also affect integration site selection. However the decisive evidence that these marks play a role in integration site selection has yet to be revealed. Moreover, other factors which affect integration site selection have been identified. Knockdown of the T-cell lineage-specific chromatin organizer, SATB1 (special AT-rich sequence-binding protein-1), reduces HIV-1 integration in the vicinity of SATB1-binding sites (Kumar, Mehta et al. 2007). Consequently, SATB1 seems to be implicated in integration site selection by an unknown mechanism. Lastly, it has been shown that the cellular protein Ku80, which is present in the preintegration complex, directs integration to chromatin domains prone to silencing (Li, Olvera et al. 2001; Masson, Bury-Mone et al. 2007). In contrast to HIV-1, integration of MLV-based and ASV-based vectors does not seem to be determined by LEDGF/p75 (Mitchell, Beitzel et al. 2004; Narezkina, Taganov et al. 2004). It is still unknown what controls ASV integration site selection. While in the case of MLV, a study using HIV chimeras with MLV genes demonstrated that MLV IN appears to be the major director for integration site selection (Lewinski, Yamashita et al. 2006). Furthermore, Gag-derived proteins play an auxiliary role in the integration selection process, as an HIV-1 chimera with MLV Gag demonstrated other site preferences different from both HIV and MLV (Lewinski, Yamashita et al. 2006). All the previous data support a different mechanism of integration site selection for MLV versus HIV.
In conclusion, current data has promoted our understating of the retroviral site selection process and demonstrates a major role of host cell proteins in the process. Yet, the process is not entirely understood, and there will likely be new determinate members involved in the retroviral integration site selection process revealed in the near future.
4. Integration site selection and gene therapy
MLV and HIV-1 vectors are the two most widely used vectors in gene therapy. It was hypothesized that even if a retroviral vector integrates in the "wrong spot", it may not necessarily lead to the development of a tumor (Hahn and Weinberg 2002; Baum, Kustikova et al. 2006). However, this hypothesis was challenged when serious adverse effects emerged in gene therapy trials involving children to treat X-linked severe combined immunodeficiency (SCID-X1) (Hacein-Bey-Abina, Von Kalle et al. 2003; Alexander, Ali et al. 2007; Bushman 2007; Deichmann, Hacein-Bey-Abina et al. 2007; Faschinger, Rouault et al. 2008). In one these trials, which used an MLV-based vector, 4 out of 11 patients developed T cell leukemia. Moreover, in another SCID-X1 gene therapy trial, it has been reported that a patient, of 10 patients enrolled, developed leukemia (Alexander, Ali et al. 2007; Schwarzwaelder, Howe et al. 2007; Thrasher and Gaspar 2008). Using sequencing analysis, T cells from two of the patients in the first trial who developed leukemia, showed an insertion of the vector near (and subsequent activation of) Lin-1, IsI-1, Mec-3 (LIM) domain only-2 (LMO2) protooncogene by the long terminal repeat (LTR) enhancer of the vector (Hacein-Bey-Abina, Von Kalle et al. 2003). Also, in the second trial, the vector insertion was in the vicinity of the LMO2 protooncogene (Thrasher and Gaspar 2008). These striking data demonstrate that vector integration at a dangerous spot of the human genome could lead to cancer development. It is also true that there could be other unknown factors that contributed to the leukemia development. Proposed factors that may have been involved are expression of the transgenes and chromosomal rearrangement (Hacein-Bey-Abina, Von Kalle et al. 2003; Pike-Overzet, de Ridder et al. 2006; Thrasher, Gaspar et al. 2006; Woods, Bottero et al. 2006). A follow-up analysis of the patients of these gene therapy trials exhibited a nonrandom distribution of integration sites
In conclusion, integration of a retroviral vector into the human genome contributed to the development of leukemia both in animal models and human patients. Nevertheless, these insertions may not be directly involved in cancer development, few patients of gene therapy trials developed malignancies (Hacein-Bey-Abina, Von Kalle et al. 2003; Dave, Jenkins et al. 2004). These cases emphasize the need for further improvements of retroviral vector designs to obtain vectors with low preferences for “wrong spots” to increase the safety margin in gene therapy applications.
5. Retargeting integration
The hypothetical need for integration targeting was realized even prior to the adverse events described above. Thus, attempts to target integration were made in the last decade of the 20th century. These attempts involved attaching a specific DNA binding domain (binding to a known DNA sequence) to the retroviral integrase protein. It had been shown that these fusion proteins target integration
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