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).
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
Aiuti A. Cassani B. et al. 2007"Multilineage hematopoietic reconstitution without clonal selection in ADA-SCID patients treated with stem cell gene therapy." J Clin Invest 117 8 2233 40.
Aiyar A. Hindmarsh P. et al. 1996"Concerted integration of linear retroviral DNA by the avian sarcoma virus integrase in vitro: dependence on both long terminal repeat termini." J Virol 70 6 3571 80.
Alexander B. L. Ali R. R. et al. 2007"Progress and prospects: gene therapy clinical trials (part 1)." Gene Ther 14 20 1439 47.
Anderson W. F. Blaese R. M. et al. 1990"The ADA human gene therapy clinical protocol: Points to Consider response with clinical protocol, July 6, 1990." Hum Gene Ther 1 3 331 62.
Ariumi Y. Serhan F. et al. 2006"The integrase interactor 1 (INI1) proteins facilitate Tat-mediated human immunodeficiency virus type 1 transcription." Retrovirology 3: 47.
Baum C. Kustikova O. et al. 2006"Mutagenesis and oncogenesis by chromosomal insertion of gene transfer vectors." Hum Gene Ther 17 3 253 63.
Beitzel B. Bushman F. 2003"Construction and analysis of cells lacking the HMGA gene family." Nucleic Acids Res 31 17 5025 32.
Blaese R. M. Culver K. W. et al. 1993"Treatment of severe combined immunodeficiency disease (SCID) due to adenosine deaminase deficiency with CD34+ selected autologous peripheral blood cells transduced with a human ADA gene. Amendment to clinical research project, Project 90-C-195, January 10, 1992." Hum Gene Ther 4 4 521 7.
Boese A. Sommer P. et al. 2004"Ini1/hSNF5 is dispensable for retrovirus-induced cytoplasmic accumulation of PML and does not interfere with integration." FEBS Lett 578 3 291 6.
Bushman F. Lewinski M. et al. 2005"Genome-wide analysis of retroviral DNA integration." Nat Rev Microbiol 3 11 848 58.
Bushman F. D. 1994"Tethering human immunodeficiency virus 1 integrase to a DNA site directs integration to nearby sequences." Proc Natl Acad Sci U S A 91 20 9233 7.
Bushman F. D. 2007"Retroviral integration and human gene therapy." J Clin Invest 117 8 2083 6.
Busschots K. Vercammen J. et al. 2005"The interaction of LEDGF/ 75with integrase is lentivirus-specific and promotes DNA binding." J Biol Chem 280(18): 17841-7.
Cherepanov P. Devroe E. et al. 2004"Identification of an evolutionarily conserved domain in human lens epithelium-derived growth factor/transcriptional co-activator 75LEDGF/p75) that binds HIV-1 integrase." J Biol Chem 279(47): 48883-92.
Cherepanov P. Maertens G. et al. 2003"HIV-1 integrase forms stable tetramers and associates with LEDGF/ 75protein in human cells." J Biol Chem 278(1): 372-81.
Ciuffi A. Llano M. et al. 2005"A role for LEDGF/ 75in targeting HIV DNA integration." Nat Med 11(12): 1287-9.
Coffin J. M. Hughes S. H. et al. 1997Retroviruses. Plainview, NY, Cold Spring Harbor Laboratory Press.
Crise B. Li Y. et al. 2005"Simian immunodeficiency virus integration preference is similar to that of human immunodeficiency virus type 1." J Virol 79 19 12199 204.
Daniel R. Katz R. A. et al. 1999"A role for DNA-PK in retroviral DNA integration." Science 284 5414 644 7.
Daniel R. Smith J. A. 2008"Integration site selection by retroviral vectors: molecular mechanism and clinical consequences." Hum Gene Ther 19 6 557 68.
Dave U. P. Jenkins N. A. et al. 2004"Gene therapy insertional mutagenesis insights." Science 303(5656): 333.
Deichmann A. Hacein-Bey-Abina S. et al. 2007"Vector integration is nonrandom and clustered and influences the fate of lymphopoiesis in SCID-X1 gene therapy." J Clin Invest 117 8 2225 32.
Derse D. Crise B. et al. 2007"Human T-cell leukemia virus type 1 integration target sites in the human genome: comparison with those of other retroviruses." J Virol 81 12 6731 41.
Breckpot "Lentiviral vectors in gene therapy: their current status and future potential." Arch Immunol Ther Exp (Warsz) Escors D. K. 58 582 2 107 19.
Farnet C. M. Bushman F. D. 1997"HIV-1 cDNA integration: requirement of HMG I(Y) protein for function of preintegration complexes in vitro." Cell 88 4 483 92.
Faschinger A. Rouault F. et al. 2008"Mouse mammary tumor virus integration site selection in human and mouse genomes." J Virol 82 3 1360 7.
Ferris A. L. Wu X. et al. 2010"Lens epithelium-derived growth factor fusion proteins redirect HIV-1 DNA integration." Proc Natl Acad Sci U S A 107 7 3135 40.
Flint S. E. Racaniello V. R. et al. 2004Principles of Virology. Washington, D.C., ASM Press.
Gijsbers R. Ronen K. et al. 2010"LEDGF hybrids efficiently retarget lentiviral integration into heterochromatin." Mol Ther 18 3 552 60.
Goulaouic H. Chow S. A. 1996"Directed integration of viral DNA mediated by fusion proteins consisting of human immunodeficiency virus type 1 integrase and Escherichia coli LexA protein." J Virol 70 1 37 46.
Hacein-Bey-Abina S. Von C. Kalle et. al 2003"LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1." Science 302 5644 415 9.
Hahn W. C. Weinberg R. A. 2002"Rules for making human tumor cells." N Engl J Med 347 20 1593 603.
Hematti P. Hong B. K. et al. 2004"Distinct genomic integration of MLV and SIV vectors in primate hematopoietic stem and progenitor cells." PLoS Biol 2(12): e423.
Hindmarsh P. Ridky T. et al. 1999"HMG protein family members stimulate human immunodeficiency virus type 1 and avian sarcoma virus concerted DNA integration in vitro." J Virol 73 4 2994 3003.
Holman A. G. Coffin J. M. 2005"Symmetrical base preferences surrounding HIV-1, avian sarcoma/leukosis virus, and murine leukemia virus integration sites." Proc Natl Acad Sci U S A 102 17 6103 7.
Kaiser J. 2009"Gene therapy. Beta-thalassemia treatment succeeds, with a caveat." Science 326 5959 1468 9.
Kalpana G. V. Marmon S. et al. 1994"Binding and stimulation of HIV-1 integrase by a human homolog of yeast transcription factor SNF5." Science 266 5193 2002 6.
Kang Y. Moressi C. J. et al. 2006"Integration site choice of a feline immunodeficiency virus vector." J Virol 80 17 8820 3.
Katz R. A. Skalka A. M. 1994"The retroviral enzymes." Annu Rev Biochem 63 133 73.
Kumar P. P. Mehta S. et al. 2007"SATB1-binding sequences and Alu-like motifs define a unique chromatin context in the vicinity of human immunodeficiency virus type 1 integration sites." J Virol 81 11 5617 27.
Lau A. Swinbank K. M. et al. 2005"Suppression of HIV-1 infection by a small molecule inhibitor of the ATM kinase." Nat Cell Biol 7 5 493 500.
Lee M. S. Craigie R. 1998"A previously unidentified host protein protects retroviral DNA from autointegration." Proc Natl Acad Sci U S A 95 4 1528 33.
Levine F. Friedmann T. 1991"Gene therapy techniques." Curr Opin Biotechnol 2 6 840 4.
Lewinski M. K. Yamashita M. et al. 2006"Retroviral DNA integration: viral and cellular determinants of target-site selection." PLoS Pathog 2(6): e60.
Li L. Olvera J. M. et al. 2001"Role of the non-homologous DNA end joining pathway in the early steps of retroviral infection." Embo J 20 12 3272 81.
Li Z. Dullmann J. et al. 2002"Murine leukemia induced by retroviral gene marking." Science 296(5567): 497.
Lin C. W. Engelman A. 2003"The barrier-to-autointegration factor is a component of functional human immunodeficiency virus type 1 preintegration complexes." J Virol 77 8 5030 6.
Llano M. Saenz D. T. et al. 2006"An essential role for LEDGF/ 75in HIV integration." Science 314(5798): 461-4.
Llano M. Vanegas M. et al. 2004"LEDGF/ 75determines cellular trafficking of diverse lentiviral but not murine oncoretroviral integrase proteins and is a component of functional lentiviral preintegration complexes." J Virol 78(17): 9524-37.
Llano M. Vanegas M. et al. 2006"Identification and characterization of the chromatin-binding domains of the HIV-1 integrase interactor LEDGF/ 75" J Mol Biol 360(4): 760-73.
Mac Neil. A. Sankale J. L. et al. 2006"Genomic sites of human immunodeficiency virus type 2 (HIV-2) integration: similarities to HIV-1 in vitro and possible differences in vivo." J Virol 80 15 7316 21.
Maertens G. Cherepanov P. et al. 2003"LEDGF/ 75is essential for nuclear and chromosomal targeting of HIV-1 integrase in human cells." J Biol Chem 278(35): 33528-39.
Mahmoudi T. Parra M. et al. 2006"The SWI/SNF chromatin-remodeling complex is a cofactor for Tat transactivation of the HIV promoter." J Biol Chem 281 29 19960 8.
Mann R. Mulligan R. C. et al. 1983"Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus." Cell 33 1 153 9.
Masson C. Bury-Mone S. et al. 2007"Ku80 participates in the targeting of retroviral transgenes to the chromatin of CHO cells." J Virol 81 15 7924 32.
Maxfield L. F. Fraize C. D. et al. 2005"Relationship between retroviral DNA-integration-site selection and host cell transcription." Proc Natl Acad Sci U S A 102 5 1436 41.
Mitchell R. S. Beitzel B. F. et al. 2004"Retroviral DNA integration: ASLV, HIV, and MLV show distinct target site preferences." PLoS Biol 2(8): E234.
Modlich U. Kustikova O. S. et al. 2005"Leukemias following retroviral transfer of multidrug resistance 1 (MDR1) are driven by combinatorial insertional mutagenesis." Blood 105 11 4235 46.
Montini E. Cesana D. et al. 2006"Hematopoietic stem cell gene transfer in a tumor-prone mouse model uncovers low genotoxicity of lentiviral vector integration." Nat Biotechnol 24 6 687 96.
Mooslehner K. Karls U. et al. 1990"Retroviral integration sites in transgenic Mov mice frequently map in the vicinity of transcribed DNA regions." J Virol 64 6 3056 8.
Narezkina A. Taganov K. D. et al. 2004"Genome-wide analyses of avian sarcoma virus integration sites." J Virol 78 21 11656 63.
Ott M. G. Schmidt M. et al. 2006"Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertional activation of MDS1-EVI1, PRDM16 or SETBP1." Nat Med 12 4 401 9.
Pike-Overzet K. de Ridder D. et al. 2006"Gene therapy: is IL2RG oncogenic in T-cell development?" Nature 443(7109): E5; discussion E 6 7.
Pruss D. Bushman F. D. et al. 1994"Human immunodeficiency virus integrase directs integration to sites of severe DNA distortion within the nucleosome core." Proc Natl Acad Sci U S A 91 13 5913 7.
Pruss D. Reeves R. et al. 1994"The influence of DNA and nucleosome structure on integration events directed by HIV integrase." J Biol Chem 269 40 25031 41.
Pryciak P. M. Muller H. P. et al. 1992"Simian virus 40 minichromosomes as targets for retroviral integration in vivo." Proc Natl Acad Sci U S A 89 19 9237 41.
Pryciak P. M. Sil A. et al. 1992"Retroviral integration into minichromosomes in vitro." EMBO J 11 1 291 303.
Pryciak P. M. Varmus H. E. 1992"Nucleosomes, DNA-binding proteins, and DNA sequence modulate retroviral integration target site selection." Cell 69 5 769 80.
Recchia A. Bonini C. et al. 2006"Retroviral vector integration deregulates gene expression but has no consequence on the biology and function of transplanted T cells." Proc Natl Acad Sci U S A 103 5 1457 62.
Scherdin U. Rhodes K. et al. 1990"Transcriptionally active genome regions are preferred targets for retrovirus integration." J Virol 64 2 907 12.
Schroder A. R. Shinn P. et al. 2002"HIV-1 integration in the human genome favors active genes and local hotspots." Cell 110 4 521 9.
Schwarzwaelder K. Howe S. J. et al. 2007"Gammaretrovirus-mediated correction of SCID-X1 is associated with skewed vector integration site distribution in vivo." J Clin Invest 117 8 2241 9.
Shun M. C. Raghavendra N. K. et al. 2007"LEDGF/ 75functions downstream from preintegration complex formation to effect gene-specific HIV-1 integration." Genes Dev 21(14): 1767-78.
"Modification of integration site preferences of an HIV-1-based vector by expression of a novel synthetic protein." Hum Gene Ther Silvers R. M. Smith J. A. et al. . 21 213 3 337 49.
Sutherland H. G. Newton K. et al. 2006"Disruption of Ledgf/Psip1 results in perinatal mortality and homeotic skeletal transformations." Mol Cell Biol 26 19 7201 10.
Taganov K. D. Cuesta I. et al. 2004"Integrase-specific enhancement and suppression of retroviral DNA integration by compacted chromatin structure in vitro." J Virol 78 11 5848 55.
Thrasher A. J. Gaspar H. B. 2008"Severe adverse event in clinical trial of gene therapy for X-SCID." Volume, DOI:
Thrasher A. J. Gaspar H. B. et al. 2006"Gene therapy: X-SCID transgene leukaemogenicity." Nature 443(7109): E 5 6; discussion E6-7.
Treand C. du I. Chene et. al 2006"Requirement for SWI/SNF chromatin-remodeling complex in Tat-mediated activation of the HIV-1 promoter." Embo J 25 8 1690 9.
Turlure F. Maertens G. et al. 2006"A tripartite DNA-binding element, comprised of the nuclear localization signal and two AT-hook motifs, mediates the association of LEDGF/ 75with chromatin in vivo." Nucleic Acids Res 34(5): 1653-65.
Vanegas M. Llano M. et al. 2005"Identification of the LEDGF/ 75HIV-1 integrase-interaction domain and NLS reveals NLS-independent chromatin tethering." J Cell Sci 118(Pt 8): 1733-43.
Weidhaas J. B. Angelichio E. L. et al. 2000"Relationship between retroviral DNA integration and gene expression." J Virol 74 18 8382 9.
Woods N. B. Bottero V. et al. 2006"Gene therapy: therapeutic gene causing lymphoma." Nature 440(7088): 1123.
Wu X. Li Y. et al. 2003"Transcription start regions in the human genome are favored targets for MLV integration." Science 300 5626 1749 51.