The Long Terminal Repeat Negative Control Region Is a Critical Element

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SHORT COMMUNICATION The long terminal repeat negative control region is a critical element for insertional oncogenesis after gene transfer into hematopoietic progenitors with Moloney murine leukemia viral vectors

Y Ikawa1,2, T Uchiyama1,3, GJ Jagadeesh1 and F Candotti1,4

Integrating vectors based on γ-retroviruses and containing full-length long terminal repeats (LTRs) have been associated with activation of oncogene expression and leukemogenesis in human gene therapy trials. Identification of the specific molecular elements of the LTRs that have a role in insertional oncogenesis events is important as it can lead to the development of safer gene transfer vectors. The negative control region (NCR) of the LTR is a particularly well-conserved sequence among mammalian γ-retroviruses with demonstrated regulatory activity of gene transcription in hematopoietic cells, which led us to hypothesize that this region may have a role in insertional oncogenesis after γ-retroviral vector (GV)-mediated gene transfer into hematopoietic progenitors. We used an in vitro assay of murine bone marrow cell immortalization to compare the immortalization capabilities of a series of GVs carrying murine leukemia virus (MLV) LTR deletion mutants. Compared with GV carrying the full-length MLV LTR, deletion of the complete LTR enhancer sequence showed significant reduction of immortalization rates. However, the use of a mutant LTR deleted of the enhancer sequence, with exception of the NCR, did not affect immortalization. Importantly, the inclusion of an LTR mutant devoid only of the NCR did show significant reduction of immortalization rates compared with the full LTR sequence. Therefore, our data point to the NCR as a key element for immortalization and justify additional studies to evaluate its specific role in MLV-mediated insertional oncogenesis.

Gene Therapy (2016) 23, 815–818; doi:10.1038/gt.2016.51; published online 4 August 2016

INTRODUCTION and the embryonal long terminal repeat-binding protein.8–11 The Gene transfer into hematopoietic stem cells has been used deletion of the NCR sequence attenuates the transcription activity 11 successfully to treat a variety of human genetic diseases.1–3 of GVs in hematopoietic cells. Moreover, a point mutation Although positive clinical results have been achieved, the success abolishing binding of NFAT has been reported to reduce the LTR 11 of clinical trials has been tempered by adverse events in which the enhancer activity. integration of gene transfer vectors including enhancer regions In this study, GVs carrying LTR deletion mutants were generated from viral long terminal repeats (LTRs) resulted in increased with the aim of studying the role of the NCR element in the transcription of cancer-related genes and thereby contributed to phenomenon of in vitro cell immortalization induced by GV development of leukemia.4,5 Previously, we have studied the integration into murine hematopoietic progenitor cells. Lentivirus- differences in cell immortalization ability of γ-retroviral vectors (LV) and foamy virus- (FV) based constructs were also evaluated (GVs) either carrying the full-length murine leukemia virus (MLVﬂ) for comparison using the same immortalization assay. LTR or a self-inactivating LTR mutant (MLV-SIN) deleted of most of Our results indicate that the NCR is an important element for the U3 enhancer element.6 Such experiments showed that there hematopoietic cell immortalization and therefore suggest that it was no statistical difference in cell immortalization rate between may be an ideal target for the generation of gene transfer vectors the full-length MLV LTR vector and MLV-SIN vector, suggesting maintaining the favorable characteristics of GVs, but less prone to that the remaining sequences of the MLV-SIN-deleted LTR or other causing insertional oncogenesis. components of our MLV-SIN vector (e.g. the PGK internal promoter) might contain elements with gene transcription- enhancing capabilities.6,7 RESULTS AND DISCUSSION The MLV negative control region (NCR) is a highly conserved We used the in vitro assay of murine bone marrow (BM) cell negative regulatory element located near the 5′ end of the MLV immortalization that we have previously reported6 to compare the LTR.8 Its sequence features binding sites for the transcription effects of the MLVﬂ to four different variants featuring either (1) factor Ying-Yang 1, the nuclear factor of activating T cells (NFAT) the deletion of most of the two 75-bp repeats associated with the

1Genetics and Molecular Biology Branch, National Human Genome Research Institute (NHGRI), Bethesda, MD, USA; 2Department of Pediatrics, Kanazawa University Hospital, Kanazawa, Japan; 3Department of Human Genetics, National Research Institute for Child Health and Development, Tokyo, Japan and 4Division of Immunology and Allergy, University Hospital of Lausanne, Lausanne, Switzerland. Correspondence: Professor F Candotti, Division of Immunology and Allergy, University Hospital of Lausanne, Rue du Bugnon 46, BH10.527, Lausanne 1011, Switzerland. E-mail: [email protected] Received 6 January 2016; revised 11 May 2016; accepted 27 May 2016; published online 4 August 2016 Key role of NCR in insertional oncogenesis Y Ikawa et al 816 immortalization and that NFAT binding to the region is not indispensable for the occurrence of such phenomenon. As additional controls, we performed a series of transductions with LV and FV vectors (57 and 34 cultures, respectively), which failed to generate any immortalized lines (Figure 2a). These findings were consistent with the notion that LV and FV backbones constitute safer gene transfer vector alternatives to GVs.13–16 However, it remained unclear whether the lack of cell immortalization capabilities had to be attributed to the reduced enhancer activities of their LTRs17,18 or to their known different integration profile compared with GVs.19,20 To begin to dissect these possibilities, we generated an LV construct carrying the U3 region from the murine stem cell virus (MSCV) as an internal promoter (LV/U3). Transductions of 128 cultures with the LV/U3 vector resulted only in two immortalized lines, which was not statistically different from the results obtained with LV vector carrying the UCOE internal promoter (Figure 2a). These results indicate that, when integrating in the context of an LV, the immortalization potentials of GV-derived enhancers are markedly reduced and suggest that the integration pattern of LVs negatively affects the GV enhancer capabilities to deregulate the expression of genes that are responsible for murine hematopoietic progenitor cell immortalization. We speculate that the reduced propensity of LVs to integrate near-gene transcription start sites, Figure 1. Schematic representation of GVs and MLV LTR mutants as well as cancer-related genes,16 is an important contributing used in this study. All vectors carried an eGFP expression cassette factor to these observations. under the transcriptional control of the UCOE promoter. The location Because robust transgene expression is generally a desirable of the TATA and CCAAT homology sequences and of relevant restriction endonuclease sites (NheI, PvuII, XbaI and SacI) are shown. outcome, we compared EGFP expression levels obtained after ELP, embryonal long terminal repeat-binding protein; C/EBP, ccaat- gene transfer with different constructs by dividing the EGFP mean enhancer-binding protein; UCOE, ubiquitously acting chromatin fluorescence intensity by vector copy number observed in the opening element; YY1, Yin Yang 1. transduced cell populations (Figure 2b). GV mutants revealed relatively higher expression level compared with LV and FV constructs, which can reduce the overall effectiveness of these MLV viral enhancer (delE1),6 (2) the complete deletion of the two safer gene transfer vectors. However, the LV/U3 vector showed 75-bp repeats and of the NCR (delE2), (3) the deletion of the NCR similar expression levels as the delNCR construct. These data only (delNCR) or (4) carrying a previously described12 deleterious suggest that the LV/U3 vector design could represent a suitable mutation of the NCR NFAT binding motif (ΔNFAT) (Figure 1). compromise between safety features and gene expression We also studied the immortalization effects of an LV- and an FV- potential. based construct. All vectors carried an internal expression cassette Taken together, our results confirm that GVs are prone to including the eGFP gene under the control of a UCOE promoter causing immortalization of hematopoietic cells and indicate that, (Figure 1). Real-time PCR was performed to verify that exposure to while resulting in significantly lower immortalization rate, the different vectors resulted in comparable vector copy numbers in deletion of the whole viral enhancer sequences does not transduced BM cells. As described, in our assay, derived murine completely eliminate the insertional oncogenesis risk. hematopoietic cell lines were considered immortalized based on Importantly, our data point to the NCR as a crucial element for their ability to continue to grow in vitro for more than 6 weeks in immortalization. Previous studies11 have demonstrated that 6 the presence of interleukin-3 and stem cell factor. deletion of the NCR sequence attenuated the transcription activity Transduction of 92 murine hematopoietic stem cells cultures of GV in hematopoietic cells. In addition, reduction of NFAT with the MLVfl vector gave rise to 37 immortalized lines (40%). binding the NCR has been shown to reduce the LTR enhancer Exposure of 108 cultures to the delE1 vector resulted also in 37 activity.11 Therefore, we can speculate that our constructs devoid immortalized lines and an immortalization rate of 34% (Figure 2a). of the NCR have similar reductions in the LTR enhancer activity, The difference in immortalization rate between these two vectors which may reduce transactivation of nearby genes and thus was not statistically significant, which confirms our previous cellular immortalization. 6 observations. The delE2, delNCR and ΔNFAT vectors were used to However, the precise mechanisms by which the MLV NCR transduce 60, 36 and 35 cultures, and resulted in 10, 3 and 10 increases cell immortalization within GVs remain unclear. Similarly, immortalized lines with immortalization rates of 17%, 8.3% and whether or not similar effects are elicited by NCRs from other viral 29%, respectively. The rate of immortalization induced by the LTRs that have been involved in insertional oncogenesis delE2 vector was significantly lower compared with that (e.g. spleen focus-forming virus) is unknown and deserves to be generated by the MLVfl and delE1 vectors (Po0.01, Po0.05). explored, which would allow to determine if our findings are Similarly, the immortalization events resulting from transductions restricted to MLV or can be generalized to other γ-retroviruses. with the delNCR vector were significantly lower compared with Despite these limitations, our observations identify the NCR as a those generated by the MLVfl and delE1 vectors (Po0.01, possible target for safety modification of gene transfer vectors Po0.01). On the contrary, we did not observe significant that may use the MLV LTR. differences between the immortalization rates induced by the Finally, our results confirm that vectors based on LV backbones MLVfl and ΔNFAT vectors (Figure 2a). These data indicate that the carrying strong internal promoter may be a safe and effective NCR element is important for GV-mediated hematopoietic cell alternative for clinical gene transfer into hematopoietic stem cells.

Figure 2. (a) Results of murine bone marrow cell immortalization assay performed with different GVs, LVs and FVs. The number of transduced cultures, transduction efficiencies and immortalization rates are indicated. *Po0.01; **Po0.05; NS, not significant. (b) EGFP transgene expression levels obtained from different GVs, LVs and FVs. The number of transduced cultures and vector copy numbers are as in panel a. MFI (mean fluorescent intensity) values are indicated.

MATERIALS AND METHODS Amicon Ultra-15 100 kDa Ultracel-100 filter columns (Millipore, Vector design Bedford, MA, USA), aliquoted and stored at − 80 °C, until used. FV All the GV mutants were generated starting from the pMSCVNeo vector particles were produced by calcium phosphate transfection vector (Clontech Laboratories, Mountain View, CA, USA), which of 293T cells with the resultant FV.UCOE.GFP gene transfer vector ′ ′ and the three helper plasmids (pCinES, pCinGS and pCinPS), as carries the 5 -LTR of MSCV and the 3 -LTR of MLV. The GV mutants 14 with deletion enhancer portions were constructed as indicated in described previously. The culture supernatant was harvested fi Figure 1. All the LV mutants were generated starting from the pHR after 48 and 72 h and concentrated by lter column. Titers of all ′SINcPPT-SE lentiviral vector, which was a kind gift from Dr Adrian the mutants were measured by GFP expression in NIH3T3 cells J Thrasher (University College London, London, UK). The FV.UCOE. 48 h after transduction with serial dilutions of vectors. GFP vector was generated from the pΔФ foamy viral vector plasmid. FV plasmids, pΔФ, pCinES, pCinGS and pCinPS, were Isolation of lineage-negative BM cells and transduction protocol kindly provided by Dr David W Russell (University of Washington, BM cells were isolated from tibias and femurs of donor C57BL6 Seattle, WA, USA). A 2.6 kb fragment of the ubiquitously acting mice (8–20 weeks old). Upon red blood cells lysis (ACK lysis buffer; chromatin-opening element from the human HNRPAB1-CBX3 Biofluids, Rockville, MD, USA), lineage-negative BM cells were locus (A2UCOE) was also a kind gift from Dr Adrian J Thrasher isolated by negative selection using magnetic sorting and lineage- (University College London). A 631 bp BspE1 and TthIIII restriction specific antibodies (Miltenyi Biotec, Auburn, CA, USA). Lineage- fragment of such a region (UCOE) with unidirectional transcrip- negative BM cells were plated at a concentration of 1 × 106/ml and tional activity14 was inserted into all the GV, LV and FV mutants as prestimulated for 48 h in Dulbecco’s modified Eagle’s medium an internal promoter upstream of the enhanced green fluores- (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal cence protein (GFP) cDNA (Addgene Inc., Cambridge, MA, USA). bovine serum (Gemini Bioproducts, Woodland, CA, USA), PvuII and AscI restriction fragments of the MSCV U3 region were 2 mmol l − 1 glutamine (Invitrogen), 50 μgml− 1 gentamicin (Invi- isolated from the 5′-LTR of pMSCVNeo (Clontech Laboratories) and trogen) in the presence of human interleukin-6 (10 ng ml − 1), inserted into pHR′SINcPPT-SE as an internal promoter upstream of murine interleukin-3 (6 ng ml − 1) and murine stem cell factor the GFP cDNA sequence. (100 ng ml − 1) (all from Peprotech, Rocky Hill, NJ, USA). Virus preloading was carried out on 6-well plates coated with Retro- − 2 Production of vector supernatants Nectin (rFN-CH-296) (10 μgcm ; Takara Bio USA, Madison, WI, Ecotropic GV supernatants were produced as previously described USA). Plates were spun in a centrifuge prewarmed to 32 °C for 2 h 21 at 32 °C (1500 g) to facilitate binding of virus particles to using GP+E86 packaging cell line. Lentiviral vectors pseudo- 5 typed with the vesicular stomatitis G protein were generated by RetroNectin. Prestimulated lineage-negative BM cells (5 × 10 /ml) transfection with Lipofectamine 2000 (Life Technologies, Rockville, were plated on preloaded plates and centrifuged again for 10 min MD, USA) into 293T cells of the pCMVDR8.91 packaging construct, at 32 °C at 300 g. the pMD.G plasmid expressing the vesicular stomatitis G protein envelope, and the LV transfer vectors as described previously.19 Culture conditions Culture medium was collected at 48 and 72 h after transfection, Two days after transduction, BM cells were collected, plated in 12- pooled, 0.45 μM filtered, concentrated ~ 50-fold by Millipore well plates and resuspended in ‘long-term culture medium’,

© 2016 Macmillan Publishers Limited, part of Springer Nature. Gene Therapy (2016) 815 – 818 Key role of NCR in insertional oncogenesis Y Ikawa et al 818 consisting of Iscove’s modified Dulbecco’s medium supplemented 3 Candotti F, Shaw KL, Muul L, Carbonaro D, Sokolic R, Choi C et al. Gene therapy for − with 20% horse serum (Life Technologies), 2 mmol l 1 glutamine, adenosine deaminase-deficient severe combined immune deficiency: clinical 50 ng ml − 1 gentamicin, murine interleukin-3 (10 ng ml − 1) and comparison of retroviral vectors and treatment plans. Blood 2012; 120: murine stem cell factor (100 ng ml − 1), as described previously.20 3635–3646. BM cells were passaged every 3–4 days and cytokines were added 4 Braun CJ, Boztug K, Paruzynski A, Witzel M, Schwarzer A, Rothe M et al. Gene — fi to the media. At each passage, 80% of the cells were discarded therapy for wiskott-Aldrich syndrome ong-term ef cacy and genotoxicity. Sci Transl Med 2014; 6: 227ra33. and 20% of cells were kept in culture. After 6 weeks of culture, the 5 Hacein-Bey-Abina S, Von Kalle C, Schmidt M, McCormack MP, Wulffraat N, phenotype of immortalized BM cells was evaluated by Leboulch P et al. LMO2-associated clonal T cell proliferation in two patients after fluorescence-activated cell sorting. Transduction efficiency was gene therapy for SCID-X1. Science 2003; 302: 415–419. calculated by fluorescence-activated cell sorter analysis of GFP 6 Bosticardo M, Ghosh A, Du Y, Jenkins NA, Copeland NG, Candotti F. expression. All procedures using experimental animals were Self-inactivating retroviral vector-mediated gene transfer induces oncogene carried out according to approved guidelines of the National activation and immortalization of primary murine bone marrow cells. Mol Ther Human Genome Institutes Animal Care and Use Committee. 2009; 17: 1910–1918. 7 McBurney MW, Sutherland LC, Adra CN, Leclair B, Rudnicki MA, Jardine K. 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