Toward Position-independent Retroviral Vector Expression in Pluripotent Stem Cells

Transgene silencing has remained an important obstacle to efficient retroviral gene transfer into pluripotent stem cells (PSCs) and their progeny, despite considerable efforts to identify and delete vector elements responsible for this process.1Ellis J Silencing and variegation of gammaretrovirus and lentivirus vectors.Hum Gene Ther. 2005; 16: 1241-1246Crossref PubMed Scopus (284) Google Scholar,2Barklis E Mulligan RC Jaenisch R Chromosomal position or virus mutation permits retrovirus expression in embryonal carcinoma cells.Cell. 1986; 47: 391-399Abstract Full Text PDF PubMed Scopus (128) Google Scholar One approach to this problem is the incorporation of genetic elements into the vector that are capable of shielding proviral DNA from molecular and epigenetic reactions associated with silencing. In this issue of Molecular Therapy, Rival-Gervier et al., from James Ellis's group in Toronto,3Rival-Gervier S Lo MYM Khattak S Pasceri P Lorincz MC Ellis J Kinetics and epigenetics of retroviral silencing in mouse embryonic stem cells defined by deletion of the D4Z4 element.Mol Ther. 2013; 21: 1536-1550Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar describe a ∼1-kilobase (kb) region in the 3′ end of the D4Z4 insulator element, which, when combined with the chicken β-globin hypersensitive site 4 (cHS4) insulator, very effectively prevents retroviral transgene silencing in PSCs. Insertion site and chromatin analysis further demonstrated that this effect was not explained by the targeting of vector integrations into favorable epigenetic domains but was instead caused by preventing the spread of heterochromatin marks such as DNA methylation and repressive histone modifications, thus directly protecting the transgene from silencing. Therefore, the study not only identifies a domain within the D4Z4 element crucial for its activity but also demonstrates how it can be combined with another element to develop a strategy for the prevention of retroviral transgene silencing in PSCs. Although PSCs are promising tools for disease modeling, developmental studies, drug testing, regenerative medicine, and cell-based gene therapy strategies, development of efficient and safe methods of gene correction in PSCs remains a challenging hurdle. Integration of gammaretroviral and lentiviral vectors has been widely used to genetically manipulate PSCs, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). However, transcriptional silencing by epigenetic mechanisms is frequently observed with retroviral vectors1Ellis J Silencing and variegation of gammaretrovirus and lentivirus vectors.Hum Gene Ther. 2005; 16: 1241-1246Crossref PubMed Scopus (284) Google Scholar,2Barklis E Mulligan RC Jaenisch R Chromosomal position or virus mutation permits retrovirus expression in embryonal carcinoma cells.Cell. 1986; 47: 391-399Abstract Full Text PDF PubMed Scopus (128) Google Scholar and can substantially compromise long-term transgene expression. First-generation gammaretroviral vectors containing intact long terminal repeats (LTRs) are particularly prone to silencing in PSCs, as was elegantly exploited in Yamanaka's seminal reprogramming work4Takahashi K Yamanaka S Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell. 2006; 126: 663-676Abstract Full Text Full Text PDF PubMed Scopus (18864) Google Scholar to extinguish expression of the exogenous reprogramming factors following establishment of the pluripotent state. Retroviral silencing is dependent on both chromosomal position effects introduced by the semirandom nature of retroviral integration and intrinsic vector sequences, such as the LTRs or the primer binding site (PBS), which corresponds to the RNA element to which transfer RNA binds to initiate reverse transcription. In PSCs, silencing occurs following deposition of repressive histone marks. These result from complex molecular and epigenetic interactions involving the recruitment of repressive complexes and factors including Trim28 (Kap1), histone methyltransferases, the nucleosome and histone deacetylase complex, or the nuclear receptor corepressor complex 1 as well as DNA methylation of CpG-rich sequences by methyltransferases Dnmt3a/3b (refs. 1Ellis J Silencing and variegation of gammaretrovirus and lentivirus vectors.Hum Gene Ther. 2005; 16: 1241-1246Crossref PubMed Scopus (284) Google Scholar, 3Rival-Gervier S Lo MYM Khattak S Pasceri P Lorincz MC Ellis J Kinetics and epigenetics of retroviral silencing in mouse embryonic stem cells defined by deletion of the D4Z4 element.Mol Ther. 2013; 21: 1536-1550Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 5Yao S Sukonnik T Kean T Bharadwaj RR Pasceri P Ellis J Retrovirus silencing, variegation, extinction, and memory are controlled by a dynamic interplay of multiple epigenetic modifications.Mol Ther. 2004; 10: 27-36Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). This highly complex process is not stationary, however, and, particularly after retroviral integration, it can progress to neighboring regions, a phenomenon called heterochromatin spread. In this context, Wolf and Goff—more than a decade after the initial observation of silencing and the first tentative link of specific vector regions to this effect6Grez M Akgun E Hilberg F Ostertag W Embryonic stem cell virus, a recombinant murine retrovirus with expression in embryonic stem cells.Proc Natl Acad Sci USA. 1990; 87: 9202-9206Crossref PubMed Scopus (168) Google Scholar—demonstrated that a repressive TRIM28 complex is recruited to the retroviral PBS as the primary determinant of retroviral silencing in PSCs.7Wolf D Goff SP TRIM28 mediates primer binding site-targeted silencing of murine leukemia virus in embryonic cells.Cell. 2007; 131: 46-57Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar To decrease the likelihood of silencing, known silencer elements such as the PBS or parts of the viral LTRs have in recent decades been deleted from viral vectors.2Barklis E Mulligan RC Jaenisch R Chromosomal position or virus mutation permits retrovirus expression in embryonal carcinoma cells.Cell. 1986; 47: 391-399Abstract Full Text PDF PubMed Scopus (128) Google Scholar,6Grez M Akgun E Hilberg F Ostertag W Embryonic stem cell virus, a recombinant murine retrovirus with expression in embryonic stem cells.Proc Natl Acad Sci USA. 1990; 87: 9202-9206Crossref PubMed Scopus (168) Google Scholar However, even self-inactivating retroviral vectors that combine large deletions in the promoter/enhancer regions of the LTR and a modified PBS remain subject to silencing. This argues for a substantial role of the chromosomal position of the integrated vector in the silencing process. It further demonstrates the need for regulatory elements such as insulators to protect the vector cassette from negative positional effects of the chromatin microenvironment and heterochromatin spread (for an overview on strategies for how to avoid chromosomal position effects, see Table 1). Currently, the 250–base pair (bp) cHS4 insulator is the best characterized one; it acts as a blocker of enhancer activity (enhancer blocker) as well as a barrier element to prevent heterochromatin spread.8Ghirlando R Giles K Gowher H Xiao T Xu Z Yao H et al.Chromatin domains, insulators, and the regulation of gene expression.Biochim Biophys Acta. 2012; 1819: 644-651Crossref PubMed Scopus (87) Google Scholar Another interesting insulator is found in the subtelomeric 4q35 locus, a region frequently affected in patients with the muscle disease facioscapulohumeral dystrophy. This element, called D4Z4, is derived from a 3.3-kb macrosatellite sequence and contains three elements: an enhancer, the insulator-typical CTCF and A-type lamin binding sequence, and the open reading frame of the Dux4 gene (Figure 1).9Ottaviani A Rival-Gervier S Boussouar A Foerster AM Rondier D Sacconi S et al.The D4Z4 macrosatellite repeat acts as a CTCF and A-type lamins-dependent insulator in facio-scapulo-humeral dystrophy.PLoS Genet. 2009; 5: e1000394Crossref PubMed Scopus (83) Google Scholar,10Ottaviani A Schluth-Bolard C Gilson E Magdinier F D4Z4 as a prototype of CTCF and lamins-dependent insulator in human cells.Nucleus. 2010; 1: 30-36Crossref PubMed Scopus (25) Google Scholar D4Z4 acts as an insulator that interferes with enhancer–promoter communication and protects transgenes from position effects. Unexpectedly, and unlike cHS4, D4Z4 blocks position effects even when inserted on only one side of the transgene.3Rival-Gervier S Lo MYM Khattak S Pasceri P Lorincz MC Ellis J Kinetics and epigenetics of retroviral silencing in mouse embryonic stem cells defined by deletion of the D4Z4 element.Mol Ther. 2013; 21: 1536-1550Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar,9Ottaviani A Rival-Gervier S Boussouar A Foerster AM Rondier D Sacconi S et al.The D4Z4 macrosatellite repeat acts as a CTCF and A-type lamins-dependent insulator in facio-scapulo-humeral dystrophy.PLoS Genet. 2009; 5: e1000394Crossref PubMed Scopus (83) Google ScholarTable 1Strategies to avoid chromosomal position effects and to achieve stable gene expressionElementStrategyReferences3'D4Z43'D4Z4 barrier element linked to internal promoter in SIN vectors; ideally in combination with cHS4 insulator to fully block silencing3Rival-Gervier S Lo MYM Khattak S Pasceri P Lorincz MC Ellis J Kinetics and epigenetics of retroviral silencing in mouse embryonic stem cells defined by deletion of the D4Z4 element.Mol Ther. 2013; 21: 1536-1550Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 10Ottaviani A Schluth-Bolard C Gilson E Magdinier F D4Z4 as a prototype of CTCF and lamins-dependent insulator in human cells.Nucleus. 2010; 1: 30-36Crossref PubMed Scopus (25) Google ScholarcHS4 insulatorInsulator and barrier element to prevent silencing8Ghirlando R Giles K Gowher H Xiao T Xu Z Yao H et al.Chromatin domains, insulators, and the regulation of gene expression.Biochim Biophys Acta. 2012; 1819: 644-651Crossref PubMed Scopus (87) Google ScholarucoeUbiquitously acting chromatin opening element to prevent silencing11Zhang F Frost AR Blundell MP Bales O Antoniou MN Thrasher AJ A ubiquitous chromatin opening element (UCOE) confers resistance to DNA methylation-mediated silencing of lentiviral vectors.Mol Ther. 2010; 18: 1640-1649Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 12Zhang F Thornhill SI Howe SJ Ulaganathan M Schambach A Sinclair J et al.Lentiviral vectors containing an enhancer-less ubiquitously acting chromatin opening element (UCOE) provide highly reproducible and stable transgene expression in hematopoietic cells.Blood. 2007; 110: 1448-1457Crossref PubMed Scopus (140) Google Scholar, 13Pfaff N Lachmann N Ackermann M Kohlscheen S Brendel C Maetzig T et al.A ubiquitous chromatin opening element prevents transgene silencing in pluripotent stem cells and their differentiated progeny.Stem Cells. 2013; 31: 488-499Crossref PubMed Scopus (60) Google Scholar, 14Brendel C Müller-Kuller U Schultze-Strasser S Stein S Chen-Wichmann L Krattenmacher A et al.Physiological regulation of transgene expression by a lentiviral vector containing the A2UCOE linked to a myeloid promoter.Gene Ther. 2012; 19: 1018-1029Crossref PubMed Scopus (48) Google Scholars/marScaffold matrix attachment element for transcriptional “augmentation”16Bode J Benham C Knopp A Mielke C Transcriptional augmentation: modulation of gene expression by scaffold/matrix-attached regions (S/MAR elements).Crit Rev Eukaryot Gene Expr. 2000; 10: 73-90Crossref PubMed Google ScholarlcrLocus control region17Dean A In the loop: long range chromatin interactions and gene regulation.Brief Funct Genomics. 2011; 10: 3-10Crossref PubMed Scopus (63) Google Scholar“Safe harbor”Molecular “scissors” to induce double-strand break by zinc- finger nucleases, TALENs, CRISPR/Cas9 in safe harbor loci (e. g., AAVS1, Rosa26, CCR5) and repair by homologous recombination; optionally also to be combined with recombinase-mediated cassette exchange (RMCE) strategies18Wirt SE Porteus MH Development of nuclease-mediated site-specific genome modification.Curr Opin Immunol. 2012; 24: 609-616Crossref PubMed Scopus (20) Google Scholar, 19Mussolino C Cathomen T TALE nucleases: tailored genome engineering made easy.Curr Opin Biotechnol. 2012; 23: 644-650Crossref PubMed Scopus (168) Google Scholar, 20Jinek M Chylinski K Fonfara I Hauer M Doudna JA Charpentier E A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.Science. 2012; 337: 816-821Crossref PubMed Scopus (9327) Google Scholar, 21Turan S Zehe C Kuehle J Qiao J Bode J Recombinase-mediated cassette exchange (RMCE)—a rapidly-expanding toolbox for targeted genomic modifications.Gene. 2013; 515: 1-27Crossref PubMed Scopus (105) Google ScholarAAVS1, adeno-associated virus integration site 1; CCR5, chemokine receptor type 5; SIN, self-inactivating; TALENS, transcription activator-like effector nucleases. Open table in a new tab AAVS1, adeno-associated virus integration site 1; CCR5, chemokine receptor type 5; SIN, self-inactivating; TALENS, transcription activator-like effector nucleases. The current work from Ellis's group extends previous studies on the D4Z4 element. The authors show that a defined ∼1-kb region from its 3′ end missing the insulator-typical CTCF and A-type lamin binding sequence (3′D4Z4; Figure 1) mediates persistent but variable retroviral expression for up to 5 months. Additional flanking of the viral expression cassette with two copies of the cHS4 insulator completely abrogated retroviral transgene silencing, thus arguing for functional cooperation between these two insulator elements. Interestingly, the engineered vector fully escaped long-term silencing at a variety of different retroviral integration sites. By flanking the 3′D4Z4 element with FLP recognition target sites, the authors convincingly demonstrate that, upon FLP recombinase-mediated excision of 3′D4Z4, retroviral silencing could be “reestablished” at many (but not all) integration sites. Moreover, utilizing this excision system the authors uncovered three distinct classes of silencing kinetics (rapid, gradual, or not silenced), implying a concerted action of multiple epigenetic pathways that contribute variably to silencing at distinct sites. As the authors suggest, these findings support a model in which 3′D4Z4 functions by blocking the spread of heterochromatin and repressive marks on the surrounding chromatin. Retroviral vectors are particularly sensitive to silencing during the early phase of PSC differentiation (most likely due to multiple epigenetic modifications that characterize this stage of the differentiation process), so it is encouraging that the 3′D4Z4–cHS4 combination maintained EF1a promoter-driven expression during differentiation into embryoid bodies. Furthermore, Rival-Gervier et al. demonstrate that placement of the 3′D4Z4 element 5′ of the EF1a promoter almost completely prevented CpG methylation of the promoter. Other groups have demonstrated similar activities for ubiquitously acting chromatin opening elements (UCOEs) when these elements are placed immediately upstream of internal promoters in retroviral constructs,11Zhang F Frost AR Blundell MP Bales O Antoniou MN Thrasher AJ A ubiquitous chromatin opening element (UCOE) confers resistance to DNA methylation-mediated silencing of lentiviral vectors.Mol Ther. 2010; 18: 1640-1649Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar,12Zhang F Thornhill SI Howe SJ Ulaganathan M Schambach A Sinclair J et al.Lentiviral vectors containing an enhancer-less ubiquitously acting chromatin opening element (UCOE) provide highly reproducible and stable transgene expression in hematopoietic cells.Blood. 2007; 110: 1448-1457Crossref PubMed Scopus (140) Google Scholar and recently Pfaff et al. demonstrated robust activity of UCOEs to prevent silencing in murine and human iPSCs and ESCs.13Pfaff N Lachmann N Ackermann M Kohlscheen S Brendel C Maetzig T et al.A ubiquitous chromatin opening element prevents transgene silencing in pluripotent stem cells and their differentiated progeny.Stem Cells. 2013; 31: 488-499Crossref PubMed Scopus (60) Google Scholar This study further showed that UCOEs display antisilencing activity not only at early phases of PSC differentiation but also during terminal hematopoietic and neuronal differentiation. This is an important feature still to be demonstrated for the 3′D4Z4 element. Interestingly, Brendel et al., of Grez's group in Frankfurt, Germany, recently showed that UCOEs can be used to protect tissue-specific promoters from silencing.14Brendel C Müller-Kuller U Schultze-Strasser S Stein S Chen-Wichmann L Krattenmacher A et al.Physiological regulation of transgene expression by a lentiviral vector containing the A2UCOE linked to a myeloid promoter.Gene Ther. 2012; 19: 1018-1029Crossref PubMed Scopus (48) Google Scholar Given that Rival-Gervier and co-workers show that the 3′D4Z4 element has no intrinsic transcriptional activity, it will probably be possible to fuse 3′D4Z4 to other transcriptional regulatory elements while maintaining their activity. Further work will be needed to better define the antisilencing activity of the 3′D4Z4 element and to dissect the factors blocking the spread of repressive epigenetic marks. In this respect, comparisons with UCOEs may help to define functionally similar pathways. Furthermore, the ability to transfer protection against silencing should be tested for a wider range of promoters, including ubiquitously active, regulated, or lineage-specific promoters. It will also be interesting to test the performance of the 3′D4Z4 in human iPSCs and to assess its activity during terminal differentiation. Last but not least, posttranscriptional issues need to be addressed. Thus, the 3′D4Z4 element should be carefully checked for cryptic splice sites and polyadenylation motifs to ensure vector integrity on the posttranscriptional level, as recently described for UCOEs.15Knight S Zhang F Mueller-Kuller U Bokhoven M Gupta A Broughton T et al.Safer, silencing-resistant lentiviral vectors: optimization of the ubiquitous chromatin-opening element through elimination of aberrant splicing.J Virol. 2012; 86: 9088-9095Crossref PubMed Scopus (36) Google Scholar In summary, the 3′D4Z4 element in conjunction with the cHS4 insulator represents an exciting new addition to the retroviral vector toolkit. The element possesses substantial promise to direct stable transgene expression in PSCs and their differentiated progeny, and it offers a potential solution to the dilemma of silencing and chromatin position effects in PSCs.3Rival-Gervier S Lo MYM Khattak S Pasceri P Lorincz MC Ellis J Kinetics and epigenetics of retroviral silencing in mouse embryonic stem cells defined by deletion of the D4Z4 element.Mol Ther. 2013; 21: 1536-1550Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar Furthermore, the study by Rival-Gervier et al. convincingly demonstrates cooperative effects between distinct (epi)genetically active elements. In this respect, this work may constitute an important blueprint for future approaches in regenerative medicine and transgenesis to tackle this longstanding problem.

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