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• W3186171807 abstract "To develop safer retroviral murine leukemia virus (MLV)-based vectors, we previously mutated and re-engineered the MLV integrase: the W390A mutation abolished the interaction with its cellular tethering factors, BET proteins, and a retargeting peptide (the chromodomain of the CBX1 protein) was fused C-terminally. The resulting BET-independent MLVW390A-CBX was shown to integrate efficiently and more randomly, away from typical retroviral markers. In this study, we assessed the functionality and stability of expression of the redistributed MLVW390A-CBX vector in more depth, and evaluated safety using a clinically more relevant vector design encompassing a self-inactivated (SIN) LTR and a weak internal elongation factor 1α short (EFS) promoter. MLVW390A-CBX-EFS produced like MLVWT and efficiently transduced laboratory cells and primary human CD34+ hematopoetic stem cells (HSC) without transgene silencing over time, while displaying a more preferred, redistributed, and safer integration pattern. In a human mesoangioblast (MAB) stem cell model, the myogenic fusion capacity was hindered following MLVWT transduction, while this remained unaffected when applying MLVW390A-CBX. Likewise, smooth muscle cell differentiation of MABs was unaltered by MLVW390A-CBX-EFS. Taken together, our results underscore the potential of MLVW390A-CBX-EFS as a clinically relevant viral vector for ex-vivo gene therapy, combining efficient production with a preferable integration site distribution profile and stable expression over time. To develop safer retroviral murine leukemia virus (MLV)-based vectors, we previously mutated and re-engineered the MLV integrase: the W390A mutation abolished the interaction with its cellular tethering factors, BET proteins, and a retargeting peptide (the chromodomain of the CBX1 protein) was fused C-terminally. The resulting BET-independent MLVW390A-CBX was shown to integrate efficiently and more randomly, away from typical retroviral markers. In this study, we assessed the functionality and stability of expression of the redistributed MLVW390A-CBX vector in more depth, and evaluated safety using a clinically more relevant vector design encompassing a self-inactivated (SIN) LTR and a weak internal elongation factor 1α short (EFS) promoter. MLVW390A-CBX-EFS produced like MLVWT and efficiently transduced laboratory cells and primary human CD34+ hematopoetic stem cells (HSC) without transgene silencing over time, while displaying a more preferred, redistributed, and safer integration pattern. In a human mesoangioblast (MAB) stem cell model, the myogenic fusion capacity was hindered following MLVWT transduction, while this remained unaffected when applying MLVW390A-CBX. Likewise, smooth muscle cell differentiation of MABs was unaltered by MLVW390A-CBX-EFS. Taken together, our results underscore the potential of MLVW390A-CBX-EFS as a clinically relevant viral vector for ex-vivo gene therapy, combining efficient production with a preferable integration site distribution profile and stable expression over time. Integrating retroviral vectors have proven to be a powerful tool for long-term correction of genetic defects in a variety of severe blood and immune disorders.1Scott C.T. DeFrancesco L. Gene therapy’s out-of-body experience.Nat. Biotechnol. 2016; 34: 600-607Google Scholar, 2Kaufmann K.B. Büning H. Galy A. Schambach A. Grez M. Gene therapy on the move.EMBO Mol. Med. 2013; 5: 1642-1661Google Scholar, 3Cicalese M.P. Aiuti A. Clinical applications of gene therapy for primary immunodeficiencies.Hum. Gene Ther. 2015; 26: 210-219Google Scholar, 4Cavazzana-Calvo M. Fischer A. Hacein-Bey-Abina S. Aiuti A. Gene therapy for primary immunodeficiencies: Part 1.Curr. Opin. Immunol. 2012; 24: 580-584Google Scholar, 5Aiuti A. Bacchetta R. Seger R. Villa A. Cavazzana-Calvo M. Gene therapy for primary immunodeficiencies: Part 2.Curr. Opin. Immunol. 2012; 24: 585-591Google Scholar However, in the initial clinical trials, therapeutic benefits were compromised by severe adverse events in a small subset of patients in the form of acute lymphoblastic leukemia and myelodysplastic syndromes.6Cavazzana-Calvo M. Payen E. Negre O. Wang G. Hehir K. Fusil F. Down J. Denaro M. Brady T. Westerman K. et al.Transfusion independence and HMGA2 activation after gene therapy of human β-thalassaemia.Nature. 2010; 467: 318-322Google Scholar, 7Maldarelli F. Wu X. Su L. Simonetti F.R. Shao W. Hill S. Spindler J. Ferris A.L. Mellors J.W. Kearney M.F. et al.HIV latency. Specific HIV integration sites are linked to clonal expansion and persistence of infected cells.Science. 2014; 345: 179-183Google Scholar, 8Hacein-Bey-Abina S. Garrigue A. Wang G.P. Soulier J. Lim A. Morillon E. Clappier E. Caccavelli L. Delabesse E. Beldjord K. et al.Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1.J. Clin. Invest. 2008; 118: 3132-3142Google Scholar, 9Hacein-Bey-Abina S. Von Kalle C. Schmidt M. McCormack M.P. Wulffraat N. Leboulch P. Lim A. Osborne C.S. Pawliuk R. Morillon E. et al.LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1.Science. 2003; 302: 415-419Google Scholar, 10Ott M.G. Schmidt M. Schwarzwaelder K. Stein S. Siler U. Koehl U. Glimm H. Kühlcke K. Schilz A. Kunkel H. et al.Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertional activation of MDS1-EVI1, PRDM16 or SETBP1.Nat. Med. 2006; 12: 401-409Google Scholar, 11Stein S. Ott M.G. Schultze-Strasser S. Jauch A. Burwinkel B. Kinner A. Schmidt M. Krämer A. Schwäble J. Glimm H. et al.Genomic instability and myelodysplasia with monosomy 7 consequent to EVI1 activation after gene therapy for chronic granulomatous disease.Nat. Med. 2010; 16: 198-204Google Scholar These adverse events were directly attributed to the integration profile and the design of the integrated proviral genome, resulting in insertional mutagenesis.12Bushman F.D. Retroviral Insertional Mutagenesis in Humans: Evidence for Four Genetic Mechanisms Promoting Expansion of Cell Clones.Mol. Ther. 2020; 28: 352-356Google Scholar Each retroviral family displays a specific integration profile that is orchestrated via the interaction of the retroviral integrase (IN) with specific host-cell co-factors that are co-opted by the viral PIC for integration. For instance, gammaretroviruses (γRV; prototype murine leukemia virus [MLV]) and their derived viral vectors integrate near strong enhancer and promoter regions,13LaFave M.C. Varshney G.K. Gildea D.E. Wolfsberg T.G. Baxevanis A.D. Burgess S.M. MLV integration site selection is driven by strong enhancers and active promoters.Nucleic Acids Res. 2014; 42: 4257-4269Google Scholar, 14De Ravin S.S. Su L. Theobald N. Choi U. Macpherson J.L. Poidinger M. Symonds G. Pond S.M. Ferris A.L. Hughes S.H. et al.Enhancers Are Major Targets for Murine Leukemia Virus Vector Integration.J. Virol. 2014; 88: 4504-4513Google Scholar, 15Wu X. Li Y. Crise B. Burgess S.M. Transcription start regions in the human genome are favored targets for MLV integration.Science. 2003; 300: 1749-1751Google Scholar, 16Mitchell R.S. Beitzel B.F. Schroder A.R.W. Shinn P. Chen H. Berry C.C. Ecker J.R. Bushman F.D. Retroviral DNA integration: ASLV, HIV, and MLV show distinct target site preferences.PLoS Biol. 2004; 2: E234Google Scholar while lentiviruses, such as HIV and HIV-based viral vectors, prefer integration in the body of actively transcribed gene bodies.17Schröder A.R.W. Shinn P. Chen H. Berry C. Ecker J.R. Bushman F. HIV-1 integration in the human genome favors active genes and local hotspots.Cell. 2002; 110: 521-529Google Scholar Hence, insertional mutagenesis is intrinsically linked to the integration preference of the proviral vector in the proximity of proto-oncogenes and the subsequent upregulation by the strong viral promoter and enhancer elements in the flanking long-terminal repeat (LTR) elements of the retroviral vectors, normally used to drive transgene expression, leading to aberrant expression of the proto-oncogene.9Hacein-Bey-Abina S. Von Kalle C. Schmidt M. McCormack M.P. Wulffraat N. Leboulch P. Lim A. Osborne C.S. Pawliuk R. Morillon E. et al.LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1.Science. 2003; 302: 415-419Google Scholar,18Persons D.A. Baum C. Solving the problem of γ-retroviral vectors containing long terminal repeats.Mol. Ther. 2011; 19: 229-231Google Scholar,19Howe S.J. Mansour M.R. Schwarzwaelder K. Bartholomae C. Hubank M. Kempski H. Brugman M.H. Pike-Overzet K. Chatters S.J. de Ridder D. et al.Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene therapy of SCID-X1 patients.J. Clin. Invest. 2008; 118: 3143-3150Google Scholar Several efforts have been made to improve both safety and efficacy of integrating retroviral vectors by alterations in the vector design such as abolition of the strong enhancer elements from the U3 part of the LTR, resulting in self-inactivating (SIN) viral vectors.20Maetzig T. Galla M. Baum C. Schambach A. Gammaretroviral vectors: biology, technology and application.Viruses. 2011; 3: 677-713Google Scholar, 21Yu S.F. von Rüden T. Kantoff P.W. Garber C. Seiberg M. Rüther U. Anderson W.F. Wagner E.F. Gilboa E. Self-inactivating retroviral vectors designed for transfer of whole genes into mammalian cells.Proc. Natl. Acad. Sci. USA. 1986; 83: 3194-3198Google Scholar, 22Kuo C.Y. Kohn D.B. Gene Therapy for the Treatment of Primary Immune Deficiencies.Curr. Allergy Asthma Rep. 2016; 16: 39Google Scholar The deletion of promoter/enhancer activity was counterbalanced by introduction of a heterologous promoter to drive transgene expression.23Demaison C. Parsley K. Brouns G. Scherr M. Battmer K. Kinnon C. Grez M. Thrasher A.J. High-level transduction and gene expression in hematopoietic repopulating cells using a human immunodeficiency [correction of imunodeficiency] virus type 1-based lentiviral vector containing an internal spleen focus forming virus promoter.Hum. Gene Ther. 2002; 13: 803-813Google Scholar,24Yam P.Y. Li S. Wu J. Hu J. Zaia J.A. Yee J.-K. Design of HIV vectors for efficient gene delivery into human hematopoietic cells.Mol. Ther. 2002; 5: 479-484Google Scholar Nonetheless, SIN vector integration profiles remain unaltered and are still targeted to gene regulatory regions where they have the potential to disrupt or deregulate the transcription of nearby genes by other mechanisms.25Moiani A. Miccio A. Rizzi E. Severgnini M. Pellin D. Suerth J.D. Baum C. De Bellis G. Mavilio F. Deletion of the LTR enhancer/promoter has no impact on the integration profile of MLV vectors in human hematopoietic progenitors.PLoS ONE. 2013; 8: e55721Google Scholar Indeed, SIN viral vector designs still induced oncogene activation in in-vitro safety assays and resulted in a dominant myeloid clone in a β-thalassemia clinical trial.6Cavazzana-Calvo M. Payen E. Negre O. Wang G. Hehir K. Fusil F. Down J. Denaro M. Brady T. Westerman K. et al.Transfusion independence and HMGA2 activation after gene therapy of human β-thalassaemia.Nature. 2010; 467: 318-322Google Scholar,26Bosticardo M. Ghosh A. Du Y. Jenkins N.A. Copeland N.G. Candotti F. Self-inactivating retroviral vector-mediated gene transfer induces oncogene activation and immortalization of primary murine bone marrow cells.Mol. Ther. 2009; 17: 1910-1918Google Scholar,27Modlich U. Navarro S. Zychlinski D. Maetzig T. Knoess S. Brugman M.H. Schambach A. Charrier S. Galy A. Thrasher A.J. et al.Insertional transformation of hematopoietic cells by self-inactivating lentiviral and gammaretroviral vectors.Mol. Ther. 2009; 17: 1919-1928Google Scholar However, replacing the potent viral promoters for a weaker cellular version, like elongation factor 1α (EF1α) and phosphoglycerate kinase (PGK) promoters, in SIN-viral vectors greatly decreased their risk on insertional transformation.28Zychlinski D. Schambach A. Modlich U. Maetzig T. Meyer J. Grassman E. Mishra A. Baum C. Physiological promoters reduce the genotoxic risk of integrating gene vectors.Mol. Ther. 2008; 16: 718-725Google Scholar,29Montiel-Equihua C.A. Zhang L. Knight S. Saadeh H. Scholz S. Carmo M. Alonso-Ferrero M.E. Blundell M.P. Monkeviciute A. Schulz R. et al.The β-globin locus control region in combination with the EF1α short promoter allows enhanced lentiviral vector-mediated erythroid gene expression with conserved multilineage activity.Mol. Ther. 2012; 20: 1400-1409Google Scholar The specific integration preference of γRV and their derived viral vectors for strong enhancers and promoter regions is dictated by interaction of the MLV pre-integration complex (PIC) with its cellular cofactors, the bromodomain and extra-terminal domain (BET)-containing family of proteins (BRD2, BRD3, and BRD4). We and others showed that BET proteins serve as bimodal anchors on the host chromatin by binding to epigenetic chromatin marks associated with strong enhancers and promoter regions via their bromodomain on the one hand and to the MLV IN via the ET domain on the other.30De Rijck J. de Kogel C. Demeulemeester J. Vets S. El Ashkar S. Malani N. Bushman F.D. Landuyt B. Husson S.J. Busschots K. et al.The BET family of proteins targets moloney murine leukemia virus integration near transcription start sites.Cell Rep. 2013; 5: 886-894Google Scholar, 31Sharma A. Larue R.C. Plumb M.R. Malani N. Male F. Slaughter A. Kessl J.J. Shkriabai N. Coward E. Aiyer S.S. et al.BET proteins promote efficient murine leukemia virus integration at transcription start sites.Proc. Natl. Acad. Sci. USA. 2013; 110: 12036-12041Google Scholar, 32Gupta S.S. Maetzig T. Maertens G.N. Sharif A. Rothe M. Weidner-Glunde M. Galla M. Schambach A. Cherepanov P. Schulz T.F. Bromo- and extraterminal domain chromatin regulators serve as cofactors for murine leukemia virus integration.J. Virol. 2013; 87: 12721-12736Google Scholar In our previous work to improve the safety of retroviral vectors, we first uncoupled the interaction between the MLV IN and its cellular BET tethering factor, by a single substitution in the MLV IN (INW390A) to create BET-independent (Bin) MLV vectors. BinMLV vectors produced and transduced efficiently, while displaying an integration pattern that associates less with traditional markers of MLV integration.33El Ashkar S. De Rijck J. Demeulemeester J. Vets S. Madlala P. Cermakova K. Debyser Z. Gijsbers R. BET-independent MLV-based Vectors Target Away From Promoters and Regulatory Elements.Mol. Ther. Nucleic Acids. 2014; 3: e179Google Scholar In a next step, we re-engineered the MLV IN by fusing small chromatin-binding peptides to the C-terminal end of the uncoupled INW390A protein, such as a chromodomain peptide of CBX1 (referred to as INW390A-CBX). The resulting MLV vectors (referred to as MLVW390A-CBX) in turn displayed an even more promising integration profile, with an integration site preference that was more random, detargeted away from the traditional markers of MLV integration.34El Ashkar S. Van Looveren D. Schenk F. Vranckx L.S. Demeulemeester J. De Rijck J. Debyser Z. Modlich U. Gijsbers R. Engineering Next-Generation BET-Independent MLV Vectors for Safer Gene Therapy.Mol. Ther. Nucleic Acids. 2017; 7: 231-245Google Scholar Interestingly, MLVW390A-CBX produced well and transduced cells as efficiently as vectors carrying INWT (MLVWT). In addition, this vector showed a reduced transformational potential compared to MLVWT in a serial colony-forming assay, supporting an improved safety profile.34El Ashkar S. Van Looveren D. Schenk F. Vranckx L.S. Demeulemeester J. De Rijck J. Debyser Z. Modlich U. Gijsbers R. Engineering Next-Generation BET-Independent MLV Vectors for Safer Gene Therapy.Mol. Ther. Nucleic Acids. 2017; 7: 231-245Google Scholar Still, the vector used in these studies did not constitute the ideal clinical vector design, either being flanked by full MLV LTRs or carrying a potent viral spleen focus forming virus (SFFV) LTR promoter, known to deregulate genes up to 50 kb away, to drive transgene expression.27Modlich U. Navarro S. Zychlinski D. Maetzig T. Knoess S. Brugman M.H. Schambach A. Charrier S. Galy A. Thrasher A.J. et al.Insertional transformation of hematopoietic cells by self-inactivating lentiviral and gammaretroviral vectors.Mol. Ther. 2009; 17: 1919-1928Google Scholar,28Zychlinski D. Schambach A. Modlich U. Maetzig T. Meyer J. Grassman E. Mishra A. Baum C. Physiological promoters reduce the genotoxic risk of integrating gene vectors.Mol. Ther. 2008; 16: 718-725Google Scholar,34El Ashkar S. Van Looveren D. Schenk F. Vranckx L.S. Demeulemeester J. De Rijck J. Debyser Z. Modlich U. Gijsbers R. Engineering Next-Generation BET-Independent MLV Vectors for Safer Gene Therapy.Mol. Ther. Nucleic Acids. 2017; 7: 231-245Google Scholar, 35Zhou S. Fatima S. Ma Z. Wang Y.-D. Lu T. Janke L.J. Du Y. Sorrentino B.P. Evaluating the Safety of Retroviral Vectors Based on Insertional Oncogene Activation and Blocked Differentiation in Cultured Thymocytes.Mol. Ther. 2016; 24: 1090-1099Google Scholar, 36Montini E. Cesana D. Schmidt M. Sanvito F. Bartholomae C.C. Ranzani M. Benedicenti F. Sergi L.S. Ambrosi A. Ponzoni M. et al.The genotoxic potential of retroviral vectors is strongly modulated by vector design and integration site selection in a mouse model of HSC gene therapy.J. Clin. Invest. 2009; 119: 964-975Google Scholar In the present study, we assessed the functionality and the safety of the redistributed MLVW390A-CBX vector configuration compared to MLVWT using a clinically more relevant vector design combining a SIN LTR and a weak internal EF1α short (EFS) promoter driving eGFP reporter gene expression. First, we analyzed transgene expression levels and expression stability over time in laboratory cells comparing MLV SIN vectors containing the EFS promoter as internal promoter with those using an SFFV promoter. The integration profile for MLVW390A-CBX was unaffected by the internal promoter and in line with our earlier data.34El Ashkar S. Van Looveren D. Schenk F. Vranckx L.S. Demeulemeester J. De Rijck J. Debyser Z. Modlich U. Gijsbers R. Engineering Next-Generation BET-Independent MLV Vectors for Safer Gene Therapy.Mol. Ther. Nucleic Acids. 2017; 7: 231-245Google Scholar In a more detailed analysis, cells were sorted for eGFP expression prior to integration site sequencing to determine whether the more random integration pattern is retained in the active (eGFP+) population of cells. Our data showed that even when integration happens more randomly and thus also in regions with markers of silent chromatin, reporter gene expression is still supported, also when using the weaker EFS promoter. In line, MLVW390A-CBX transduction of clinically relevant human CD34+ hematopoetic stem cell (HSC) corroborated stable transgene expression over time, with a more preferable and safer integration site distribution compared to MLVWT. In a final experiment, we questioned whether MLVW390A-CBX, displaying a potentially safer integration profile, remained active following differentiation of transduced progenitor cells. In a mesoangioblast (MAB) stem cell model, MLVW390A-CBX supported stable transgene expression over time, also following differentiation into skeletal and smooth muscle cells. In addition, the altered integration profile of MLVW390A-CBX resulted in a normal myogenic capacity of transduced mesoangioblasts compared to those using MLVWT, suggesting reduced genotoxicity of MLVW390A-CBX and demonstrating its value as viral vector for ex-vivo gene therapy. In a next step toward safer retroviral vectors for gene therapy, we opted to make a comprehensive analysis of the previously generated next-generation BinMLV INW390A-CBX vector, further referred to as MLVW390A-CBX (Figure 1A; dark green), by implementing a clinically more relevant vector design.34El Ashkar S. Van Looveren D. Schenk F. Vranckx L.S. Demeulemeester J. De Rijck J. Debyser Z. Modlich U. Gijsbers R. Engineering Next-Generation BET-Independent MLV Vectors for Safer Gene Therapy.Mol. Ther. Nucleic Acids. 2017; 7: 231-245Google Scholar In parallel, we used wild-type MLV (Figure 1A; MLVWT, dark red) and the Bin MLV vector only containing the point-mutation in IN (Figure 1A; MLVW390A, yellow) as controls.33El Ashkar S. De Rijck J. Demeulemeester J. Vets S. Madlala P. Cermakova K. Debyser Z. Gijsbers R. BET-independent MLV-based Vectors Target Away From Promoters and Regulatory Elements.Mol. Ther. Nucleic Acids. 2014; 3: e179Google Scholar We combined the respective packaging constructs with a self-inactivating LTR design and an internal EFS promoter, a short intron-less version derived from human EF1α, to drive eGFP expression (Figure 1A; EFS, open bar).28Zychlinski D. Schambach A. Modlich U. Maetzig T. Meyer J. Grassman E. Mishra A. Baum C. Physiological promoters reduce the genotoxic risk of integrating gene vectors.Mol. Ther. 2008; 16: 718-725Google Scholar,37Schambach A. Bohne J. Chandra S. Will E. Margison G.P. Williams D.A. Baum C. Equal potency of gammaretroviral and lentiviral SIN vectors for expression of O6-methylguanine-DNA methyltransferase in hematopoietic cells.Mol. Ther. 2006; 13: 391-400Google Scholar As a reference, we used the same vectors carrying the potent SFFV promoter (Figure 1A; SFFV, filled bar). Vector particles pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) were produced, generating six different viral vectors: MLVWT-SFFV, MLVW390A-SFFV, MLVW390A-CBX-SFFV, MLVWT-EFS, MLVW390A-EFS, and MLVW390A-CBX-EFS, respectively. All viral vectors produced efficiently, as corroborated by comparable reverse transcriptase (RT) activities (Figure 1B; SFFV-driven filled bars, EFS-driven open bars) and transducing titers, with 293T titers reaching 5 × 107 transducing units (TU)/mL (Figure 1C). Following efficient MLV vector production, we assessed transduction of laboratory cell lines, SupT1 cells and K562 cells. Cells were transduced at three different multiplicities of infection (MOI) for the different vector configurations following normalization for RT activity. Transduction efficiencies were monitored over time by flow cytometry as %eGFP-positive (eGFP+) cells. The respective vectors reached similar transduction efficiencies for different MOIs in both cell lines (Figures S1A and S1B). When monitoring the stability of expression over time, %eGFP+ cells slightly increased between days 3 and day 6 and was thereafter stable over time for both SFFV- and EFS-driven vector configurations (full and dashed lines, respectively) in SupT1 cells (Figure 2A; between 30.6% and 34.5% for SFFV vectors and between 37.9% and 41.6% for EFS vectors) as well as in K562 cells (Figure 2B; between 34.6% and 36.5% for SFFV vectors and between 38.8% and 43.0% for EFS vectors). Although transduction efficiencies were comparable for the respective vectors, mean fluorescence intensity (MFI) levels, serving as an indicator for eGFP transgene expression, showed to be different. In SupT1 cells an overall 1.6-fold difference was observed between SFFV-driven viral vectors and their EFS-driven counterparts (Figure 2C; Figure S1C). When comparing the respective vector configurations, MFI levels were 1.5-fold lower for MLVW390A and MLVW390A-CBX compared to MLVWT equipped with the same promoter (Figure 2C; Figure S1C), which may be explained by a retargeted integration profile. While differences in MFI were not that different in SupT1 cells, MFI levels in K562 cells of EFS-containing vectors were on average 5-fold lower compared to the same vectors equipped with SFFV as an internal promoter (Figure 2D; Figure S1D). Interestingly, considering conditions with the same percentage of eGFP+ cells, eGFP expression levels (MFI) of SFFV- and EFS-containing viral vectors were substantially higher in K562 cells compared to SupT1 cells (25-fold and 8-fold, respectively), indicating that both promoters are more functional in K562 cells. Taken together, we assessed three different vector configurations (MLVWT, MLVW390A, and MLVW390A-CBX) that are all expected to distribute differently in the target cell genome. All three vectors efficiently transduced both SupT1 and K562 cells and resulted in stable reporter gene expression over time. Even though vectors equipped with SFFV displayed higher eGFP expression levels compared to those carrying a weaker EFS promoter, still expression over time was constant for both the EFS and SFFV vector designs. In a next step, we determined the integration profile of the MLV vectors in SupT1 cells. Integration sites were recovered by ligation-mediated PCR (LM-PCR) followed by high-throughput Illumina sequencing and mapped to the human genome (hg18 assembly) by a previously described bioinformatic pipeline, yielding a total of 19,385 unique integration sites.38Sherman E. Nobles C. Berry C.C. Six E. Wu Y. Dryga A. Malani N. Male F. Reddy S. Bailey A. et al.INSPIIRED: A Pipeline for Quantitative Analysis of Sites of New DNA Integration in Cellular Genomes.Mol. Ther. Methods Clin. Dev. 2016; 4: 39-49Google Scholar,39Berry C.C. Nobles C. Six E. Wu Y. Malani N. Sherman E. Dryga A. Everett J.K. Male F. Bailey A. et al.INSPIIRED: Quantification and Visualization Tools for Analyzing Integration Site Distributions.Mol. Ther. Methods Clin. Dev. 2016; 4: 17-26Google Scholar As an initial analysis, we examined the frequency of integration near typical markers associated with MLV integration (Figure 3A). γRV integration is traditionally enriched near active enhancer and promoter regions.13LaFave M.C. Varshney G.K. Gildea D.E. Wolfsberg T.G. Baxevanis A.D. Burgess S.M. MLV integration site selection is driven by strong enhancers and active promoters.Nucleic Acids Res. 2014; 42: 4257-4269Google Scholar,14De Ravin S.S. Su L. Theobald N. Choi U. Macpherson J.L. Poidinger M. Symonds G. Pond S.M. Ferris A.L. Hughes S.H. et al.Enhancers Are Major Targets for Murine Leukemia Virus Vector Integration.J. Virol. 2014; 88: 4504-4513Google Scholar In line, MLVWT integration was enriched near transcription start sites (TSS; 20%), CpG islands (CpG; 18%) and DNaseI hypersensitive sites (DHS; 40%) irrespective of the promoter driving the transgene expression (Figure 3A, dark red filled and open bars), whereas MLVW390A showed a profile that was significantly detargeted from these features (Figure 3A, yellow filled and open bars; ∗∗∗p < 0.001; two-tailed χ2 test compared to MLVWT), which is in line with earlier data.33El Ashkar S. De Rijck J. Demeulemeester J. Vets S. Madlala P. Cermakova K. Debyser Z. Gijsbers R. BET-independent MLV-based Vectors Target Away From Promoters and Regulatory Elements.Mol. Ther. Nucleic Acids. 2014; 3: e179Google Scholar,34El Ashkar S. Van Looveren D. Schenk F. Vranckx L.S. Demeulemeester J. De Rijck J. Debyser Z. Modlich U. Gijsbers R. Engineering Next-Generation BET-Independent MLV Vectors for Safer Gene Therapy.Mol. Ther. Nucleic Acids. 2017; 7: 231-245Google Scholar Integration preferences for MLVW390A-CBX-SFFV and -EFS (dark green filled and open bars, respectively) near these features were shifted even more, as reported earlier.34El Ashkar S. Van Looveren D. Schenk F. Vranckx L.S. Demeulemeester J. De Rijck J. Debyser Z. Modlich U. Gijsbers R. Engineering Next-Generation BET-Independent MLV Vectors for Safer Gene Therapy.Mol. Ther. Nucleic Acids. 2017; 7: 231-245Google Scholar In a more elaborate analysis, integration frequencies near a selection of genomic features and a collection of epigenetic marks known to associate with transcriptionally active and silent chromatin were determined and represented as a genomic and an epigenetic heatmap, respectively (Figures 3B and 3C). Here, tile color depicts the correlation for an integration dataset with the respective genomic or epigenetic feature (indicated at the left side) relative to matched random controls (MRCs), as indicated by the colored receiver operating characteristic (ROC) curve area scale at the bottom of the panel. Pink tiles indicate that integration for a specific dataset is enriched in these features relative to MRC, while blue tiles indicate that integration is disfavored. A near random (MRC) distribution would result in a gray tile. Again, both MLVWT-SFFV and -EFS preferentially integrated in transcriptionally active chromatin (Figure 3C; tiles color dark pink) and disfavoring silent chromatin (Figure 3C; tiles color blue and gray). Uncoupling BET interaction (MLVW390A) resulted in an integration profile that associates less with the above-mentioned features (tiles color less pink), whereas MLVW390A-CBX viral vectors showed an integration profile with an even more pronounced shift toward random (Figure 3B). In addition, MLVW390A-CBX-SFFV and -EFS integration correlated less with histone modifications generally associated with active transcription, such as all acetylations and some histone methylations (Figure 3C; tile colors shift more toward gray), whereas integration occurred more frequently near H3K9me2 and H3K9me3, histone modifications associated with transcriptionally silent chromatin and described to be bound by CBX1 chromodomains40Kaustov L. Ouyang H. Amaya M. Lemak A. Nady N. Duan S. Wasney G.A. Li Z. Vedadi M. Schapira M. et al.Recognition and specificity determinants of the human cbx chromodomains.J. Biol. Chem. 2011; 286: 521-529Google Scholar (Figure 3C; tile colors shift to gray for MLVW390A and MLVW390A-CBX). Asterisks indicate statistical significance of the integration site distributions of the respective MLV vectors relative to that of MLVWT-SFFV (Figure S2). Although differences were detected for some features, the overall integration profile of EFS-containing MLV vector configurations was similar to that of their SFFV-driven counterparts, indicating that the endogenous promoter had no effect on the integration profile. Additionally, we determined integr" @default.
• W3186171807 created "2021-08-02" @default.
• W3186171807 creator A5004661448 @default.
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• W3186171807 date "2021-07-01" @default.
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• W3186171807 title "Improved Functionality and Potency of Next Generation BinMLV Viral Vectors towards Safer Gene Therapy" @default.
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• W3186171807 doi "https://doi.org/10.1016/j.omtm.2021.07.003" @default.
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