Volume 16, Issue 4, Pages 718-725 (April 2008) Physiological Promoters Reduce the Genotoxic Risk of Integrating Gene Vectors  Daniela Zychlinski, Axel Schambach, Ute Modlich, Tobias Maetzig, Johann Meyer, Elke Grassman, Anjali Mishra, Christopher Baum  Molecular Therapy  Volume 16, Issue 4, Pages 718-725 (April 2008) DOI: 10.1038/mt.2008.5 Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 1 Transient transfection assays reveal low enhancer activity of cellular promoters and reduced enhancer activity of self-inactivating (SIN) vector in comparison with long-terminal repeat (LTR) vectors. (a) The reporter construct encodes a codon-optimized luciferase that is devoid of at least 300 known transcription factor binding sites. A synthetic polyadenylation (syn. pA) prevents potential readthrough from the enhancer–promoter cassette (Enh-P) cloned downstream of the termination site of the luciferase cassette. Alternatively, entire provirus sequences of LTR or SIN vectors were introduced in sense or antisense orientation. (b) Schema of transcription factor binding sites predicted by computer algorithms and functional data, revealing a dense clustering of binding sites in the SFFV-U3 region, in contrast to the cellular promoter elongation factor-1α (EF1α). (c) The retroviral Enh-P sequences derived from myeloproliferative sarcoma virus (MPSV) and spleen focus–forming virus (SFFV) show a significant induction of two different minimal promoters, in contrast to the cellular promoters [EFS and phosphoglycerate kinase (PGK)]. No, lack of Enh-P 3′ of the luciferase cassette. Error bars indicate standard deviations of four experiments. (d) When compared with LTR vectors, SIN vectors show a greatly decreased enhancer activity, even when they contain the same Enh-P. Enhancer activity is also more dependent on orientation (sense or antisense to the luciferase cassette) in the SIN configuration. CMV, cytomegalovirus; EGFP, enhanced green fluorescent protein; no, SIV vector lacking an interval Enh-P TK, thymidine kinase; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element. Molecular Therapy 2008 16, 718-725DOI: (10.1038/mt.2008.5) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 2 Stably integrated “minigene” reporter constructs reveal the low enhancer activity of the cellular promoters tested. (a) The retroviral self inactivating (SIN) vector contains a minigene cassette in the U3 region of the long-terminal repeat (LTR), consisting of a minimal promoter followed by a truncated CD34 surface marker gene (tCD34). Termination is mediated by signals in the R region of the LTR. The same vectors were also tested with 1×HS4 insulator element inserted upstream of the minigene cassette. Enh-P sequences were introduced as indicated. A control vector contained no minimal promoter (ΔCMV). (b) In SC1 fibroblasts and lineage-negative primary murine hematopoietic cells, only the internal spleen focus–forming virus (SFFV) promoter led to a significant activation of the minigene cassette. Background levels are reflected in the tCD34 expression of the construct that lacks a minimal promoter. Data obtained from SC1 cells are shown in white columns and lineage-negative cells in grey ones. The insets show representative flow cytometry data (SC1 cells) for illustration of the assay, CD34 expression shown on the y-axis. Potential activation of the minigene by transcriptional readthrough could be ruled out (Supplementary Figure S2). CMV, cytomegalovirus; EFS, short form of EF1α EGFP, enhanced green fluorescent protein; PGK, phosphoglycerate kinase. Molecular Therapy 2008 16, 718-725DOI: (10.1038/mt.2008.5) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 3 The clonal dominance assay shows a significant reduction in transformed cells following the use of a self-inactivating (SIN) vector with a physiological cellular promoter (EFS). The ratio of replating frequency (determined by limiting dilution cloning) per vector copy number (detected by real-time PCR) 4 days after transduction, is a measure of the degree of transformation. A SIN vector with an internal EFS promoter shows no transforming capacity. The efficiency of transformation is significantly reduced in comparison to an unmodified or insulated SIN vector with an internal retroviral promoter. All seven experiments conducted with SIN.EFS vectors yielded results that were below the detection limit, indicated as a horizontal line. For negative values, calculations are based on the assumption that a replating clone would have been detected when plating 97 instead of 96 wells. Molecular Therapy 2008 16, 718-725DOI: (10.1038/mt.2008.5) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 4 Self-inactivating (SIN) vectors containing an internal retrovirus-derived enhancer–promoter (SF) and insulator sequences (HS4 250 base pair core element) in the otherwise deleted U3 region of the long-terminal repeat. (a) The vector with a single copy of the HS4 element (SIN.SF.GFP.1×HS4) has identical titers and expression levels (in SC1 fibroblasts) as the unmodified counterpart (SIN.SF.GFP) does. In contrast, a vector with a tandem repeat of the HS4 element had significantly reduced titers, indicative of genetic instability (SIN.SF.GFP.2xHS4). (b) Histogram of enhanced green fluorescent protein (EGFP) expression achieved with the different vectors under similar multiplicity of infection (SC1 fibroblasts). Differences in the replating frequency cannot be explained by variations in EGFP expression levels. t.u., transducing units. Molecular Therapy 2008 16, 718-725DOI: (10.1038/mt.2008.5) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 5 Molecular characterization of cultures and replating clones. (a) Southern blot analysis of replating clones shows multiple vector insertion sites when using vectors SIN.SF.GFP or SIN.SF.GFP.1×HS4. (b) Northern blot revealing transcriptional upregulation of Evi1 in replating clones. Mock-transduced Lin− bone marrow cells cultured for >14 days served as negative control, and 18S RNA served as loading control. (c) Induction of Evi1 transcripts can be detected as early as 6 days after gene transfer, thereby suggesting a quick outgrowth of mutants. In contrast, cultures transduced with a vector containing the EFS internal promoter showed no detectable induction of Evi1 at any of the time points. MOI, multiplicity of infection; SIN, self-inactivating. Molecular Therapy 2008 16, 718-725DOI: (10.1038/mt.2008.5) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 6 Cytology of cultures before replating (May-Grünwald–Giemsa stain of cytospin preparations). Mock-treated and SIN.EFS-transduced cultures both show a predominance of mature myeloid cells, whereas numerous blasts are present in cultures transduced with vectors SIN.SF.GFP or SIN.SF.GFP.1×HS4. SIN, self-inactivating. Molecular Therapy 2008 16, 718-725DOI: (10.1038/mt.2008.5) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions