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251. TALENs Facilitate Targeted Genome Editing in Human Cells With High Specificity and Low Cytotoxicity
GENOME EDITING & GENE TARGETING TECHNOLOGIES
To identify off-target cleavage at genomic sites with insertions or deletions compared to the guide strand, we systematically added a base into or removed a base from sgRNAs at different positions throughout the guide sequence to model insertions (DNA bulges, Fig. A) or deletions (RNA bulges, Fig. B) at off-target sequences. RNA-guided Cas9 cleavage activity at endogenous loci in HEK293T cells transfected with plasmids encoding Cas9 and sgRNA variants was quantified as NHEJ-induced mutation rates. We found that offtarget cleavage resulting from the sgRNA variants occurred with a DNA bulge or a sgRNA bulge at multiple positions in the guide strands, sometimes at levels comparable to or even higher than those of original sgRNAs. We also examined the Cas9-mediated mutagenesis at potential offtarget loci in the human genome carrying single-base DNA bulges or sgRNA bulges together with a range of base mismatches, and confirmed off-target sites with mutation frequencies up to 45.5%. Our results indicate that off-target effects may limit the applications of Cas9-mediated gene modification, especially in large mammalian genomes that contain multiple DNA sequences differing by only a few bases due to mismatches, insertions or deletions. Future design of specific sgRNAs would require a search for genomic sites with base-pair mismatches, insertions and deletions, such as with our newly developed program COSMID, to fully analyze CRISPR/Cas9 off-target activity.
250. Helper-Dependent Ad5/35 Vectors for ZFNMediated Gene Editing in Hematopoietic Stem Cells
Kamola Saydaminova,1 Hongjie Wang,1 Eirini Papapetrou,1 Michael Holmes,2 Philip Gregory,2 Donna Palmer,3 Philip Ng,3 Anja Ehrhardt,4 Andre Lieber.1 1 University of Washington, Seattle; 2Sangamo Biosciences, Richmond; 3Baylor College of Medicine, Houston; 4University Witten/Herdecke, Witten, Germany.
Ad5/35 vectors efficiently transduce hematopoietic stem/progenitor cells (HSCs) in vitro, and as we have recently shown also in vivo after HSC mobilization. In contrast to first-generation Ad5/35 vectors, helper-dependent (HD) Ad5/35 do not affect the clonogenic and engraftment potential of transduced HSCs. A problem with the production of HD Ad5/35 vectors expressing ZFNs is that they kill the Ad producer cells most potentially due to excessively high expression levels during viral replication. To generate HD Ad5/35 vectors that would express ZFNs in human hematopoietic CD34+ stem cells, we used a miRNA-regulated gene expression system. If the RNA of a transgene contains a target site for a miRNA that is expressed at high levels in HD-Ad5/35 producer (293-Cre) cells, the transgene mRNA will be degraded in this cell type. Using microarrays, we established the miRNA expression profiles in Ad5/35-infected 293-Cre and human CD34+ cells. We then selected two miRNAs (has-miR1835p and has-miR218-5p), which had the highest expression levels in 293-Cre cells and that were absent in CD34+ cells. Deep sequencing of these miRNA revealed miRNA species that, in addition to the 22nt canonical sequence, contained additional nucleotides but with lower frequency. We then inserted 4 target sites with 100% homology to the selected miRNAs into the 3’UTR of a GFP gene, which was under the control of an EF1alpha promoter, a promoter that is highly active in CD34+ cells. The GFP expression cassettes were inserted into an Ad5/35 vector. The vectors also contained a PGK promoter-driven mCherry expression cassette that was not regulated by the selected Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy
miRNAs. We then transduced human CD34+ cells and 293-Cre cells with the vectors and analyzed GFP and mCherry expression 48 hours later by flow cytometry. In 293-Cre cells, mCherry expression levels were comparable for all vectors, while GFP expression was suppressed with vectors that contained the miRNA target sites. Based on the ratio of GFP to mCherry levels, the greatest suppression was achieved with vector that contained target sequences of miR183 and miR218 including three additional nucleotides at their 3’ end. Both GFP and mCherry expression were comparably high in CD34+ cells for all vectors. By using the miRNA-183/218 regulated gene expression system, we were able to produce an HD-Ad5/35 vector that expresses a ZFN specific to human CCR5. This vector conferred efficient ZFN expression in CD34+ cells resulting in site-specific DNA cleavage in >20% of CCR5 alleles as detected by mismatchsensitive surveyor nuclease PCR. We are currently testing the persistence of site-specific gene modification after HSC transduction in vitro and in vivo. Our data show i) that considering heterogeneous miRNA processing and optimizing the target site design can increase the specificity of a miRNA regulated gene expression and ii) that miRNA based suppression of ZFN expression in 293-Cre cells allows for the production of HD-Ad vectors at high titers.
251. TALENs Facilitate Targeted Genome Editing in Human Cells With High Specificity and Low Cytotoxicity Claudio Mussolino,1,2 Jamal Alzubi,1,2 Eli J. Fine,3 Robert Morbitzer,4 Thomas J. Cradick,3 Thomas Lahaye,4,5 Gang Bao,3 Toni Cathomen.1,2 1 Institute for Cell and Gene Therapy & Center for Chronic Immunodeficiency, University Medical Center Freiburg, Freiburg, Germany; 2Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany; 3Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA; 4Institute of Genetics, LudwigMaximilians-University Munich, Munich, Germany; 5Center for Plant Molecular Biology, Eberhard-Karls-University, Tübingen, Germany.
Designer nucleases have been successfully employed to modify the genomes of various model organisms and human cell types. While the specificity of zinc-finger nucleases (ZFNs) and RNAguided endonucleases (RGNs) has been assessed to some extent, little data is available for TALE-based nucleases (TALENs). Here, we have engineered TALEN pairs targeting three human loci (CCR5, AAVS1, IL2RG) and performed a detailed analysis of their activity, toxicity, and specificity. The TALENs showed comparable activity to benchmark ZFNs, with allelic gene disruption frequencies of 1530% in human cells. Notably, TALEN expression was overall marked by a low cytotoxicity and the absence of cell cycle aberrations. Bioinformatics-based analysis of designer nuclease specificity confirmed partly substantial off-target activity of ZFNs targeting CCR5 and AAVS1 at six known and five novel sites, respectively. In contrast, only marginal off-target cleavage activity was detected at four out of 49 predicted off-target sites for CCR5 and AAVS1specific TALENs. The rational design of a CCR5-specific TALEN pair decreased off-target activity at the closely related CCR2 locus considerably, consistent with fewer genomic rearrangements between the two loci. In conclusion, our results link nuclease-associated toxicity to off-target cleavage activity and corroborate TALENs as a highly specific platform for future clinical translation.
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To identify off-target cleavage at genomic sites with insertions or deletions compared to the guide strand, we systematically added a base into or removed a base from sgRNAs at different positions throughout the guide sequence to model insertions (DNA bulges, Fig. A) or deletions (RNA bulges, Fig. B) at off-target sequences. RNA-guided Cas9 cleavage activity at endogenous loci in HEK293T cells transfected with plasmids encoding Cas9 and sgRNA variants was quantified as NHEJ-induced mutation rates. We found that offtarget cleavage resulting from the sgRNA variants occurred with a DNA bulge or a sgRNA bulge at multiple positions in the guide strands, sometimes at levels comparable to or even higher than those of original sgRNAs. We also examined the Cas9-mediated mutagenesis at potential offtarget loci in the human genome carrying single-base DNA bulges or sgRNA bulges together with a range of base mismatches, and confirmed off-target sites with mutation frequencies up to 45.5%. Our results indicate that off-target effects may limit the applications of Cas9-mediated gene modification, especially in large mammalian genomes that contain multiple DNA sequences differing by only a few bases due to mismatches, insertions or deletions. Future design of specific sgRNAs would require a search for genomic sites with base-pair mismatches, insertions and deletions, such as with our newly developed program COSMID, to fully analyze CRISPR/Cas9 off-target activity.
250. Helper-Dependent Ad5/35 Vectors for ZFNMediated Gene Editing in Hematopoietic Stem Cells
Kamola Saydaminova,1 Hongjie Wang,1 Eirini Papapetrou,1 Michael Holmes,2 Philip Gregory,2 Donna Palmer,3 Philip Ng,3 Anja Ehrhardt,4 Andre Lieber.1 1 University of Washington, Seattle; 2Sangamo Biosciences, Richmond; 3Baylor College of Medicine, Houston; 4University Witten/Herdecke, Witten, Germany.
Ad5/35 vectors efficiently transduce hematopoietic stem/progenitor cells (HSCs) in vitro, and as we have recently shown also in vivo after HSC mobilization. In contrast to first-generation Ad5/35 vectors, helper-dependent (HD) Ad5/35 do not affect the clonogenic and engraftment potential of transduced HSCs. A problem with the production of HD Ad5/35 vectors expressing ZFNs is that they kill the Ad producer cells most potentially due to excessively high expression levels during viral replication. To generate HD Ad5/35 vectors that would express ZFNs in human hematopoietic CD34+ stem cells, we used a miRNA-regulated gene expression system. If the RNA of a transgene contains a target site for a miRNA that is expressed at high levels in HD-Ad5/35 producer (293-Cre) cells, the transgene mRNA will be degraded in this cell type. Using microarrays, we established the miRNA expression profiles in Ad5/35-infected 293-Cre and human CD34+ cells. We then selected two miRNAs (has-miR1835p and has-miR218-5p), which had the highest expression levels in 293-Cre cells and that were absent in CD34+ cells. Deep sequencing of these miRNA revealed miRNA species that, in addition to the 22nt canonical sequence, contained additional nucleotides but with lower frequency. We then inserted 4 target sites with 100% homology to the selected miRNAs into the 3’UTR of a GFP gene, which was under the control of an EF1alpha promoter, a promoter that is highly active in CD34+ cells. The GFP expression cassettes were inserted into an Ad5/35 vector. The vectors also contained a PGK promoter-driven mCherry expression cassette that was not regulated by the selected Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy
miRNAs. We then transduced human CD34+ cells and 293-Cre cells with the vectors and analyzed GFP and mCherry expression 48 hours later by flow cytometry. In 293-Cre cells, mCherry expression levels were comparable for all vectors, while GFP expression was suppressed with vectors that contained the miRNA target sites. Based on the ratio of GFP to mCherry levels, the greatest suppression was achieved with vector that contained target sequences of miR183 and miR218 including three additional nucleotides at their 3’ end. Both GFP and mCherry expression were comparably high in CD34+ cells for all vectors. By using the miRNA-183/218 regulated gene expression system, we were able to produce an HD-Ad5/35 vector that expresses a ZFN specific to human CCR5. This vector conferred efficient ZFN expression in CD34+ cells resulting in site-specific DNA cleavage in >20% of CCR5 alleles as detected by mismatchsensitive surveyor nuclease PCR. We are currently testing the persistence of site-specific gene modification after HSC transduction in vitro and in vivo. Our data show i) that considering heterogeneous miRNA processing and optimizing the target site design can increase the specificity of a miRNA regulated gene expression and ii) that miRNA based suppression of ZFN expression in 293-Cre cells allows for the production of HD-Ad vectors at high titers.
251. TALENs Facilitate Targeted Genome Editing in Human Cells With High Specificity and Low Cytotoxicity Claudio Mussolino,1,2 Jamal Alzubi,1,2 Eli J. Fine,3 Robert Morbitzer,4 Thomas J. Cradick,3 Thomas Lahaye,4,5 Gang Bao,3 Toni Cathomen.1,2 1 Institute for Cell and Gene Therapy & Center for Chronic Immunodeficiency, University Medical Center Freiburg, Freiburg, Germany; 2Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany; 3Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA; 4Institute of Genetics, LudwigMaximilians-University Munich, Munich, Germany; 5Center for Plant Molecular Biology, Eberhard-Karls-University, Tübingen, Germany.
Designer nucleases have been successfully employed to modify the genomes of various model organisms and human cell types. While the specificity of zinc-finger nucleases (ZFNs) and RNAguided endonucleases (RGNs) has been assessed to some extent, little data is available for TALE-based nucleases (TALENs). Here, we have engineered TALEN pairs targeting three human loci (CCR5, AAVS1, IL2RG) and performed a detailed analysis of their activity, toxicity, and specificity. The TALENs showed comparable activity to benchmark ZFNs, with allelic gene disruption frequencies of 1530% in human cells. Notably, TALEN expression was overall marked by a low cytotoxicity and the absence of cell cycle aberrations. Bioinformatics-based analysis of designer nuclease specificity confirmed partly substantial off-target activity of ZFNs targeting CCR5 and AAVS1 at six known and five novel sites, respectively. In contrast, only marginal off-target cleavage activity was detected at four out of 49 predicted off-target sites for CCR5 and AAVS1specific TALENs. The rational design of a CCR5-specific TALEN pair decreased off-target activity at the closely related CCR2 locus considerably, consistent with fewer genomic rearrangements between the two loci. In conclusion, our results link nuclease-associated toxicity to off-target cleavage activity and corroborate TALENs as a highly specific platform for future clinical translation.
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