5 results on '"Firth, Mike"'
Search Results
2. Universal toxin-based selection for precise genome engineering in human cells
- Author
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Li, Songyuan; https://orcid.org/0000-0003-2085-9253, Akrap, Nina, Cerboni, Silvia, Porritt, Michelle J; https://orcid.org/0000-0003-1700-0085, Wimberger, Sandra, Lundin, Anders; https://orcid.org/0000-0003-3131-8022, Möller, Carl, Firth, Mike, Gordon, Euan, Lazovic, Bojana, Sieńska, Aleksandra, Pane, Luna Simona, Coelho, Matthew A; https://orcid.org/0000-0003-3737-2468, Ciotta, Giovanni, Pellegrini, Giovanni; https://orcid.org/0000-0001-9593-5578, Sini, Marcella, Xu, Xiufeng, Mitra, Suman; https://orcid.org/0000-0002-3426-371X, Bohlooly-Y, Mohammad, Taylor, Benjamin J M; https://orcid.org/0000-0001-6101-3786, Sienski, Grzegorz; https://orcid.org/0000-0002-2730-7710, Maresca, Marcello; https://orcid.org/0000-0003-0796-661X, Li, Songyuan; https://orcid.org/0000-0003-2085-9253, Akrap, Nina, Cerboni, Silvia, Porritt, Michelle J; https://orcid.org/0000-0003-1700-0085, Wimberger, Sandra, Lundin, Anders; https://orcid.org/0000-0003-3131-8022, Möller, Carl, Firth, Mike, Gordon, Euan, Lazovic, Bojana, Sieńska, Aleksandra, Pane, Luna Simona, Coelho, Matthew A; https://orcid.org/0000-0003-3737-2468, Ciotta, Giovanni, Pellegrini, Giovanni; https://orcid.org/0000-0001-9593-5578, Sini, Marcella, Xu, Xiufeng, Mitra, Suman; https://orcid.org/0000-0002-3426-371X, Bohlooly-Y, Mohammad, Taylor, Benjamin J M; https://orcid.org/0000-0001-6101-3786, Sienski, Grzegorz; https://orcid.org/0000-0002-2730-7710, and Maresca, Marcello; https://orcid.org/0000-0003-0796-661X
- Abstract
Prokaryotic restriction enzymes, recombinases and Cas proteins are powerful DNA engineering and genome editing tools. However, in many primary cell types, the efficiency of genome editing remains low, impeding the development of gene- and cell-based therapeutic applications. A safe strategy for robust and efficient enrichment of precisely genetically engineered cells is urgently required. Here, we screen for mutations in the receptor for Diphtheria Toxin (DT) which protect human cells from DT. Selection for cells with an edited DT receptor variant enriches for simultaneously introduced, precisely targeted gene modifications at a second independent locus, such as nucleotide substitutions and DNA insertions. Our method enables the rapid generation of a homogenous cell population with bi-allelic integration of a DNA cassette at the selection locus, without clonal isolation. Toxin-based selection works in both cancer-transformed and non-transformed cells, including human induced pluripotent stem cells and human primary T-lymphocytes, as well as it is applicable also in vivo, in mice with humanized liver. This work represents a flexible, precise, and efficient selection strategy to engineer cells using CRISPR-Cas and base editing systems.
- Published
- 2021
3. In vivo phage display identifies novel peptides for cardiac targeting.
- Author
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Ivanova A, Kohl F, González-King Garibotti H, Chalupska R, Cvjetkovic A, Firth M, Jennbacken K, Martinsson S, Silva AM, Viken I, Wang QD, Wiseman J, and Dekker N
- Subjects
- Humans, Animals, Mice, Peptide Library, Hep G2 Cells, Cell Surface Display Techniques methods, Drug Delivery Systems, High-Throughput Nucleotide Sequencing, Heart Failure metabolism, Heart Failure therapy, Myocytes, Cardiac metabolism, Peptides metabolism, Induced Pluripotent Stem Cells metabolism, Induced Pluripotent Stem Cells cytology
- Abstract
Heart failure remains a leading cause of mortality. Therapeutic intervention for heart failure would benefit from targeted delivery to the damaged heart tissue. Here, we applied in vivo peptide phage display coupled with high-throughput Next-Generation Sequencing (NGS) and identified peptides specifically targeting damaged cardiac tissue. We established a bioinformatics pipeline for the identification of cardiac targeting peptides. Hit peptides demonstrated preferential uptake by human induced pluripotent stem cell (iPSC)-derived cardiomyocytes and immortalized mouse HL1 cardiomyocytes, without substantial uptake in human liver HepG2 cells. These novel peptides hold promise for use in targeted drug delivery and regenerative strategies and open new avenues in cardiovascular research and clinical practice., (© 2024. The Author(s).)
- Published
- 2024
- Full Text
- View/download PDF
4. Rapid target validation in a Cas9-inducible hiPSC derived kidney model.
- Author
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Shamshirgaran Y, Jonebring A, Svensson A, Leefa I, Bohlooly-Y M, Firth M, Woollard KJ, Hofherr A, Rogers IM, and Hicks R
- Subjects
- A549 Cells, Animals, Cell Differentiation, Cells, Cultured, Doxycycline pharmacology, Gene Knockout Techniques, HEK293 Cells, Humans, Kidney cytology, Organoids drug effects, Polycystic Kidney, Autosomal Dominant drug therapy, RNA, Guide, CRISPR-Cas Systems genetics, Swine, TRPP Cation Channels genetics, CRISPR-Cas Systems, Drug Discovery methods, Gene Editing methods, Induced Pluripotent Stem Cells drug effects, Molecular Targeted Therapy, Polycystic Kidney, Autosomal Dominant genetics
- Abstract
Recent advances in induced pluripotent stem cells (iPSCs), genome editing technologies and 3D organoid model systems highlight opportunities to develop new in vitro human disease models to serve drug discovery programs. An ideal disease model would accurately recapitulate the relevant disease phenotype and provide a scalable platform for drug and genetic screening studies. Kidney organoids offer a high cellular complexity that may provide greater insights than conventional single-cell type cell culture models. However, genetic manipulation of the kidney organoids requires prior generation of genetically modified clonal lines, which is a time and labor consuming procedure. Here, we present a methodology for direct differentiation of the CRISPR-targeted cell pools, using a doxycycline-inducible Cas9 expressing hiPSC line for high efficiency editing to eliminate the laborious clonal line generation steps. We demonstrate the versatile use of genetically engineered kidney organoids by targeting the autosomal dominant polycystic kidney disease (ADPKD) genes: PKD1 and PKD2. Direct differentiation of the respective knockout pool populations into kidney organoids resulted in the formation of cyst-like structures in the tubular compartment. Our findings demonstrated that we can achieve > 80% editing efficiency in the iPSC pool population which resulted in a reliable 3D organoid model of ADPKD. The described methodology may provide a platform for rapid target validation in the context of disease modeling., (© 2021. The Author(s).)
- Published
- 2021
- Full Text
- View/download PDF
5. In vivo CRISPR editing with no detectable genome-wide off-target mutations.
- Author
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Akcakaya P, Bobbin ML, Guo JA, Malagon-Lopez J, Clement K, Garcia SP, Fellows MD, Porritt MJ, Firth MA, Carreras A, Baccega T, Seeliger F, Bjursell M, Tsai SQ, Nguyen NT, Nitsch R, Mayr LM, Pinello L, Bohlooly-Y M, Aryee MJ, Maresca M, and Joung JK
- Subjects
- Animals, CRISPR-Associated Proteins genetics, Female, Humans, INDEL Mutation, Male, Mice, Mice, Inbred C57BL, Proprotein Convertase 9 genetics, Transgenes genetics, CRISPR-Associated Proteins metabolism, CRISPR-Cas Systems genetics, Gene Editing methods, Gene Editing standards, Genome genetics, Mutation, Substrate Specificity genetics
- Abstract
CRISPR-Cas genome-editing nucleases hold substantial promise for developing human therapeutic applications
1-6 but identifying unwanted off-target mutations is important for clinical translation7 . A well-validated method that can reliably identify off-targets in vivo has not been described to date, which means it is currently unclear whether and how frequently these mutations occur. Here we describe 'verification of in vivo off-targets' (VIVO), a highly sensitive strategy that can robustly identify the genome-wide off-target effects of CRISPR-Cas nucleases in vivo. We use VIVO and a guide RNA deliberately designed to be promiscuous to show that CRISPR-Cas nucleases can induce substantial off-target mutations in mouse livers in vivo. More importantly, we also use VIVO to show that appropriately designed guide RNAs can direct efficient in vivo editing in mouse livers with no detectable off-target mutations. VIVO provides a general strategy for defining and quantifying the off-target effects of gene-editing nucleases in whole organisms, thereby providing a blueprint to foster the development of therapeutic strategies that use in vivo gene editing.- Published
- 2018
- Full Text
- View/download PDF
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