TRIAMF: A New Method for Delivery of Cas9 Ribonucleoprotein Complex to Human Hematopoietic Stem Cells
Abstract CRISPR/Cas9 mediated gene editing of patient-derived hematopoietic stem and progenitor cells (HSPCs) ex vivo followed by autologous transplantation of the edited HSPCs back to the patient can provide a potential cure for monogenic blood disorders such as β-hemoglobinopathies. One challenge...
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doaj-7126e75f7f804ba7a87cf878647861232020-12-08T04:57:57ZengNature Publishing GroupScientific Reports2045-23222018-11-018111110.1038/s41598-018-34601-6TRIAMF: A New Method for Delivery of Cas9 Ribonucleoprotein Complex to Human Hematopoietic Stem CellsJonathan Yen0Michael Fiorino1Yi Liu2Steve Paula3Scott Clarkson4Lisa Quinn5William R. Tschantz6Heath Klock7Ning Guo8Carsten Russ9Vionnie W. C. Yu10Craig Mickanin11Susan C. Stevenson12Cameron Lee13Yi Yang14Chemical Biology and Therapeutics, Novartis Institutes for BioMedical ResearchNIBR Informatics, Novartis Institutes for BioMedical ResearchChemical Biology and Therapeutics, Novartis Institutes for BioMedical ResearchChemical Biology and Therapeutics, Novartis Institutes for BioMedical ResearchChemical Biology and Therapeutics, Novartis Institutes for BioMedical ResearchBiotherapeutic and Analytical Tech, Novartis Institutes for BioMedical ResearchBiotherapeutic and Analytical Tech, Novartis Institutes for BioMedical ResearchBiotherapeutics & Biotechnology, The Genomics Institute of the Novartis Research FoundationChemical Biology and Therapeutics, Novartis Institutes for BioMedical ResearchChemical Biology and Therapeutics, Novartis Institutes for BioMedical ResearchChemical Biology and Therapeutics, Novartis Institutes for BioMedical ResearchChemical Biology and Therapeutics, Novartis Institutes for BioMedical ResearchChemical Biology and Therapeutics, Novartis Institutes for BioMedical ResearchGlobal Discovery Chemistry, Novartis Institutes for BioMedical ResearchChemical Biology and Therapeutics, Novartis Institutes for BioMedical ResearchAbstract CRISPR/Cas9 mediated gene editing of patient-derived hematopoietic stem and progenitor cells (HSPCs) ex vivo followed by autologous transplantation of the edited HSPCs back to the patient can provide a potential cure for monogenic blood disorders such as β-hemoglobinopathies. One challenge for this strategy is efficient delivery of the ribonucleoprotein (RNP) complex, consisting of purified Cas9 protein and guide RNA, into HSPCs. Because β-hemoglobinopathies are most prevalent in developing countries, it is desirable to have a reliable, efficient, easy-to-use and cost effective delivery method. With this goal in mind, we developed TRansmembrane Internalization Assisted by Membrane Filtration (TRIAMF), a new method to quickly and effectively deliver RNPs into HSPCs by passing a RNP and cell mixture through a filter membrane. We achieved robust gene editing in HSPCs using TRIAMF and demonstrated that the multilineage colony forming capacities and the competence for engraftment in immunocompromised mice of HSPCs were preserved post TRIAMF treatment. TRIAMF is a custom designed system using inexpensive components and has the capacity to process HSPCs at clinical scale.https://doi.org/10.1038/s41598-018-34601-6Hematopoietic Stem And Progenitor Cells (HSPCs)Purified Cas9 ProteinHSPC ExpansionNeon ElectroporationSyringe Connector |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Jonathan Yen Michael Fiorino Yi Liu Steve Paula Scott Clarkson Lisa Quinn William R. Tschantz Heath Klock Ning Guo Carsten Russ Vionnie W. C. Yu Craig Mickanin Susan C. Stevenson Cameron Lee Yi Yang |
spellingShingle |
Jonathan Yen Michael Fiorino Yi Liu Steve Paula Scott Clarkson Lisa Quinn William R. Tschantz Heath Klock Ning Guo Carsten Russ Vionnie W. C. Yu Craig Mickanin Susan C. Stevenson Cameron Lee Yi Yang TRIAMF: A New Method for Delivery of Cas9 Ribonucleoprotein Complex to Human Hematopoietic Stem Cells Scientific Reports Hematopoietic Stem And Progenitor Cells (HSPCs) Purified Cas9 Protein HSPC Expansion Neon Electroporation Syringe Connector |
author_facet |
Jonathan Yen Michael Fiorino Yi Liu Steve Paula Scott Clarkson Lisa Quinn William R. Tschantz Heath Klock Ning Guo Carsten Russ Vionnie W. C. Yu Craig Mickanin Susan C. Stevenson Cameron Lee Yi Yang |
author_sort |
Jonathan Yen |
title |
TRIAMF: A New Method for Delivery of Cas9 Ribonucleoprotein Complex to Human Hematopoietic Stem Cells |
title_short |
TRIAMF: A New Method for Delivery of Cas9 Ribonucleoprotein Complex to Human Hematopoietic Stem Cells |
title_full |
TRIAMF: A New Method for Delivery of Cas9 Ribonucleoprotein Complex to Human Hematopoietic Stem Cells |
title_fullStr |
TRIAMF: A New Method for Delivery of Cas9 Ribonucleoprotein Complex to Human Hematopoietic Stem Cells |
title_full_unstemmed |
TRIAMF: A New Method for Delivery of Cas9 Ribonucleoprotein Complex to Human Hematopoietic Stem Cells |
title_sort |
triamf: a new method for delivery of cas9 ribonucleoprotein complex to human hematopoietic stem cells |
publisher |
Nature Publishing Group |
series |
Scientific Reports |
issn |
2045-2322 |
publishDate |
2018-11-01 |
description |
Abstract CRISPR/Cas9 mediated gene editing of patient-derived hematopoietic stem and progenitor cells (HSPCs) ex vivo followed by autologous transplantation of the edited HSPCs back to the patient can provide a potential cure for monogenic blood disorders such as β-hemoglobinopathies. One challenge for this strategy is efficient delivery of the ribonucleoprotein (RNP) complex, consisting of purified Cas9 protein and guide RNA, into HSPCs. Because β-hemoglobinopathies are most prevalent in developing countries, it is desirable to have a reliable, efficient, easy-to-use and cost effective delivery method. With this goal in mind, we developed TRansmembrane Internalization Assisted by Membrane Filtration (TRIAMF), a new method to quickly and effectively deliver RNPs into HSPCs by passing a RNP and cell mixture through a filter membrane. We achieved robust gene editing in HSPCs using TRIAMF and demonstrated that the multilineage colony forming capacities and the competence for engraftment in immunocompromised mice of HSPCs were preserved post TRIAMF treatment. TRIAMF is a custom designed system using inexpensive components and has the capacity to process HSPCs at clinical scale. |
topic |
Hematopoietic Stem And Progenitor Cells (HSPCs) Purified Cas9 Protein HSPC Expansion Neon Electroporation Syringe Connector |
url |
https://doi.org/10.1038/s41598-018-34601-6 |
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