Engineered human Diamond-Blackfan anemia disease model confirms therapeutic effects of clinically applicable lentiviral vector at single-cell resolution
Yang Liu,
Ludwig Schmiderer,
Martin Hjort,
Stefan Lang,
Tyra Bremborg,
Anna Rydström,
Axel Schambach,
Jonas Larsson,
Stefan Karlsson
Affiliations
Yang Liu
Division of Molecular Medicine and Gene Therapy, Lund Stem Cell Center, Lund University, Lund 22184, Sweden; Department of Medicine, Huddinge, Karolinska Institutet, 14157 Stockholm
Ludwig Schmiderer
Division of Molecular Medicine and Gene Therapy, Lund Stem Cell Center, Lund University, Lund 22184
Martin Hjort
Chemical Biology and Therapeutics, Department of Experimental Medical Science, Lund University, 22100 Lund, Sweden; Navan Technologies, MBC Biolabs, San Carlos, CA 94070; NanoLund, Lund University, Box 118, 22100 Lund
Stefan Lang
Division of Molecular Hematology and Stem Cell Center, Lund University, Lund
Tyra Bremborg
Division of Molecular Medicine and Gene Therapy, Lund Stem Cell Center, Lund University, Lund 22184
Anna Rydström
Division of Molecular Medicine and Gene Therapy, Lund Stem Cell Center, Lund University, Lund 22184
Axel Schambach
Institute of Experimental Hematology, Hannover Medical School, Hannover 30625, Germany; Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School
Jonas Larsson
Division of Molecular Medicine and Gene Therapy, Lund Stem Cell Center, Lund University, Lund 22184
Stefan Karlsson
Division of Molecular Medicine and Gene Therapy, Lund Stem Cell Center, Lund University, Lund 22184
Diamond-Blackfan anemia is a rare genetic bone marrow failure disorder which is usually caused by mutations in ribosomal protein genes. In the present study, we generated a traceable RPS19-deficient cell model using CRISPR-Cas9 and homology-directed repair to investigate the therapeutic effects of a clinically applicable lentiviral vector at single-cell resolution. We developed a gentle nanostraw delivery platform to edit the RPS19 gene in primary human cord bloodderived CD34+ hematopoietic stem and progenitor cells. The edited cells showed expected impaired erythroid differentiation phenotype, and a specific erythroid progenitor with abnormal cell cycle status accompanied by enrichment of TNFα/NF-κB and p53 signaling pathways was identified by single-cell RNA sequencing analysis. The therapeutic vector could rescue the abnormal erythropoiesis by activating cell cycle-related signaling pathways and promoted red blood cell production. Overall, these results establish nanostraws as a gentle option for CRISPR-Cas9- based gene editing in sensitive primary hematopoietic stem and progenitor cells, and provide support for future clinical investigations of the lentiviral gene therapy strategy.
