Engineered human Diamond-Blackfan anemia disease model confirms therapeutic effects of clinically applicable lentiviral vector at single-cell resolution

Abstract

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.

Figure 1.

Genome editing via homologous recombination using electroporation leads to strongly decreased cell viability. (A) Schematic overview of RPS19 editing strategy. The RPS19 gene is targeted with Cas9, and gene fluorescent protein (GFP)-encoding adeno-associated virus (AAV) with homology arms flanking the cut site. Successful integration of the homology-directed repair (HDR) template leads to disruption of RPS19 expression, which allows traceable GFP expression. (B) Timeline of experiment. (C and D) Representative FACS plots of cell viability and GFP expression of cells on (C) day 1 and (D) day 4 after electroporation. (E) Percentage of live cell recovery relative to completely untreated cells on days 1 and 4 after electroporation (*P<0.05 by t-test, N=3). (F) Relative percentage of GFP⁺ cells compared to total recovered live cell numbers of the untreated condition, on days 1 and 4 after electroporation (*P<0.05, **P<0.01, ****P<0.0001 by one-way ANOVA test, N=3). LHA: left homology arm; RHA: right homology arm; HSPC: hemotopoietic stem and progenitor cells.

Figure 2.

Nanostraw-mediated Cas9 mRNA delivery enables CD45 knockout in human hematopoietic stem and progenitor cells. (A) Schematic overview and false-colored scanning electron microscope picture of the nanostraw delivery system. (B) Timeline of experiment. (C) Representative FACS plots of cell viability (7-AAD-/Annexin-) on day 1 (D1). (D) Representative FACS plots of edited cells on day 4. (E) Percentage of live cell recovery compared to completely untreated cells on days 1 and 4 upon Cas9 mRNA and RNP delivery to knockout CD45 (*P<0.05, **P<0.01, ***P<0.001 by one-way ANOVA test, N=3). (F) Efficiency of CD45 knockout in live (7-AAD) cells on day 4 (***P<0.001, ****P<0.0001 by one-way ANOVA test, N=3). HSPC: hematopoietic stem and progenitor cells.

Figure 3.

Delivery of Cas9 mRNA with nanostraws enables the recovery of heterozygous GFP⁺ RPS19-deficient hematopoietic stem and progenitor cells with reduced cell viability. (A) Timeline of experiment. (B) Representative FACS plots of cell viability and GFP⁺ cells on (left) day 1 and (right) day 4. (C) Percentage of live cell recovery compared to completely untreated cells on days 1 (D1) and 4 upon using nanostraw to deliver Cas9 mRNA (*P<0.05 by t-test, N=3). (D) Relative percentage of GFP⁺ cells compared to total recovered live cell numbers of the untreated condition, using a nanostraw to deliver Cas9 mRNA on days 1 and 4 (**P<0.01, ***P<0.001 by t-test, N=3). (E) Number of colonies for BFU-E, CFU-G/M/GM and CFU-GEMM in each dish after culture with methylcellulose media for 14 days (^####P<0.0001, ^#P<0.05 compared with the same colony category in the RPS19-deficient group by unpaired Mann-Whitney test, N=12 dishes in each group). (F) Ratio of edited allele (HDR-RPS19-GFP) to reference gene (APOE) by ddPCR (a total of 20 colonies in the mock group, and 100 colonies in the RPS19-deficient group were analyzed). HSPC: hematopoietic stem and progenitor cells.

Figure 4.

RPS19-deficient cells showed impaired erythroid differentiation ability which can be rescued by the lentiviral EFS-RPS19 vector. (A) Schematic overview of erythroid differentiation analysis of RPS19-deficient CD34⁺ cord blood hematopoietic stem and progenitor cells (HSPC). (B) Expression of endogenous RPS19 (*P <0.05, **P <0.01, ***P <0.001 by two-way ANOVA test, N=3). (C) Transgene RPS19 (coRPS19) expression (****P<0.0001 by two-way ANOVA test, N=3). (D) Representative FACS plots of GFP⁺ cells for erythroid differentiation on day (D) 10 in each group. (E) Statistical analysis of each population during erythroid differentiation from stage I (day 6) to stage II (day 10) (^#P<0.001 compared to the CD34 and the Cas9 only groups; **P<0.01 compared to the RPS19-D group; ***P<0.001 compared to the RPS19-D group; ****P<0.0001 compared to the RPS19-D group, by two-way ANOVA test, N=3). (F) Formation of red blood cell pellets on day 21 in each group. EM: expansion medium; ED: erythroid differentiation.

Figure 5.

Clustering analysis and differentiation trajectory during erythroid differentiation. (A) Heatmap of the mean expression value of manually selected marker genes for each cluster. CMP: common myeloid progenitors; GMP: granulocyte-macrophage progenitors; MKP: megakaryocyte progenitors; EEP: early erythroid progenitors; LEP: late erythroid progenitors. (B) UMAP plot colored by (left) cluster and (middle) UMAP plot split by tissue. (Right) Frequency of each cluster in CD34, RPS19-D, and LV-RPS19 groups. (C) Partition-based approximate graph abstraction (PAGA) initialized embedding and PAGA graph of the differentiation trajectory. Size of dots is proportional to number of cells in the clusters.

Figure 6.

RPS19-deficient cells show abnormal cell cycle with activation of inflammatory signaling pathway that can be rescued by EFS-RPS19. (A) Donut plots showing percentages of cells in G1, S, and G2M phase in erythroid progenitor (EP) clusters. (B) Bubble plot showing abnormal significantly enriched signaling pathways (NOM: P<0.05) in the DBA EP 1 cluster from the RPS19-deficient group compared with the LEP 1 cluster from the CD34 group and Rescued EP cluster from the LV-RPS19 group. Pathways are from the Hallmark gene sets of the Molecular Signatures Database. (C) Dot plot showing the 20 top differentially expressed genes in the indicated clusters.