Gene therapy restores the transcriptional program of hematopoietic stem cells in Fanconi anemia

Abstract

Clinical trials have shown that lentiviral-mediated gene therapy can ameliorate bone marrow failure (BMF) in nonconditioned Fanconi anemia (FA) patients resulting from the proliferative advantage of corrected FA hematopoietic stem and progenitor cells (HSPC). However, it is not yet known if gene therapy can revert affected molecular pathways in diseased HSPC. Single-cell RNA sequencing was performed in chimeric populations of corrected and uncorrected HSPC co-existing in the BM of gene therapy-treated FA patients. Our study demonstrates that gene therapy reverts the transcriptional signature of FA HSPC, which then resemble the transcriptional program of healthy donor HSPC. This includes a down-regulated expression of TGF-β and p21, typically up-regulated in FA HSPC, and upregulation of DNA damage response and telomere maintenance pathways. Our results show for the first time the potential of gene therapy to rescue defects in the HSPC transcriptional program from patients with inherited diseases; in this case, in FA characterized by BMF and cancer predisposition.

Figure 1.

Experimental design of scRNAseq analyses performed in Fanconi anemia group A patients 2-5 years afer lentiviral-mediated gene therapy. Four Fanconi anemia group A (FA-A) patients who had been treated with ex vivo lentiviral-mediated gene therapy in the absence of conditioning were included in this study. At 2-5 years post gene therapy, these patients harbored a chimeric population of corrected and uncorrected hematopoietic stem and progenitor cells (HSPC) in their bone marrow (BM). Aliquots of BM CD34⁺ cells from these patients were sorted and processed for single-cell RNA-seq. Bioinformatic analyses comparatively investigated changes in the transcriptional program of corrected versus uncorrected HSPC, co-existing in each of the gene therapy-treated patients. HSC: hematopoietic stem cell.

Figure 2.

scRNAseq analysis of corrected and uncorrected hematopoietic stem and progenitor cells (CD34+ cells) from Fan-coni anemia group A patients 2-5 years after lentiviral-mediated gene therapy. (A) UMAP plot showing the clustering analysis for BM CD34⁺ cells from a Fanconi anemia group A (FA-A) patient previously treated by gene therapy (FA-02006 patient as an example; see Online Supplementary Figure S1 for patients FA-02002, FA-02004 and FA-02008). A total of 12 clusters were identified, spanning the different hematopoietic stem and progenitor cell (HSPC) CD34⁺ subpopulations. Identified clusters include an HSC cluster (hematopoietic stem cell; brown). Clusters with megakaryocytic-erythroid identity include MEP (mega-karyocyte-erythroid progenitor; purple), erythroid (erythroid progenitor; pink), and basophils (basophil progenitor; light pink). Clusters with lympho-myeloid identity include LMPP (lymphoid-primed multipotent progenitor; light blue), Cycling-LMPP (blue), CLP (common lymphoid progenitor; orange), GMP1 and GMP2 (granulocyte-monocyte progenitor; light green and green), monocytes (monocyte progenitor; red), DC (dendritic cell progenitor; nude), and PreB (B-cell progenitor; light purple). (B) Same UMAP as shown in (A), highlighting the distribution of FANCA⁺ cells (FANCA mRNA detectable; red) versus FANCA- cells (FANCA mRNA levels are below detection limit; blue). (C) Barplot showing the total number of cells in the different HSPC CD34⁺ populations corresponding to the four gene therapy-treated patients. In each case, the number of FANCA⁺ (red) and FANCA-(blue) cells is shown. (D) Boxplot of integrated and normalized FANCA gene expression in FANCA⁺, depicted by cell type and FA individual (N=3). (E) Barplot representation of the differential expression analysis between FANCA⁺ and FANCA- for each FA patient (N=4) and for each of the HSPC CD34⁺ populations. In each case, the number of up-regulated and down-regulated genes is shown. Up-regulated genes were defined as logFC>0.25 (orange), up-regulated and significant genes were defined as a logFC>0.25 and adjusted P-value <0.05 (red), down-regulated genes were defined as a logFC<-0.25 (light blue) and down-regulated and significant genes were defined as logFC<-0.25 and adjusted P-value <0.05 (dark blue). The number of genes with the same behavior (up-regulated or down-regulated) in the four individuals is shown below each HSPC (labeled as “shared”).

Figure 3.

Comparisons of the gene expression signature between corrected and uncorrected hematopoietic stem and progenitor cells co-existing in gene therapy treated Fanconi anemia patients. (A) Left, large panel: UMAP plot showing the clustering analysis of CD34⁺ bone marrow (BM) cells after integration of data from a gene therapy-treated Fanconi anemia group A (FA-A) patient and a healthy donor (HD). (FA-02006 is included as a representative example; see Online Supplementary Figure S3 for the other individuals). Right, small panel: the same UMAP as shown in left panel but highlighting the distribution of HD hematopoietic stem and progenitor cells (HSPC) (yellow), FANCA⁺ HSPC (red) and FANCA-HSPC (blue). Cluster identification as in Figure 1. (B) Boxplot representation of normalized single-cell FANCA expression of FANCA⁺ cells (red) and HD cells (yellow) by HSPC cluster. For each cell type, differences in the expression levels between the ectopic expression of FANCA from corrected FA CD34⁺ cells and expression levels corresponding to HD CD34⁺ cells are shown. *Adjusted P<0.05; **adjusted P<0.01; ***adjusted P<0.001. (C) Results associated to three differential expression contrasts: FANCA⁺ versus FANCA- HSPC from GT-treated FA patients; HD HSPC versus FANCA- HSPC from GT-treated patients; and HD HSPC versus FANCA⁺ HSPC from GT-treated patients. Second row shows the twelve different CD34⁺ cell types; third row shows the sample identification from each of the four GT-treated patients. Up-regulated genes (logFC>0) are shown in orange; those with significant upregulation in red (logFC>0.25 and adjusted P<0.05). Down-regulated genes (logFC<0) are shown in light blue and those with significant downregulation in dark blue (logFC<-0.25 and adjusted P<0.05). Unsupervised hierarchical clustering using Pearson distance and average linkage method was applied for gene classification. Genes included in the heatmap are those that for at least one cell type are identified as differentially expressed (abs(logFC)>0.25 and adjusted P<0.05) in “at least three patients”, and “showing the same direction of the change for the three patients”, when considering the contrast FANCA⁺ versus FANCA- (N=152; entire list available in Online Supplementary Table S2). FANCA was excluded from the list.

Figure 4.

Enrichment score of restored gene expression pathways associated to the ectopic expression of FANCA in hematopoietic stem and progenitor cells from gene therapy-treated Fanconi anemia patients. Dotplot depicts the outcome of the Gene Set Enrichment Analysis for a selected set of pathways. For each patient, paired comparisons between FANCA⁺ versus FANCA^- hematopoietic stem and progenitor cells (HSPC) from gene therapy (GT)-treated Fanconi anemia (FA) patients and healthy donor (HD) HSPC versus FANCA^- HSPC are shown. The dotplot depicts the statistical significance (color) and enrichment score (size). The complete name of the pathways with numbers are: (i) telomere maintenance via semi-conservative replication, (ii) positive regulation of G2/M transition of mitotic cell cycle, (iii) negative regulation of G2/M transition of mitotic cell cycle, (iv) double-strand break repair via nonhomologous end joining, (v) double-strand break repair via homologous recombination, and (vi) DNA damage response, detection of DNA damage.

Figure 5.

Restored gene expression of key pathways of CD34⁺ cells commited to the monocyte lineage in corrected hematopoietic stem and progenitor cells from gene therapy-treated Fanconi anemia patients. (A) Up-regulated and down-regulated genes implicated in cell cycle control. The figure represents the logFC associated to two contrasts (see Contrast box): FANCA⁺ versus FANCA^-hematopoietic stem and progenitor cells (HSPC) from gene therapy (GT)-treated Fanconi anemia (FA) patients (internal crowns), and healthy donor (HD) HSPC versus FANCA^- HSPC from GT-treated patients (external crowns). Each external and internal crown is divided in four parts (see Sample box), each of them representing one FA patient. Up-regulated genes are shown in red and down-regulated genes in blue. (B) Same representation as in (A) showing changes in the expression of genes participating in the FA/BRCA pathway.

Figure 6.

Functional implications associated to the restoration of DNA repair and telomere biology pathways. (A) Survival to mitomycin C (MMC; 10 nM) of bone marrow (BM) colony forming cells (CFC) from Fanconi anemia group A complementation group (FA-A) patients shown in Figures 1-5 prior and after infusion of gene-corrected cells. (B) Percentage of peripheral blood (PB) T cells with chromosomal aberrations induced by diepoxibutane (DEB), prior to and after gene therapy. (C) Analysis of the telomere length of PB cells from FA-A patients shown in Figures 1-5 at 15^th day post infusion, when corrected cells were still undetectable, to the 2^nd-5^th year post infusion of gene-corrected cells. As a negative control, three FA patients with no significant engraftment of genecorrected cells are included. Dashed lines correspond to telomere lengths from healthy donor (HD) age-matched PB cells. Time points of the MMC, DEB and telomere analyses are the same or the closest ones corresponding to the scRNAseq analyses shown in Figures 1-5. Percentages of survival in (A) are deduced from colony counts in cultures grown in the absence of MMC. Dashed lines (A) and (B) correspond to mean values determined in samples from HD. GT: gene therapy; d: days; y: years.