Myelodysplasia after clonal hematopoiesis with APOBEC3-mediated CYBB inactivation in retroviral gene therapy for X-CGD

Abstract

Stem cell gene therapy using the MFGS-gp91^phox retroviral vector was performed on a 27-year-old patient with X-linked chronic granulomatous disease (X-CGD) in 2014. The patient's refractory infections were resolved, whereas the oxidase-positive neutrophils disappeared within 6 months. Thirty-two months after gene therapy, the patient developed myelodysplastic syndrome (MDS), and vector integration into the MECOM locus was identified in blast cells. The vector integration into MECOM was detectable in most myeloid cells at 12 months after gene therapy. However, the patient exhibited normal hematopoiesis until the onset of MDS, suggesting that MECOM transactivation contributed to clonal hematopoiesis, and the blast transformation likely arose after the acquisition of additional genetic lesions. In whole-genome sequencing, the biallelic loss of the WT1 tumor suppressor gene, which occurred immediately before tumorigenesis, was identified as a potential candidate genetic alteration. The provirus CYBB cDNA in the blasts contained 108 G-to-A mutations exclusively in the coding strand, suggesting the occurrence of APOBEC3-mediated hypermutations during the transduction of CD34-positive cells. A hypermutation-mediated loss of oxidase activity may have facilitated the survival and proliferation of the clone with MECOM transactivation. Our data provide valuable insights into the complex mechanisms underlying the development of leukemia in X-CGD gene therapy.

Keywords: APOBEC3; CGD; MECOM; WT1; hypermutation; insertional mutagenesis; retroviral vector.

Graphical abstract

Figure 1

Study design and clinical results (A) Scheme used for the procedures. G-CSF was administered for 5 days continuously, and mobilized peripheral blood cells were collected twice over 2 days. After their isolation, CD34⁺ cells were transduced three times from days −3 to −1. The resultant cells were infused into the patient after 12 infusions of busulfan. (B) Flow cytometric analysis of gp91^phox expression and DHR oxidation in neutrophils. Six months after infusion, the expression level was <1%. The percentage of DHR-positive cells was reduced, in agreement with the decrease in gp91^phox expression. (C) Positron emission tomography (PET)-computed tomography (CT) performed before and at 4 months after gene therapy. The pathological uptake in the tympanum, paranasal sinuses, and anterior mediastinum was improved. (D) Hematological findings after gene therapy. A decrease in platelet counts and the emergence of blasts were observed at approximately 30 months after gene therapy. WBC, white blood cells; Hb, hemoglobin. (E) Appearance of blast cells in the peripheral blood at 32 months after gene therapy.

Figure 2

Insertional mutagenesis of the MFGS-gp91^phox vector in the patient (A) Neutrophil vector copy number (VCN) at each time point. VCN was calculated using primers/probes against the CYBB cDNA or the vector packaging signal (Ψ). The VCN based on CYBB (VCN-CYBB) decreased throughout the clinical course, whereas the VCN based on the packaging signal (Ψ) (VCN-Ψ) re-increased after 6 months. (B) Vector integration-site analysis using LTR capture. Frequency of targeted genes with vector integration in CD34⁺ cells after retrovirus transduction and neutrophils at each time point. The size corresponds to the frequency of the genes into (or near) which the retroviral vector was integrated. Vector integration into the MECOM locus became dominant at 12 months after gene therapy. (C) Structure of the vector integration sites. At 3 months after gene therapy, myeloid-lineage cells contained two integrations into IVS1 and IVS2 at the MECOM locus. These two integrations disappeared thereafter, and another two integrations into the IVS2 of the MDS1 locus, one of which was detected in the blasts, appeared at 6 months after gene therapy. (D) Frequency of the integration into the MECOM locus in the neutrophils of the patient. Integration-site-specific droplet-digital PCR (IS-ddPCR) revealed the monoclonal proliferation of the clone that exhibited vector integration into MECOM starting at 6 months after gene therapy. (E) Quantitative determination of the MECOM and total EVI1 transcripts. The transcription levels of MECOM and EVI1 relative to the TATA box binding protein (TBP) gene were determined in peripheral blood mononuclear cells at 12 months (Pt-12M PBMC) and in blast cells at 32 months after gene therapy (Pt-32M blast cells). As controls, CD34⁺ cells collected from the patient before transduction and PBMCs from two healthy donors were also analyzed.

Figure 3

Myeloid-lineage hematopoiesis by the clone exhibiting vector integration into MECOM (A) IS-specific ddPCR on the genomic DNA from the colonies identified in the CFU-C assays. The colonies in the CFU-C assays using CD34⁺ cells isolated from the bone marrow at 12 months after gene therapy were analyzed for vector integration into the MECOM locus. The colonies from all myeloid lineages showed vector integration into the MECOM locus. CFU-G/M, colony-forming unit-granulocyte/macrophage; BFU-E, burst-forming unit-erythroid; CFU-E, colony-forming unit-erythroid. (B) Vector integration into the MECOM locus in the hematopoietic subsets at 18 months after gene therapy. IS-specific ddPCR revealed vector integration into MECOM in the myeloid-lineage subsets (granulocytes and monocytes), indicating that the clone with vector integration into the MECOM locus maintained the potential for myeloid-lineage hematopoiesis. CD15⁺gra, granulocytes; CD14⁺mono, monocytes; MNC-32M, mononuclear cells at the onset of MDS (32 months). (C) Principal-component analysis (PCA) of the transcript profiles from the blast cells at 32 months and monocytes at 18 months after gene therapy, as well as monocytes from healthy donors. (D) Sample heatmap and hierarchical clustering. (E) Distribution of the upregulated or downregulated genes in a scatterplot analysis of the samples. Blasts, blast cells collected from the patient at 32 months after gene therapy; 18M, monocytes collected from the patient at 18 months after gene therapy; CTRL, monocytes collected from healthy donors.

Figure 4

CYBB inactivation mechanism in blast cells (A) Bisulfite sequencing was performed to analyze the methylation pattern at CpG islands within the promoter and enhancer regions of the LTR in blast cells and neutrophils at 12 months after gene therapy. Despite the loss of CYBB expression, few methylation events were observed in the neutrophils at 12 months after gene therapy. The filled and open circles indicate methylated and non-methylated CpG sites, respectively. The pie charts showed the ratio at each time point. E, enhancer; P, promoter. (B) Presence of multiple G-to-A mutations in the CYBB cDNA. Blast cells exhibited a total of 108 mutations in proviral CYBB, all of which were observed in the blood sample collected 6 months after gene therapy. (C) G-to-A hypermutations in endogenous genes. The frequency of G-to-A mutation in endogenous genes that appeared in blast cells was much lower than that detected in proviral CYBB. Peripheral blood collected at 12 months after gene therapy also exhibited hypermutations. However, their frequencies were also much lower than those observed in proviral CYBB. (D) G-to-A hypermutations in proviral CYBB at 6 months after gene therapy. Eight proviruses, one of which corresponded to that detected in blast cells, were sequenced. Six proviruses were integrated at sites other than the MECOM locus, and all proviruses other than that in EXPSC7 exhibited multiple G-to-A mutations. The numbers on the vertical axis indicate the nucleotide position in CYBB. The blank areas indicate positions that could not be analyzed.

Figure 5

G-to-A hypermutation in the proviral sequence during the transduction of CD34⁺ cells (A) G-to-A mutations in the TA cloning of the proviral CYBB cDNA sequence from c.340 to c.500 in the CD34⁺ cells of the patient undergoing transduction. G-to-A mutations could be observed in 7.8% of the total clones from the transduced CD34⁺ cells, whereas the amplicon from the vector plasmid and vector-producing cells (293SPA-gp91^phox) showed no mutations. (B) Determination of the timeline of the occurrence of the G-to-A hypermutations in proviral CYBB. The vector-derived CYBB gene was sequenced in the 293SPA-gp91phox virus producer line, reverse-transcribed cDNA from viral RNA, and the patient’s CD34⁺ cells undergoing transduction. (C) Mutation-specific ddPCR using locked nucleic acid (LNA) probes. Mutation-specific ddPCR using LNA probes was performed to detect G-to-A mutations at c.318, c.474, c.1140, and c.1702 in the proviral CYBB gene. The genomic DNA from the 293SPA-gp91^phox virus producer cells, cDNA reverse transcribed from clinical viral RNA, and the CD34⁺ cells of the patient undergoing transduction were analyzed. (D) Frequencies of G-to-A mutations detected in the NGS analysis. Deep sequencing using NGS showed an increase in the frequency of G-to-A mutations at each position and the mean frequency in the CD34⁺ cells of the patient. ∗Statistically significant.

Figure 6

Possible involvement of virus-producing cell-derived APOBEC3 proteins in G-to-A hypermutations (A) Transcription levels of APOBEC3 families in the CD34⁺ cells of the patient, 293SPA-gp91^phox virus producer cells, and 293 cells. (B) APOBC3C transcription in 293SPA-gp91^phox virus producer cells after RNAi using a short hairpin RNA and targeted destruction using the CRISPR-Cas9 system. (C) Frequencies of G-to-A mutations in cord-blood CD34⁺ cells transduced with the viral supernatant from APOBEC3C-targeted 293SPA-gp91^phox cells. A mutation-specific ddPCR analysis revealed that RNAi and Cas9-mediated targeting of the APOBEC3C gene in 293SPA-gp91^phox cells decreased the number of G-to-A mutations (at c.318, c.474, c.1140, and c.1702). (D) The NGS deep-sequencing analysis also revealed that APOBEC3C knockdown in virus-producing cells decreased the frequency of G-to-A changes in target cells. 293SPA, CD34⁺ cells transduced with the virus from 293SPA-gp91^phox cells; 3C KD, CD34⁺ cells transduced with the virus from APOBEC3C-targeted 293SPA-gp91^phox cells. ∗Statistically significant.

Figure 7

Cytotoxicity caused by the aberrant expression of CYBB and possible secondary genetic alteration (A) Apoptotic status, as assessed by annexin V staining. Cord-blood CD34⁺ cells transduced with MFGS-mock vector and MFGS-gp91phox were analyzed for annexin V expression. (B) γH2AX level in transduced cells. Transduction with MFGS-gp91phox yielded increased levels of γH2AX, which indicated the accumulation of DNA damage. (C) The occurrence of a 6.7 Mb deletion on chromosome 11, including WT1, and a 23 kb deletion within WT1 was identified in blast cells. Both deletions appeared immediately before the onset of the myelodysplastic syndrome. (D) Changes in the expression profile of WT1-target genes. A microarray analysis revealed significant changes in the WT1-target genes between blast cells at 32 months and monocytes at 18 months. ∗Statistically significant.