Recipient Engraftment and Acute Lymphoblastic Leukemia Assay

Table 1. Table 1. Key Laboratory Values before and after Transplantation.

Neutrophil and platelet engraftment occurred on days 13 and 27, respectively, after transplantation (see the Methods: Allogeneic Transplantation section in the Supplementary Appendix). Lymphocyte counts and T-lymphocyte subsets increased after transplantation, accompanied by the recovery of the CD4+ cell count to 592.94×106 per liter in month 6 and its stabilization in a normal range (Table 1). Although the platelet count transiently decreased at month 3, the count spontaneously recovered and stabilized in a normal range (Figure 1A). Full donor chimerism was achieved at week 4 after transplantation and persisted through the most recent time point, 19 months after transplantation.

The acute lymphoblastic leukemia was in morphologic complete remission at week 4 after transplantation; this remission continued over the 19-month follow-up period. In addition, minimal residual disease remained undetectable for leukemia-associated phenotypes on the basis of flow cytometry. The expression level of the Wilms’ tumor gene (WT1) normalized to the Abelson gene (ABL) — an increase in which predicts relapse — was less than 0.5% after transplantation, which was a level unchanged from before transplantation (Table 1).13,14

CCR5 Gene Editing

The donor-derived, sorted HSPCs (CD34+ cells) were edited with the use of CRISPR–Cas9, resulting in CCR5 insertion or deletion (indel) efficiency of 17.8% as indicated by sequencing. In case the edited HSPCs did not result in long-term engraftment, the gene-edited HSPCs were transplanted with the CD34-depleted cells, which contained 28.8% of total CD34+ cells. Consequently, the proportion of CCR5 ablation in the genome of bone marrow karyocytes ranged between 5.20% and 8.28% during the 19-month long-term engraftment (Figure 1B). The representative types of CCR5 gene mutation are shown in Figure S1 in the Supplementary Appendix. Deep sequencing was used to determine the gene-ablation efficiencies in multiple hematopoietic lineages, including CD4+, CD8+, CD19+, CD33+, and CD235a+ cells. CCR5 ablation was detected in multiple blood lineages, and a similar or slightly higher level of editing efficiency was observed in CD19+, CD33+, and CD235a+ cells, as compared with that in total peripheral-blood karyocytes (Figure 1C). The levels of CCR5 ablation in CD4+ and CD8+ cells were not as high as in other cell subsets, possibly owing to the long-term persistence of T cells in the coinfused CD34-depleted cells.15 These results collectively showed that CRISPR-edited HSPCs successfully engrafted and differentiated into multiple lineages that retained the gene editing.

Safety Analysis

The patient presented with predictable side effects after preconditioning, including anemia (hemoglobin level, 79 g per liter at day 22), neutropenia (undetectable neutrophils at day 0), and thrombocytopenia (platelet count, 12×109 per liter at day 6). No acute immune response was observed after the infusion of donor cells. Febrile neutropenia (grade 3) and bacteremia (Staphylococcus epidermidis; grade 3) developed in the first 2 weeks, which resolved with the use of standard antibiotic therapy. Acute graft-versus-host disease (of the skin; grade 1), urinary frequency and urgency (grade 2), cytomegalovirus viremia (grade 3), and herpes simplex reactivation occurred in month 2. Intermittent exotropia of the left eye, influenza-like symptoms, and an increased alanine aminotransferase level were observed successively from month 5 after transplantation to the latest time point of the follow-up. All these events resolved. No adverse events that were related to CCR5 gene editing were noted (Table S2 in the Supplementary Appendix).

To examine the off-target effects of the gene editing, we performed high-throughput genomewide sequencing at 100× coverage to analyze the genome of modified HSPCs that were sampled from the prerelease product. We first analyzed the previously predicted off-target site in our system (chr4:18476075–18476173); DNA cleavage was not detected at this site in this study.10 To identify other potential off-target sites, we used a computerized tool to predict 1997 loci to be candidates by comparing the two single-guide RNAs in our system with the human genome.16 Using the genomewide sequencing data, we excluded candidate sites with no indels near the prediction sites in modified HSPCs, with indels detected in the donor sample, or with previously identified indels, and we obtained 26 candidate sites including the 2 on-target loci (Table S3 in the Supplementary Appendix). We further performed deep sequencing to validate these 24 candidate off-target sites. Indels were detected in only 14 sites, which were all 1-bp length variance on nucleotide repeats and thus were not considered to be true off-target events.

To determine whether any off-target editing appeared after engraftment, we conducted a whole-genome sequencing assay on bone marrow blood samples that were obtained at week 15, month 12, and month 19 after transplantation. No off-target site was identified in any of these samples. Moreover, no chromosomal rearrangements or long-range deletions were identified in any of the four whole-genome sequencing data sets.

Measurements of HIV-1 Viral Load

Previous work has shown that the infusion of autologous CCR5-edited T cells may decrease the viral load in patients during a 4-week period of interruption of antiretroviral therapy.17 This finding suggests that CCR5 deletion may have the potential to mitigate the use of long-term antiretroviral therapy. To determine whether CCR5-edited stem cells could lead to an analogous benefit in our patient, an interruption of antiretroviral therapy was proposed. After we obtained a separate written informed consent from the patient, which was specifically related to the temporary cessation of antiretroviral therapy, a planned analytic interruption was performed 7 months after transplantation when the patient’s CD4+ cell count increased to a value in the normal range and the HIV RNA copies in plasma remained undetectable.

Figure 2. Figure 2. Clinical Outcomes during and after the Interruption of Antiretroviral Therapy. Shown are the results of assessments for viral load (Panel A), the CD4+ cell counts and the ratio of CD4+ cells to CD8+ cells (Panel B), and the CCR5 gene-disruption efficiency in CD4+ cells (Panel C) from month 3 to month 12 after transplantation. Antiretroviral therapy was interrupted from day 221 to day 249.

The serum viral load increased to 3×107 copies per milliliter at week 4 during the interruption of antiretroviral therapy, and the drugs were then resumed (Figure 2A). The viral load gradually decreased to an undetectable level during the following months. During the interruption of antiretroviral therapy, the peripheral CD4+ cell count decreased from 575×106 per liter to 250×106 per liter, and the same trend was observed in the ratio of CD4+ cells to CD8+ cells. In addition, the level of CCR5 disruption in peripheral CD4+ cells before the interruption was 2.96%. The level of CCR5 disruption in CD4+ cells peaked (4.39%) during the interruption, at a level that was 1.6 times as great as the mean level, and was accompanied by a decrease in the CD4+ cell count (360×106 per liter). Moreover, immune-cell counts were evaluated before and after transplantation. CD4+ T-lymphopenia developed before transplantation, at which time the cell count was 201.31×106 per liter (Table 1), and the CD4+ cell count increased to a value in a normal range at month 6 after transplantation (Figure 2C) and while the HIV-1 infection was under control with antiretroviral therapy.

To identify HIV tropism after transplantation, we tested peripheral-blood samples at months 8 and 19 after transplantation, and the virus tropism was still CCR5. In addition, the peripheral reservoir of HIV-1 was evaluated by the detection of HIV-1 DNA copies in peripheral CD4+ cells. The levels of total and integrated HIV-1 DNA were 734 and 72.5 copies per million CD4+ cells, respectively, after transplantation (Fig. S2 in the Supplementary Appendix). Their quick rebound after the interruption of antiretroviral therapy coincided with the appearance of a measurable viral load.