Background In China, it is estimated that there are 15,000-30,000 patients with β Thalassemia Major (β-TM). Our recent Cross-sectional survey of β-TM patients’ conditions in China found that only 7% of patients with β-TM expect to live beyond age 18, and only a fraction of patients have received allogeneic hematopoietic stem cell transplantation. Higher level of fetal hemoglobin (HbF) by downregulation of BCL11A during erythroid differentiation has been reported to ameliorate the clinical severity of the patients with β-TM. Disruption of GATAA motif in DHS h+58 region of BCL11A erythroid enhancer (BEE) by gene editing in CD34+ cells has been shown to significantly induce the production of HbF in patients. An adjacent motif, i.e., the CTG motif within TAL1-GATA1 binding site could potentially also be such a gene-editing target due to naturally occurring SNPs associated with elevated HbF levels (eg rs1427407). ET-01 is an investigational cell therapy product comprised of autologous CD34+ cells that have undergone CRISPR/Cas9-mediated ex vivo editing targeting this motif (Fig 1).

Methods The EDI-001 trial (NCT04390971) aimed to evaluate the safety and efficacy of ET-01 in patients with transfusion dependent thalassemia (TDT). Patients (aged 6 to 35 years) with TDT whose total hemoglobin level fell within the range of 4.5g/dL and 7g/dL were eligible. One patient was enrolled and CD34+ cells were collected from the patient by apheresis following mobilization with lenograstim and plerixafor. ET-01 was manufactured by editing at BEE with Cas9 mRNA and a specific single-guide RNA via electroporation. The ET-01 product was infused following myeloablative conditioning on date 18 Nov 2020 (patient age at 12). Afterwards, the patient was followed up per schedule defined in the Protocol, monitoring safety and efficacy endpoints.

Results cGMP manufacturing process at clinical scale was developed for ET-01. High editing rate was achieved with stemness well maintained (Fig 2A). Indels pattern analysis showed that in ET-01 four indel types were most prevalent (Fig 2B). Moreover, the percentage of HbF+ cells were significantly increased compared with the unedited CD34+ cells in erythroid differentiation assay (Fig 2C). IND-enabling studies showed that no tumorigenicity nor other toxicities associated with ET-01 were observed in over 400 mice at up to 40 weeks of observation.

One patient (β0/β+) was enrolled, for which ET-01 was manufactured under cGMP process and infused at a dose of 23.19 X 10^6 CD34+ cells/kg. Successful hematopoietic reconstitution was observed, in which neutrophil engraftment occurred on day 24 and platelet engraftment on day 37 post ET-01 infusion.

The patient experienced a significant increase of HbF production and total Hb, in which the levels of HbF increased from 3.35 g/L at baseline to 89.87 g/L, and the total hemoglobin level reached 110 g/L at month 18, respectively (Fig 3). The patient received her last pRBCs transfusion on day 87 after ET-01 infusion, and achieved transfusion-free for over 15 months. Moreover, we observed that the prevalence of indels changed during the initial few weeks and remained stable in both peripheral blood and bone marrow (Fig 4A). NGS analysis of 42 potential off-target sites, discovered by in silico prediction and DiGenome-seq, showed editing efficiencies below 1%, and no significant change was observed in any of the 42 sites throughout visits (Fig 4B).

The patient experienced 62 cases of adverse events, most of which were Grade 1 or 2 in severity. Of these, only one Grade 1 AE was attributed to the ET-01 drug product, caused by the ET-01 preparation component DMSO during infusion, and was gradually relieved by adjusting the infusion rate. Two Grade 3 serious adverse events (SAE) unrelated to ET-01 were reported: enterocolitis and febrile neutropenia, caused by inappropriate diet after apheresis and busulfan conditioning, respectively.

Conclusions These data demonstrated that a single-dose of ET-01 composed of autologous CRISPR/Cas9 mediated CD34+ cells led to the timely hematopoietic engraftment, significant and durable increases in HbF production and total Hb, and transfusion-independent for 15 months at the time of data cut-off. Safety profile of ET-01 is consistent with that of autologous HSC transplant including busulfan myeloablative conditioning. These preliminary results support further experimental testing of ET-01 to treat patients with β-TM.

Fang:EdiGene Inc.: Current Employment. Shi:EdiGene Inc.: Current Employment. Zhang:EdiGene Inc.: Current Employment. Yang:EdiGene Inc.: Current Employment. Shi:EdiGene (GuangZhou) Inc.: Current Employment. Liu:EdiGene (GuangZhou) Inc.: Current Employment. Wang:EdiGene (GuangZhou) Inc.: Current Employment. Yang:EdiGene Inc.: Current Employment. Wang:EdiGene Inc.: Current Employment. Li:EdiGene (GuangZhou) Inc.: Current Employment. Shi:EdiGene Inc.: Current Employment. Zhang:EdiGene Inc.: Current Employment. Yuan:EdiGene Inc.: Current Employment. Wei:EdiGene Inc.: Current Employment.

Author notes


Asterisk with author names denotes non-ASH members.

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