Pre-Clinical Development of a Highly Efficient TALEN ^®-Based Correction of the β-Globin Gene in Patient-Derived Hematopoietic Stem and Progenitor Cells (HSPCs) to Treat Sickle Cell Disease

Sickle cell disease (SCD) is one of the most common inherited diseases affecting millions of people worldwide. SCD stems from a single point mutation (A>T) in exon 1 of the HBB gene which results in sickle hemoglobin. The only available curative treatment of SCD is allogeneic hematopoietic stem cell transplant, which is only viable for ~20% of SCD patients.

Ex-vivo gene therapy approaches have shown to be a promising therapeutic option for patients. Most products currently in the clinic have focused on methods to enhance functional hemoglobin production (e.g., disruption of BCL11A to encourage β-globin gene switching or direct insertion of a functional β-globin gene into patient HSPCs).

The Cellectis approach is to directly repair the mutated HBB gene in order to restore HbA production. TALGlobin01 is an autologous HSPC-based gene therapy product designed with a TALEN ^® optimized to cleave the sickle HBB gene (TALEN-HBB01) and an AAV based engineering process leading to highly efficient HBB gene correction via endogenous homology directed repair (HDR), while mitigating potential risks of HBB gene knock-out (KO).

Use of TALGlobin01 resulted in up to 70% of HDR-mediated HBB gene correction (56% mean frequency) in homozygous sickle (HbSS) patient HSPCs with only 20% of NHEJ-dependent insertion/deletion (indels) events detected. This gene correction process did not affect cell viability, hematopoietic stem/progenitor immunophenotype or differentiation potential of corrected HSPCs. Allelic editing at clonal resolution in single BFU-E colonies showed that up to 72% (with a mean of 50%) of progenitors contained at least one corrected allele, while only 23% were either not corrected or had indels on one allele. Notably, our optimized engineering process led to only 9% of colonies harboring bi-allelic indels events.

To evaluate the ability of TALGlobin01 to prevent the sickling phenotype associated with SCD, we performed in vitro differentiation of HbSS patient-edited cells into late-stage erythroid cells and assessed HbA protein production by HPLC. We observed that HbA accounted for up to 60% (with a mean of 49%) of the total Hb with a concomitant decrease of HbS production from 90% to 19%. Interestingly, our gene correction process maintained a balanced α chain/non-α chain ratio, consistent with our genotyping results showing a low frequency of clones harboring bi-allelic indels. More importantly, efficient expression of HbA was translated into a sharp decrease of hypoxia-induced sickling rate of in vitro-generated erythrocytes when comparing unedited to TALGlobin01 edited cells (from 95% to 13%, respectively).

When injected in vivo, engineered HSPCs from non-mobilized SCD patients retained the capacity to engraft into immunodeficient NSG mice with levels of allelic correction comparable to the input (~40%) and still detectable at 16-17 weeks post-transplantation.

An unbiased genome wide approach (OCA), coupled to target enrichment high-throughput sequencing screening, confirmed the TALEN-HBB01 cleavage activity at only one off-target site located at the HBD locus. TALEN-HBB01 cleavage activity was assessed at this off-target site in patient HSPCs engineered with TALGlobin01 and found to be very low compared to the on-site cleavage activity (50.7% indels at the on-site versus 1.2% at the off-site).

Furthermore, we developed a clinical-scale TALGlobin01 manufacturing process that achieved up to 60% of HDR-mediated HBB gene correction and less than 10% of NHEJ-mediated HBB gene inactivation in healthy donor derived HSPCs.

These results are the first demonstration that a TALEN-based engineering process could be used to efficiently correct the SCD mutated HBB gene in HbSS patient-derived CD34+ HSPCs. Taken together, the high level of HbA expression and reversion of sickling phenotype, the efficient in vivo long-term engraftment potential of TALGlobin01 edited cells and the low levels of HBB KO or off-target cleavage generated by our gene correction process, warrant the clinical evaluation of TALGlobin01 to treat SCD patients.