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
Authors: Moiani Arianna , Hong Patrick , Letort Gil , Lizot Sabrina , Chirinos Alexa , Temburni-Blake Sonal , Nik Sara , Mayer Louisa , Leduc Sophie , Pinard Noemie , Foray Chloe , Boyne Alex , Kazancioglu Selena , Frati Giacomo , Felix Tristan , Chalumeau Anne , Miccio Annarita , Dusseaux Mathilde , Gouble Agnès , Frattini Mark G. , Brownstein Carrie , Duclert Aymeric , Shiffer-Mannoui Cecile , Juillerat Alexandre , Duchateau Philippe , Valton Julien 
Published: November 2021

Abstract
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&gt;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.

Disclosures
Moiani: Cellectis, S.A.: Current Employment, Current equity holder in publicly-traded company. Hong: Cellectis, Inc.: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Letort: Cellectis, S.A.: Current Employment, Current equity holder in publicly-traded company. Lizot: Cellectis, S.A.: Current Employment, Current equity holder in publicly-traded company. Chirinos: Cellectis, S.A.: Current Employment. Temburni-Blake: Cellectis, Inc.: Current Employment, Current equity holder in publicly-traded company. Mayer: Cellectis, Inc.: Current Employment, Current equity holder in publicly-traded company. Leduc: Cellectis, S.A.: Current Employment, Current equity holder in publicly-traded company. Pinard: Cellectis, S.A.: Current Employment, Current equity holder in publicly-traded company. Foray: Cellectis, S.A.: Current Employment, Current equity holder in publicly-traded company. Boyne: Cellectis, Inc.: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months, Patents & Royalties. Kazancioglu: Cellectis, Inc.: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months. Dusseaux: Cellectis, S.A.: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Gouble: Cellectis SA: Current Employment, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company. Frattini: Celgene/BMS: Current equity holder in publicly-traded company, Ended employment in the past 24 months; Cellectis, Inc.: Current Employment, Current equity holder in publicly-traded company. Brownstein: BMS: Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months, Ended employment in the past 24 months; Cellectis, Inc.: Current Employment, Current equity holder in publicly-traded company. Duclert: Cellectis, S.A.: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months. Shiffer-Mannoui: Cellectis, S.A.: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months. Juillerat: Cellectis, Inc.: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Duchateau: Cellectis, S.A.: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Valton: Cellectis, S.A.: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties.