Contributions
Abstract: S251
Type: Oral Presentation
Session title: Cellular immunotherapy and gene therapy - Experimental
Background
Sickle cell disease (SCD) is an inherited blood disorder affecting ~ 100,000 individuals in the United States. Naturally occurring mutations at the distal CCAAT-box region of the HBG1 and HBG2 promoters are associated with elevated fetal hemoglobin (HbF) expression, which has been shown to be protective against clinical manifestation of SCD.
Aims
EDIT-301 is an investigational autologous cell therapy comprising CD34+ cells genetically modified with CRISPR-Cas at the HBG1 and HBG2 promoters to promote HbF expression for the potential treatment of SCD.
Methods
Editing is achieved using a highly specific CRISPR-Cas12a enzyme, which we have shown to have a superior on-target editing profile at this site compared to S.pyogenes Cas9. Preclinical studies were conducted using mobilized peripheral blood (mPB) CD34+ cells from normal and SCD donors transfected at research-scale and/or large-scale. Editing efficiency, HbF induction, functional improvement of SCD red blood cells (RBCs), off-target editing, and engraftment of edited CD34+ cells in NOD scid gamma (NSG) mice were studied.
Results
At research scale, electroporation of mPB CD34+ cells from both normal and SCD donors resulted in on-target editing of ~90%, with more than 50% of HbF expression detected in the erythroid progeny of SCD donor cells in a pancellular fashion (~93% population). This robust HbF induction was associated with significant functional improvement of SCD donor derived RBCs under hypoxia, including reduced sickling and increased deformability. In addition, using a microfluidic assay that mimics blood flow in the microvasculature, we observed a marked improvement in the rheological behavior of SCD RBCs derived from RNP-electroporated CD34+ cells. This rheology improvement was strongly correlated with the high HbF levels achieved in edited RBCs.
We have developed a consistent large-scale process using semi-automated systems. Using this process on normal donor cells, we have achieved CD34+ cell purity >90% and editing levels >80% after RNP electroporation. Evaluation of batches from this representative process demonstrated a highly consistent editing profile and no detectable off-targets. In addition, no HBG transcript or protein variants were detected in the erythroid progeny of the RNP-edited CD34 cells, demonstrating that the editing procedure did not affect the integrity of HbF. Finally, infusion of large scale manufactured edited CD34+ cells into NSG mice led to a polyclonal engraftment, with no difference in hematopoietic reconstitution compared to unedited CD34+ cells. Editing levels of >90% were detected after long term engraftment, demonstrating robust editing of long-term-repopulating hematopoietic stem cells.
Conclusion
In summary, Cas12a-RNP editing of SCD donor mPB CD34+ cells at the HBG1 and HBG2 promoters resulted in robust HbF induction and reduced sickling as well as markedly improved rheological behavior in erythroid progeny cells. We established a current Good Manufacturing Practices (cGMP) clinical scale process to support EDIT-301 clinical manufacturing. Evaluation of process representative large-scale manufacturing batches demonstrated high and consistent editing of the HBG1 and HBG2 promoters, with no measurable off-targets, and no alteration of the HBG transcript and protein. In vivo, edited CD34+ cells led to polyclonal engraftment and reconstituted all blood cell lineages at levels comparable to unedited cells. These results support the dosing of EDIT301 in the upcoming RUBY trial, a phase 1/2 study of patients with severe SCD.
Keyword(s): Autologous hematopoietic stem cell transplantation, Hematopoietic stem cell, Hemoglobin, Sickle cell disease
Abstract: S251
Type: Oral Presentation
Session title: Cellular immunotherapy and gene therapy - Experimental
Background
Sickle cell disease (SCD) is an inherited blood disorder affecting ~ 100,000 individuals in the United States. Naturally occurring mutations at the distal CCAAT-box region of the HBG1 and HBG2 promoters are associated with elevated fetal hemoglobin (HbF) expression, which has been shown to be protective against clinical manifestation of SCD.
Aims
EDIT-301 is an investigational autologous cell therapy comprising CD34+ cells genetically modified with CRISPR-Cas at the HBG1 and HBG2 promoters to promote HbF expression for the potential treatment of SCD.
Methods
Editing is achieved using a highly specific CRISPR-Cas12a enzyme, which we have shown to have a superior on-target editing profile at this site compared to S.pyogenes Cas9. Preclinical studies were conducted using mobilized peripheral blood (mPB) CD34+ cells from normal and SCD donors transfected at research-scale and/or large-scale. Editing efficiency, HbF induction, functional improvement of SCD red blood cells (RBCs), off-target editing, and engraftment of edited CD34+ cells in NOD scid gamma (NSG) mice were studied.
Results
At research scale, electroporation of mPB CD34+ cells from both normal and SCD donors resulted in on-target editing of ~90%, with more than 50% of HbF expression detected in the erythroid progeny of SCD donor cells in a pancellular fashion (~93% population). This robust HbF induction was associated with significant functional improvement of SCD donor derived RBCs under hypoxia, including reduced sickling and increased deformability. In addition, using a microfluidic assay that mimics blood flow in the microvasculature, we observed a marked improvement in the rheological behavior of SCD RBCs derived from RNP-electroporated CD34+ cells. This rheology improvement was strongly correlated with the high HbF levels achieved in edited RBCs.
We have developed a consistent large-scale process using semi-automated systems. Using this process on normal donor cells, we have achieved CD34+ cell purity >90% and editing levels >80% after RNP electroporation. Evaluation of batches from this representative process demonstrated a highly consistent editing profile and no detectable off-targets. In addition, no HBG transcript or protein variants were detected in the erythroid progeny of the RNP-edited CD34 cells, demonstrating that the editing procedure did not affect the integrity of HbF. Finally, infusion of large scale manufactured edited CD34+ cells into NSG mice led to a polyclonal engraftment, with no difference in hematopoietic reconstitution compared to unedited CD34+ cells. Editing levels of >90% were detected after long term engraftment, demonstrating robust editing of long-term-repopulating hematopoietic stem cells.
Conclusion
In summary, Cas12a-RNP editing of SCD donor mPB CD34+ cells at the HBG1 and HBG2 promoters resulted in robust HbF induction and reduced sickling as well as markedly improved rheological behavior in erythroid progeny cells. We established a current Good Manufacturing Practices (cGMP) clinical scale process to support EDIT-301 clinical manufacturing. Evaluation of process representative large-scale manufacturing batches demonstrated high and consistent editing of the HBG1 and HBG2 promoters, with no measurable off-targets, and no alteration of the HBG transcript and protein. In vivo, edited CD34+ cells led to polyclonal engraftment and reconstituted all blood cell lineages at levels comparable to unedited cells. These results support the dosing of EDIT301 in the upcoming RUBY trial, a phase 1/2 study of patients with severe SCD.
Keyword(s): Autologous hematopoietic stem cell transplantation, Hematopoietic stem cell, Hemoglobin, Sickle cell disease