SELECTION OF GENOME EDITED HEMATOPOIETIC STEM CELLS PRIOR TO TRANSPLANTATION DECREASES THE ENGRAFTMENT POTENTIAL AND THERAPEUTIC EFFICACY

Abstract

<h3>Background</h3> Autologous hematopoietic stem cell transplantation using genome edited cells has the potential to become a definitive therapy for lysosomal diseases, including some with neurological involvement. We have previously shown that transplantation with human, genome edited hematopoietic stem and progenitor cells (HSPC) can correct phenotypic and biochemical abnormalities in mice with mucopolysaccharidosis type I (MPSI), a multi-systemic lysosomal disorder caused by alfa-L-iduronidase (IDUA) deficiency and consequent glycosaminoglycan (GAG) storage. Before, we used GFP expression to select edited cells prior to transplantation. To effectively translate this proof-of-concept study to the clinic, we tested a clinically relevant selection marker, the truncated Nerve Growth Factor Receptor (tNGFR), which is not expressed in HSPCs, is not toxic and allows antibody-mediated cell isolation. <h3>Methods</h3> Human HSPCs were CRISPR-edited in the safe harbor CCR5 to express IDUA or IDUA and tNGFR (IDUA-tNGFR). Cells edited with IDUA were transplanted as bulk preparations while IDUA-tNGFR cells were selected with magnetic beads and transplanted as positive (IDUA-tNGFR+) and negative (IDUA-tNGFR-) fractions into NSG-MPSI mice. Phenotypical, molecular and biochemical analyses were performed 18 weeks post-transplant. <h3>Results</h3> Genome editing of IDUA-tNGFR cells was significantly lower than IDUA (14% vs. 31%), possibly due to increased IDUA-tNGFR vector size comparing to IDUA only, reducing the editing efficiency. In transplanted mice, IDUA-tNGFR+ cells engrafted poorly in hematopoietic tissues comparing to IDUA or IDUA-tNGFR- cells. The reduced engraftability and consequent lower therapeutic dose observed with tNGFR-selected cells predicted a worse performance at correcting biochemical features in the MPSI mouse model, with only few mice with detectable levels of the enzyme. Contrarily, mice transplanted with bulk cell preparations had supraphysiological levels of enzyme, with significant biochemical correction even in the brain. <h3>Conclusion</h3> These data suggest that selection of edited HSPCs for transplantation reduces drastically the therapeutic potential of the product, regardless of the selection marker. Therefore, higher therapeutic efficacy is achieved when cells are transplanted as bulk preparations (edited + unedited cells), with significant expression of the therapeutic product also in the brain, constituting a promising therapy for neurodegenerative disorders.