Modified IPS Cells for Hemoglobinopathies

We have recently used CRISPR-Cas to correct the HBB gene in induced Pluripotent Stem Cells (iPSC) derived from patients with Sickle Cell Disease (SCD) and to correct the JAK3 gene in iPSC derived from patients with Severe Combine Immune Deficiency (SCID).  Off-target mutations were minimized, if not eliminated, by use of paired guide RNAs and the Cas9 nickase.  When erythroid progenitors (EP) produced from corrected SCD iPSC are transplanted into NSG mice, a complete switch from gamma- to beta-globin gene expression occurs within 24 hours and high levels of beta mRNA are synthesized; the betaA:betaS ratio is 60:40.   Hematopoietic progenitors produced from corrected SCID iPSC can be differentiated into T cell populations that express a full repertoire of T Cell Receptors (TCRs).  These results suggest that CRISPR-Cas enhanced gene replacement may provide safe and effective therapies for many erythroid and lymphoid disorders.  In an alternative approach, we electroporated a preformed, biochemical complex composed of a guide RNA (gRNA), a modified recombinant Cas9 (mrCas9) and a single stranded oligodeoxnucleotide (ssODN) into sickle iPSC or primary sickle CD34+ hematopoietic progenitors.  Sixty-five percent of sickle iPSC colonies contained at least one corrected allele.  When homozygous corrected colonies were differentiated into erythroid progenitors and transplanted into NSG mice, human erythroid cells expressed 100% HbA.  When primary sickle CD34+ hematopoietic progenitors were electroporated with the gRNA/mrCas9/ssODN complex and differentiated into erythroid cells in vitro, HbA was expressed at 30-40%.  These results suggest that primary bone marrow CD34+ cells that are isolated from patients with sickle cell disease, beta-thalassemia, SCID, DBA and other hematopoietic disorders may be electroporated with specific gRNA/mrCas9/ssODN complexes and transplanted into patients to rapidly, safely and effectively treat these debilitating disorders.