Preservation of Gene Edited Hematopoietic Stem Cells By Transient Overexpression of BCL-2 mRNA

• Published in: Blood 2016-12, Vol.128 (22), p.3636-3636
• Main Authors: Bjurström, Carmen Flores, Mojadidi, Michelle, Lomova, Anastasia, Lai, Stephen, Fitz-Gibbon, Sorel, Pellegrini, Matteo, Cooper, Aaron R, Lill, Georgia R, Kaufman, Michael, Cost, Gregory J, Holmes, Michael C., Hollis, Roger P, Kohn, Donald B
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V128.22.3636.3636

Introduction: Site-specific gene correction of the point mutation causing sickle cell disease (SCD) in hematopoietic stem cells (HSCs) constitutes a precise strategy to generate a life-long source of gene-corrected erythrocytes that do not sickle. However, low efficiency of homology-directed repair (HDR) in primitive reconstituting HSCs is currently a limit to the use of therapeutic genome editing for treatment of severe genetic blood disorders. To identify the mechanism(s) that underlie decreased HDR efficacy in primitive HSCs relative to that in more mature progenitor populations, we assessed: efficiency of gene delivery and expression after electroporation of in vitro transcribed mRNA; functional ZFN-mediated endonuclease activity; cell cycle status; gene expression of key HDR genes; and cytotoxic responses; in the following immunophenotypically-defined human cell populations: HSCs (CD34+/CD38-/CD90+CD45RA-); multipotent progenitors (MPPs) (CD34+/CD38-/CD45RA-/CD90-); and progenitor cells (CD34+/CD38+). Methods: CD34+ cells were enriched from human G-CSF-mobilized peripheral blood and cultured for 1-3 days prior to electroporation of in vitro transcribed mRNA encoding GFP or a pair of zinc finger nucleases (ZFN). The ZFNs, designed to target the sickle mutation in exon 1 of the human beta-globin gene, were co-delivered with one of the homologous donor templates containing the corrective base (A/T): an integrase-deficient lentiviral vector (IDLV) or a 101bp single-stranded oligodeoxynucleotide (oligo). Percentages of alleles containing insertions/deletions (indels) and/or HDR-mediated gene correction were analyzed by high throughput sequencing (HTS). Acute cytotoxicity was determined by flow cytometry, identifying viable cells as 7AAD/AnnexinV neg. cells. To assess HDR-mediated gene correction in vivo after three months, gene-edited cells were transplanted (>1E6 viable CD34+ cells/mouse, I.V.) one day after electroporation into irradiated (250cGy) NOD/SCID/IL2R gamma-/- (NSG) mice. Results: In HSCs, MPPs and progenitor populations, no differences were observed in delivery and expression from electroporated GFP mRNA [%GFP(+) and MFI]. To assess the activity of ZFN mRNA in the stem and progenitor populations, ZFNs were delivered to CD34+ cells through electroporation of in vitrotranscribed mRNA. The CD34+ cells were then FACS-sorted into the respective populations and HTS was used to determine the percentage of alleles containing indels; the frequencies of