“… Mobilized peripheral blood | NA (X-VIV015 media + cytokine supplements) | 500,000 | P3 Primary Cell 4D-Nucleofector kit | Lonza 4D-Nucleofector (program EO-100) | 10 μg sgRNA, 15 μg Cas9 mRNA. and 1 μg plasmid | 28 |
Mobilized peripheral BM and CB | 1–2 days cells in culture before transfection (StemSpan SFEM II media + listed cytokines) | 5 million–80 million | Human T Cell Nucleofector kit | Nucleofector 2b device (program U-014) | Cas9 protein/sgRNA or Cas9 mRNA/sgRNA + AAV6 donor vector | 2 |
Mobilized peripheral blood | 24 hr cultured cells before transfection (StemSpan SFEM + CC110 cocktail) | 100,000–200,000 | Lorita P3 solution | Lonza 4D-Nucleofector (program 00100 or ER1 OO) | 72–200 pmol Cas9 RNP + 100 μM ssDNA | 26 |
Peripheral blood of SC patients | cells expanded 7 days before transfection (StemSpan SFEM II + CC100 cytokine cocktail) | 5,000–10,000 | Human CD34 Cell Nucleofector kit | Nucleofector 2b device (program U-008) | 1st electroporation, l0μg Cas9 mRNA; 2nd electroporation, sgRNA and HDR template | 29 |
Peripheral blood and CB | 2 days in culture before transfection (StemSpan + cytokines) | 150,000–250,000 | Neon Transfection System (10 μL kit) | Neon Transfection System (1,600 V, 10 ms, 3 pulses) | 200 ng-1 μg sgRNAs + 1 μg Cas9 protein | 6 |
CB and BM of SCD patients | 2 days in culture before transfection (X-VIV015 + cytokines) | 200,000 | BTXpress buffer | BTX ECM 830 Square Wave Electroporator (250 V, 5 ms) | ZFN mRNA + oligonucleotide | 30 |
Mobilized peripheral blood | NA (not easily found) | 1 million–5 million | NA | Nucleofector 2b device (program U-008) | 2 × 10 μg DNA plasmid encoding gRNA, Cas9, and GFP | 31 |
The options available for the delivery of gene editing tools into CD34+ cells. BM, bone marrow; CB, cord blood; HDR, homology-directed repair; NA, not applicable; ssDNA, single-stranded DNA; SC, sickle cell; ZFN, zinc-finger nuclease.…”