Priming Human Repopulating Hematopoietic Stem and Progenitor Cells for Cas9/sgRNA Gene Targeting

Abstract

Engineered nuclease-mediated gene targeting through homologous recombination (HR) in hematopoietic stem and progenitor cells (HSPCs) has the potential to treat a variety of genetic hematologic and immunologic disorders. Here, we identify critical parameters to reproducibly achieve high frequencies of RNA-guided (single-guide RNA [sgRNA]; CRISPR)-Cas9 nuclease (Cas9/sgRNA) and rAAV6-mediated HR at the β-globin (HBB) locus in HSPCs. We identified that by transducing HSPCs with rAAV6 post-electroporation, there was a greater than 2-fold electroporation-aided transduction (EAT) of rAAV6 endocytosis with roughly 70% of the cell population having undergone transduction within 2 hr. When HSPCs are cultured at low densities (1 × 105 cells/mL) prior to HBB targeting, HSPC expansion rates are significantly positively correlated with HR frequencies in vitro as well as in repopulating cells in immunodeficient NSG mice in vivo. We also show that culturing fluorescence-activated cell sorting (FACS)-enriched HBB-targeted HSPCs at low cell densities in the presence of the small molecules, UM171 and SR1, stimulates the expansion of gene-edited HSPCs as measured by higher engraftment levels in immunodeficient mice. This work serves not only as an optimized protocol for genome editing HSPCs at the HBB locus for the treatment of β-hemoglobinopathies but also as a foundation for editing HSPCs at other loci for both basic and translational research.

Highly Efficient Repair of the ΔF508 Mutation in Airway Stem Cells of Cystic Fibrosis Patients with Functional Rescue of the Differentiated Epithelia

“…Using the optimal template, our correction sequence was observed in 43  5% alleles ( Figure 1B) and INDELs were observed in 38  2% of the alleles ( Figure 1B), a ratio of HR:INDEL of >1 which is consistent with prior work that when optimal amounts of donor are delivered to dividing cells, high frequencies of HR-mediated editing are possible (Charlesworth et al, 2018;Hendel et al, 2014). On the day of extraction, 67  8% of edited cells were KRT5+ and Integrin Alpha-6+ (ITGA6+) which was similar to control (mock) cells, with 60  15% ( Figure 1C-D).…”

“…Isolation and culture of UABCs: Cas9-based gene editing utilizing the homologous recombination pathway to correct mutations has been shown to be improved under conditions that promote cell proliferation (Charlesworth et al, 2018). Therefore, we first optimized cell culture conditions to both increase proliferation of basal cells and improve their survival after electroporation (also called nucleofection).…”

“…Cells were transferred to a 12 well plate coated with 5% Matrigel (density = 20,000 cells/cm 2 ) and 400 L of EN media with 10 M ROCK inhibitor was added. AAV carrying the correction template was added immediately after electroporation to maximize transduction Charlesworth et al, 2018). Multiplicity of Infection (MOI) of 10 6 particles per cell (as determined by qPCR) was optimal.…”

Global Transcriptional Response to CRISPR/Cas9-AAV6-Based Genome Editing in CD34+ Hematopoietic Stem and Progenitor Cells

“…Rather, it has been reported that the HR rates can be dramatically boosted by simply altering the cell culture density of CD34 + HSPCs in order to promote cell cycling, which is thought to be necessary for efficient HR. 51 Therefore, it would be of great interest to perform additional tran-scriptional analysis on HSPCs that have been targeted using the HR-optimized protocol.…”

Gene therapy targeting haematopoietic stem cells for inherited diseases: progress and challenges

“…It is also important to note that, in autosomal recessive haematological disorders, even the genome-editing-mediated correction of only one of the two affected alleles in a reasonably high proportion of haematopoietic stem cells (HSCs) would avoid the need for multiple copies of the integrated lentiviral vector with suboptimal transgene expression. Although HDR-based approaches for correcting haematological genetic disorders are relatively efficient in haematopoietic stem and progenitor cells (HSPCs), the frequency of stably modified, repopulating HSCs rarely exceeds 10% [149][150][151][152][153][154][155] , suggesting that stem cells might be poorly permissive to HDR (which is typical of nonquiescent cells). The inclusion of selectable markers in the donor template enables the enrichment of edited HSCs 151 ; however, cell selection would be difficult to translate into the clinic because it would yield lower numbers of HSCs than conventional approaches do.…”