Enhanced homology-directed repair for highly efficient gene editing in hematopoietic stem/progenitor cells

Abstract

Lentivector gene therapy for X-linked chronic granulomatous disease (X-CGD) has proven to be a viable approach, but random vector integration and subnormal protein production from exogenous promoters in transduced cells remain concerning for long-term safety and efficacy. A previous genome editing-based approach using Streptococcus pyogenes Cas9 mRNA and an oligodeoxynucleotide donor to repair genetic mutations showed the capability to restore physiological protein expression but lacked sufficient efficiency in quiescent CD34+ hematopoietic cells for clinical translation. Here, we report that transient inhibition of p53-binding protein 1 (53BP1) significantly increased (2.3-fold) long-term homology-directed repair to achieve highly efficient (80% gp91phox+ cells compared with healthy donor control subjects) long-term correction of X-CGD CD34+ cells.

Graphical abstract

Figure 1.

DHR assay and quantitative assessment of superoxide production highlights X-CGD carriers with reduced-frequency functioning phagocytes to produce normal level O₂^– per cell. (A) FACS DHR assay comparing ROS (blue) production in HD subjects, X-CGD carriers, and X-CGD patients with null mutation, or missense low residual ROS producing, compared with unstimulated controls (red). (B) Quantitative superoxide measurement in pooled cells from HD subjects (n = 1095), X-CGD carriers (n = 187) (pooled cells or normalized to DHR⁺ cells), and X-CGD patients (n = 128).

Figure 2.

HDR enhancement with 53BP1 inhibition is most effective with i53 mRNA. (A) Depiction of CYBB c.676 C>T mutation (top; red arrowhead) and successful repair with a 100-nucleotide ODN donor or TI of CYBB E7-13pA, CYBB E7-13pA-WPRE, or CYBB E7-13pA-SFFV-GFP delivered by AAV. (B) Percentages of GFP⁺ in HD HSPCs following TI with increasing i53 concentration (0-300 µg/mL); (n = 1; 8 replicates). (C) Percentages of GFP⁺ cells GE with Cas9 RNA or RNP and i53 or e18 enhancer (n = 4 experiments; 2 donors). (D) TI rates after GE with Cas9 RNA or RNP and increasing AAV donor (multiplicity of infection 1 = 10³/cell) ±i53 (150 µg/mL; n = 3 [1 HD, 2 X-CGD patients]). (E) TI rates after GE in the presence of i53, BRCA2, or RAD51 at indicated concentrations (n = 2 experiments). *P < .05, **P < .01, ****P < .0001, paired Student t test.

Figure 3.

Optimization of HDR-dependent GE conditions to functionally correct X-CGD HSPCs and DNA damage response triggered by AAV/DSB. (A) TI CYBB E7-13pA in X-CGD CD34⁺ HSPCs with i53 as indicated. (B) Dot plots of gp91^phox expression in myeloid-differentiated HSPCs gene edited with AAV (E7-13pA and E7-13pA WPRE) or ODN. (C) FACS histograms comparing gp91^phox expression in X-CGD E7 mutation repair by ODN (navy line) aligned with HD (shaded red) and AAV-TI CYBB E7-13pA (orange line), compared with naive control (gray). (D) Representative FACS of gp91^phox expression (top) and DHR assay (bottom) in myeloid-differentiated X-CGD CD34⁺ cells after CYBB E7 ODN mutation repair ±i53 (150 µg/mL). (E) FACS comparison of gp91^phox expression in differentiated X-CGD HSPCs GE with Cas9 RNA or RNP. (F) Percentages of gp91^phox expressing cells with ODN mutation repair of X-CGD HSPCs using Cas9 mRNA ±i53 or RNP +i53 (150 µg/mL). (G) Sequencing shows repaired alleles in HSPCs GE with Cas9 RNA or RNP and ODN ±i53 (150 µg/mL). (H) FACS for phosphorylated H2AX (γH2AX) in HSPCs exposed to AAV alone, or with DSB repaired with AAV or ODN donor at 2 hours. (I) The kinetics of γH2AX expression in HSPCs exposed to AAV with or without DSB and i53 (150 µg/mL). (J) Phosphorylation of H2AX after exposure of HSPCs to AAV (4 different donors) alone or with DSB repair by AAV or ODN. n = 4 experiments. **P < .01, ****P < .0001, paired Student t test. N, naive; SSC, side scatter.

Figure 4.

Highly efficient correction of engrafting X-CGD HSPCs by SpCas9/sg mRNA/ODN donor with i53. (A) Peripheral blood (PB) from mice transplanted with X-CGD HSPCs GE with ODN or AAV (CYBB E7-13pA) analyzed at weeks 8, 12, and 26 for engraftment (hCD45⁺). (B) Percentages of gp91^phox+ cells in hCD45⁺ PB. (C) FACS showing human CD45⁺ engraftment in mice bone marrow (BM) 18 to 26 weeks after transplant with ODN mutation-repaired X-CGD HSPCs (n = 3 patients). (D) Gp91^phox expression in myeloid-differentiated BM human CD45⁺ cells from NSG transplanted with ODN mutation–repaired X-CGD HSPCs (3 patients). (E) DHR assay showing ROS production in the myeloid-differentiated cells in panel D. (F) Lineage (CD33⁺ and CD19⁺) composition in BM, PB, and spleen of transplanted mice. (G) Gp91^phox expression in CD33⁺ myeloid cells in myeloid-differentiated BM and PB. (H) Percentages of gp91^phox+ before (in vitro) and after transplant (BM) with ODN-treated cells using Cas9 RNA (±i53) vs RNP (n = 4 experiments; 3 X-CGD patients, 11 mice). (I) Correction rates (% corrected alleles) before (input, 5 days post-electroporation, blue) vs after transplant (red) (n = 3 X-CGD patients). **P < .001.

Figure 5.

Molecular analysis of treated X-CGD HSPCs at on-target (ON) and OT sites confirm the high specificity of the SpCas9/sg/ODN genome-editing system maintained in the presence of i53. (A) Long-range PCR at CYBB E7 target site in HSPCs (naive, Cas9/sg alone, or with i53, ODN donor, or both). Genomic deletions shown by loss of coverage of sequencing reads. (B) Graph summarizing frequency of large deletions in samples treated as indicated. (C) ON and OT sites identified by circularization for high-throughput analysis of nuclease genome-wide effects by sequencing (CHANGE-seq) with mismatches as shown, with the number of reads for respective sites as indicated on the right. (D) Manhattan plot of the ON and OT sites with the sg7 sgRNA on CYBB c.676 C>T mutation X-CGD patient genomic DNA. The arrow indicates CYBB on chromosome X. (E) Indel frequency at ON and OT sites before (input) and after transplant (26 weeks) in NSG (output) of HSPCs (naive [gray]), GE with (blue) or without (red) i53 (mean ± standard deviation, ordinary one-way analysis of variance, not significant). (F) Percentages of reads with size discrepancies indicating indels for naive (N), or treated with (+) or without (-) i53 at ON and at each OT site. (G) Frequency of DSB repair events in vitro (IV) and posttransplant (fresh BM) with/without i53.

Figure 6.

FISH (left) and karyotyping (right) studies in GE HSPCs. HSPCs CD34⁺ were genome edited without i53 (top) (n = 10 cells) and with i53 (bottom) (n = 15 cells).