Previous studies of gene editing for inborn errors of immunity
| Gene . | Methodology . | Results . | Off-target analysis . | Reference . |
|---|---|---|---|---|
| IL2RG | CRISPR/Cas9 targeted insertion of IL2RG by HDR with AAV6 with i53 | Superior engraftment of gene-edited HSPCs in NSG-SGMC3 mice vs lentiviral correction (23.3% vs 8.1%). Improved correction of NKs vs lentivirus (40.7% vs 2.8%) | No notable OT sites after sgRNA optimization | 11 |
| IL2RG | CRISPR/Cas9 targeted insertion of IL2RG by HDR with AAV6 with i53 | >25% gene correction rate in engrafted marrow HSPCs in NSG mice >10% retention of gene corrected HSPCs in serial transplantation, and rescue of T-cell development in vivo. | 5 OT sites identified; reduced to background with sgRNA optimization and Hifi Cas9 | 13 |
| MAGT1 | CRISPR/Cas9 targeted insertion of MAGT1 by HDR with AAV6 with i53 | Optimization of targeted insertion of MAGT1 corrected gene in >50% of HSPCs. Normalization of NKG2D expression in GE T and NK cells. Engraftment of gene edited MAGT1 HSCPs (>11%) in NSGS-SGMC2 mice with development of corrected T, B, and NK cells. | No notable OT sites | 15 |
| CD40LG | CRISPR/Cas9 and TALEN-mediated insertion of CD40L with AAV6 in HSCs | >20% correction by CRISPR/Cas9 and >13% by TALEN in primary T cells Comparable viability and capacity of gene corrected CD34+ HSCs to support multilineage hematopoiesis in NSG mice. | 2 OT sites by TALEN, no notable OT sites by CRISPR | 10 |
| CD3D | Repair of nonsense mutation (p.R68X) in CD3D by ABE | Correction of CD3D mutation in >80% of edited Jurkat T cells Successful engraftment of corrected HSPCs in humanized mice (85% gene corrected in bone marrow) Restoration of T-cell development in ATO system. | Minimal local bystander editing by lead ABE candidate (<1.4%), and no OT sites in coding regions. | 8 |
| SH2D1A | Comparison of TALEN, CRISPR/Cas9, or Cas12 for targeted insertion of SAP cDNA | Restoration of SAP expression in >45% of primary T cells with comparable efficiency of tested systems. | 2 lower frequency OT sites (1 TALEN, 1 Cas9); none in coding regions | 14 |
| BTK | CRISPR/Cas9 targeted insertion of BTK cDNA via AAV6 | >60% correction of BTK-deficient B-cell lines with comparable BTK expression to WT. >50% integration in human peripheral blood CD34+ stem cells. | 2 OT sites, eliminated by use of engineered Cas9 variants | 9 |
| FOXP3 | CRISPR/Cas9 targeted insertion of FOXP3 via AAV6 | >30% integration of FOXP3 in HSPCs Comparable immunophenotyping of FOXP3-edited Tregs compared with WT | No OT sites in coding regions | 12 |
| Gene . | Methodology . | Results . | Off-target analysis . | Reference . |
|---|---|---|---|---|
| IL2RG | CRISPR/Cas9 targeted insertion of IL2RG by HDR with AAV6 with i53 | Superior engraftment of gene-edited HSPCs in NSG-SGMC3 mice vs lentiviral correction (23.3% vs 8.1%). Improved correction of NKs vs lentivirus (40.7% vs 2.8%) | No notable OT sites after sgRNA optimization | 11 |
| IL2RG | CRISPR/Cas9 targeted insertion of IL2RG by HDR with AAV6 with i53 | >25% gene correction rate in engrafted marrow HSPCs in NSG mice >10% retention of gene corrected HSPCs in serial transplantation, and rescue of T-cell development in vivo. | 5 OT sites identified; reduced to background with sgRNA optimization and Hifi Cas9 | 13 |
| MAGT1 | CRISPR/Cas9 targeted insertion of MAGT1 by HDR with AAV6 with i53 | Optimization of targeted insertion of MAGT1 corrected gene in >50% of HSPCs. Normalization of NKG2D expression in GE T and NK cells. Engraftment of gene edited MAGT1 HSCPs (>11%) in NSGS-SGMC2 mice with development of corrected T, B, and NK cells. | No notable OT sites | 15 |
| CD40LG | CRISPR/Cas9 and TALEN-mediated insertion of CD40L with AAV6 in HSCs | >20% correction by CRISPR/Cas9 and >13% by TALEN in primary T cells Comparable viability and capacity of gene corrected CD34+ HSCs to support multilineage hematopoiesis in NSG mice. | 2 OT sites by TALEN, no notable OT sites by CRISPR | 10 |
| CD3D | Repair of nonsense mutation (p.R68X) in CD3D by ABE | Correction of CD3D mutation in >80% of edited Jurkat T cells Successful engraftment of corrected HSPCs in humanized mice (85% gene corrected in bone marrow) Restoration of T-cell development in ATO system. | Minimal local bystander editing by lead ABE candidate (<1.4%), and no OT sites in coding regions. | 8 |
| SH2D1A | Comparison of TALEN, CRISPR/Cas9, or Cas12 for targeted insertion of SAP cDNA | Restoration of SAP expression in >45% of primary T cells with comparable efficiency of tested systems. | 2 lower frequency OT sites (1 TALEN, 1 Cas9); none in coding regions | 14 |
| BTK | CRISPR/Cas9 targeted insertion of BTK cDNA via AAV6 | >60% correction of BTK-deficient B-cell lines with comparable BTK expression to WT. >50% integration in human peripheral blood CD34+ stem cells. | 2 OT sites, eliminated by use of engineered Cas9 variants | 9 |
| FOXP3 | CRISPR/Cas9 targeted insertion of FOXP3 via AAV6 | >30% integration of FOXP3 in HSPCs Comparable immunophenotyping of FOXP3-edited Tregs compared with WT | No OT sites in coding regions | 12 |
ABE, adenine base editing; ATO, artificial thymic organoid; BTK, Bruton's tyrosine kinase; cDNA, complementary DNA; OT, off target; sgRNA, single guide RNA; WT, wild type.