Macaque CCR5 ZFN off-target activity in vitro and in vivo following delivery into HSPCs and infusion
| OT # . | Database . | Chrom . | Location . | Gene . | Exon/Intron . | ZFNBS . | In vitro 5-7 d GFP . | In vitro 5-7 d ZFN . | In vivo 6-11 d ZFN . | In vivo 189-198 d ZFN . |
|---|---|---|---|---|---|---|---|---|---|---|
| Intended | rheMac2 | chr2 | 90143599 | CCR5 | Exon | L_6_R | 0.11 ± 0.02 | 52.76 ± 9.29* | 33.55 ± 7.69* | 3.65 ± 0.39* |
| 1 | rheMac2 | chrX | 70535298 | CXCR3 | Exon | L_6_R | 0.03 ± 0.02 | 0.05 ± 0.02 | 0.47 ± 0.71 | 0.04 ± 0.03 |
| 2 | rheMac2 | chr14 | 74379519 | WNT11 | Intron | R_5_L | 0.03 ± 0.00 | 0.03 ± 0.01 | 0.02 ± 0.00 | 0.02 ± 0.01 |
| 3 | rheMac2 | chr10 | 30952397 | — | Intergenic | L_5_R | 0.00 ± 0.00 | 0.01 ± 0.00 | 0.01 ± 0.01 | 0.01 ± 0.00 |
| 4 | rheMac3 | chr20 | 2291152 | — | Unknown | R_6_L | 0.01 ± 0.01 | 0.01 ± 0.00 | 0.01 ± 0.01 | 0.01 ± 0.01 |
| 5 | rheMac2 | chr4 | 46918712 | — | Intergenic | L_5_R | 0.02 ± 0.00 | 0.01 ± 0.01 | 0.02 ± 0.01 | 0.02 ± 0.00 |
| 6 | rheMac2 | chr15 | 2710971 | — | Intergenic | L_6_R | 0.01 ± 0.01 | 0.01 ± 0.00 | 0.01 ± 0.00 | 0.02 ± 0.01 |
| 7 | rheMac2 | chr13 | 58922755 | — | Intergenic | R_5_L | 0.01 ± 0.00 | 0.02 ± 0.01 | 0.02 ± 0.01 | 0.01 ± 0.01 |
| 8 | rheMac2 | chr10 | 32119972 | C20ORF112 | Intron | L_5_R | 0.05 ± 0.01 | 0.05 ± 0.00 | 0.06 ± 0.01 | 0.04 ± 0.01 |
| 9 | rheMac3 | chr10 | 94067013 | — | Unknown | R_6_L | 0.02 ± 0.00 | 0.02 ± 0.01 | 0.02 ± 0.01 | 0.01 ± 0.00 |
| 10 | rheMac2 | chr11 | 61360349 | C12orf56 | Intron | R_6_L | 0.07 ± 0.02 | 0.09 ± 0.02 | 0.08 ± 0.02 | 0.08 ± 0.01 |
| 11 | rheMac2 | chr9 | 79520402 | — | Intergenic | R_5_L | 0.28 ± 0.03 | 0.26 ± 0.08 | 0.30 ± 0.04 | 0.27 ± 0.02 |
| 12 | rheMac2 | chr8 | 109331649 | — | Intergenic | R_6_L | 0.04 ± 0.01 | 0.05 ± 0.03 | 0.07 ± 0.03 | 0.05 ± 0.00 |
| 13 | rheMac2 | chr8 | 126618925 | FER1L6 | Intron | R_5_L | 0.02 ± 0.01 | 0.01 ± 0.00 | 0.02 ± 0.01 | 0.02 ± 0.00 |
| 14 | rheMac2 | chr16 | 39404073 | STXBP4 | Intron | L_5_R | 0.11 ± 0.03 | 0.17 ± 0.00 | 0.10 ± 0.01 | 0.08 ± 0.01 |
| 15 | rheMac2 | chr19 | 15545514 | — | Intergenic | L_5_R | 0.06 ± 0.01 | 0.05 ± 0.00 | 0.05 ± 0.02 | 0.05 ± 0.02 |
| 16 | rheMac3 | chr10 | 39382414 | — | Unknown | R_5_L | 0.02 ± 0.00 | 0.03 ± 0.02 | 0.02 ± 0.00 | 0.01 ± 0.00 |
| 17 | rheMac3 | chr19 | 13168773 | — | Unknown | L_5_R | 0.07 ± 0.03 | 0.40 ± 0.46 | 0.13 ± 0.04 | 0.23 ± 0.10 |
| 18 | rheMac2 | chr11 | 2692851 | CACNA1C | Intron | L_6_R | 0.01 ± 0.01 | 0.01 ± 0.01 | 0.01 ± 0.01 | 0.01 ± 0.00 |
| OT # . | Database . | Chrom . | Location . | Gene . | Exon/Intron . | ZFNBS . | In vitro 5-7 d GFP . | In vitro 5-7 d ZFN . | In vivo 6-11 d ZFN . | In vivo 189-198 d ZFN . |
|---|---|---|---|---|---|---|---|---|---|---|
| Intended | rheMac2 | chr2 | 90143599 | CCR5 | Exon | L_6_R | 0.11 ± 0.02 | 52.76 ± 9.29* | 33.55 ± 7.69* | 3.65 ± 0.39* |
| 1 | rheMac2 | chrX | 70535298 | CXCR3 | Exon | L_6_R | 0.03 ± 0.02 | 0.05 ± 0.02 | 0.47 ± 0.71 | 0.04 ± 0.03 |
| 2 | rheMac2 | chr14 | 74379519 | WNT11 | Intron | R_5_L | 0.03 ± 0.00 | 0.03 ± 0.01 | 0.02 ± 0.00 | 0.02 ± 0.01 |
| 3 | rheMac2 | chr10 | 30952397 | — | Intergenic | L_5_R | 0.00 ± 0.00 | 0.01 ± 0.00 | 0.01 ± 0.01 | 0.01 ± 0.00 |
| 4 | rheMac3 | chr20 | 2291152 | — | Unknown | R_6_L | 0.01 ± 0.01 | 0.01 ± 0.00 | 0.01 ± 0.01 | 0.01 ± 0.01 |
| 5 | rheMac2 | chr4 | 46918712 | — | Intergenic | L_5_R | 0.02 ± 0.00 | 0.01 ± 0.01 | 0.02 ± 0.01 | 0.02 ± 0.00 |
| 6 | rheMac2 | chr15 | 2710971 | — | Intergenic | L_6_R | 0.01 ± 0.01 | 0.01 ± 0.00 | 0.01 ± 0.00 | 0.02 ± 0.01 |
| 7 | rheMac2 | chr13 | 58922755 | — | Intergenic | R_5_L | 0.01 ± 0.00 | 0.02 ± 0.01 | 0.02 ± 0.01 | 0.01 ± 0.01 |
| 8 | rheMac2 | chr10 | 32119972 | C20ORF112 | Intron | L_5_R | 0.05 ± 0.01 | 0.05 ± 0.00 | 0.06 ± 0.01 | 0.04 ± 0.01 |
| 9 | rheMac3 | chr10 | 94067013 | — | Unknown | R_6_L | 0.02 ± 0.00 | 0.02 ± 0.01 | 0.02 ± 0.01 | 0.01 ± 0.00 |
| 10 | rheMac2 | chr11 | 61360349 | C12orf56 | Intron | R_6_L | 0.07 ± 0.02 | 0.09 ± 0.02 | 0.08 ± 0.02 | 0.08 ± 0.01 |
| 11 | rheMac2 | chr9 | 79520402 | — | Intergenic | R_5_L | 0.28 ± 0.03 | 0.26 ± 0.08 | 0.30 ± 0.04 | 0.27 ± 0.02 |
| 12 | rheMac2 | chr8 | 109331649 | — | Intergenic | R_6_L | 0.04 ± 0.01 | 0.05 ± 0.03 | 0.07 ± 0.03 | 0.05 ± 0.00 |
| 13 | rheMac2 | chr8 | 126618925 | FER1L6 | Intron | R_5_L | 0.02 ± 0.01 | 0.01 ± 0.00 | 0.02 ± 0.01 | 0.02 ± 0.00 |
| 14 | rheMac2 | chr16 | 39404073 | STXBP4 | Intron | L_5_R | 0.11 ± 0.03 | 0.17 ± 0.00 | 0.10 ± 0.01 | 0.08 ± 0.01 |
| 15 | rheMac2 | chr19 | 15545514 | — | Intergenic | L_5_R | 0.06 ± 0.01 | 0.05 ± 0.00 | 0.05 ± 0.02 | 0.05 ± 0.02 |
| 16 | rheMac3 | chr10 | 39382414 | — | Unknown | R_5_L | 0.02 ± 0.00 | 0.03 ± 0.02 | 0.02 ± 0.00 | 0.01 ± 0.00 |
| 17 | rheMac3 | chr19 | 13168773 | — | Unknown | L_5_R | 0.07 ± 0.03 | 0.40 ± 0.46 | 0.13 ± 0.04 | 0.23 ± 0.10 |
| 18 | rheMac2 | chr11 | 2692851 | CACNA1C | Intron | L_6_R | 0.01 ± 0.01 | 0.01 ± 0.01 | 0.01 ± 0.01 | 0.01 ± 0.00 |
M nemestrina CD34+ cells were BTX electroporated with the mCCR5 ZFN (32106:32118) mRNA followed by a 30°C “cold shock” to increase ZFN activity. Cells were then transplanted back into the M nemestrina (in vivo) or cultured at 37°C (in vitro) for 5 days. Cells were collected at indicated time points and subjected to gDNA purification and sequencing analysis (MiSeq) to evaluate genome modification (% indels) at each of the potential target sites indicated. Data from 3 independent experiments were combined (n = 3) except the “in vitro, 5 to 7 days, ZFN” data set (n = 2). Shown are average ± standard deviation. ZFN-dependent disruption at the on-target CCR5 locus was significantly increased relative to GFP mRNA controls (*P ≤ .05). No off-target values were significantly different from the GFP mRNA control.
Chrom, chromosome; ZFNBS, zinc-finger nuclease binding site.