Table 1 |.
Advantages and limitations of technologies for genome editing of therapeutic T cell products
| Technology | Mechanism | On-target efficacy | Limitations | Benefits | Targets | Rationale |
|---|---|---|---|---|---|---|
| ZFN | DSB; ZFN DBD + FokI endonuclease domain; operates as dimer | Up to 28% in a clinical trial212; up to 90% on a non-clinical scale213 | ZFN specificity designed by screening then tested empirically; requires pair of ZFNs | Low off-target editing | CCR5 (REFS100,212,214) | Resistance to HIV by knockout of viral co-receptor |
| CXCL4 (REFS215,216) | ||||||
| TRAC213 | Knockout of endogenous TCRa | |||||
| TRBC217 | ||||||
| TALEN | DSB; TALE DBD + FokI endonuclease domain; operates as dimer | Up to 64% in a clinical trial218; up to 80% on a non-clinical scale219 | Large protein size; can induce translocations218 | Low off-target editing | CD52 (REF.218) | Resistance to anti-CD52 antibody therapy |
| CD40L102 | Repair of damaged gene causing hyper-IgM syndrome | |||||
| CCR5 (REF.220) | Resistance to HIV by knockout of viral co-receptor | |||||
| DCK (encodes deoxycytidine kinase)219 | Resistance to purine nucleotide analogue drugs | |||||
| PDCD1 (encodes PD1)221 | Reduces T cell exhaustion | |||||
| TRAC219,221 | Knockout of endogenous TCRa | |||||
| MegaTAL | DSB; TALE DBD + meganuclease | Up to 61% on a non-clinical scale101 | Off-target editing103 | Small protein size | CCR5 (REF.103) | Resistance to HIV by knockout of viral co-receptor |
| CRISPR-Cas9 | DSB; gRNA + Endonuclease Cas9 | Up to 45% in a clinical trial10; up to 95% on a non-clinical scale222 | Target must be adjacent to PAM; high degree of off-target editing or translocations | Easy to design and screen guides, high efficiency | B2M222 | Protects universal T cells from alloreactive rejection by host |
| CCR5 (REF.223) | Resistance to HIV by knockout of viral co-receptor | |||||
| PDCD1 (REF.222) | Reduces T cell exhaustion | |||||
| RNF20 (REF.159) | Stabilizes FOXP3 in Treg cells | |||||
| TRAC85,222 | Knockout of endogenous TCRa | |||||
| TRBC222 | ||||||
| Nuclease + HDR | DSB in presence of ssDNA or dsDNA delivered by AAV or electroporation | Non-viral: up to 61% on a non-clinical scale105 Viral: 40–60% on a non-clinical scale85,224 |
Immunogenicity of DNA or AAV, off-target integration; dependent on presence of adjacent PAM | Integration of large cargoes | AAVS1 (REF.100) | Safe harbour site for genome integration |
| CD40L102 | Repair of damaged gene causing hyper-IgM syndrome | |||||
| FOXP3 (REF.75) | Converts Teff cells into Treg-like cells | |||||
| IL2RA104 | Repair of damaged gene causing autoimmunity | |||||
| IL2RG220 | Repair of damaged gene causing SCID | |||||
| TRAC85,104 | Puts CAR or other transgenes under control of endogenous promoter | |||||
| Base editing | Nucleotide deaminase + dCas9 + uracil DNA glycosylase inhibitor + single gRNA | >95% on a non-clinical scale225 | Transition mutations only, no deletions or insertions; bystander edits in editing window, dependent on adjacent PAM; may not eliminate protein expression | No DSB, no detectable translocations in triple editing | TRAC225 | Knockout of endogenous TCRa |
| CIITA225 | Protects universal T cells from alloreactive rejection by host | |||||
| B2M225 |
AAV, adeno-associated virus; CAR, chimeric antigen receptor; DBD, DNA-binding domain; dCas9, dead (catalytically inactive) Cas9; DSB, DNA double-strand break; dsDNA, double-stranded DNA; gRNA, guide RNA; HDR, homology directed repair; PAM, protospacer adjacent motif; SCID, severe combined immunodeficiency; ssDNA, single-stranded DNA; TALE, transcription activator-like effector; TALEN, transcription activator-like effector nuclease; TCR, T cell receptor; Teff cell; effector T cell; Treg cell; regulatory T cell; ZFN, zinc-finger nuclease.
a
Knockout of endogenous TCR improves surface expression of transgenic TCR or generates universal T cells.