Highly efficient homology-driven genome editing in human T cells by combining zinc-finger nuclease mRNA and AAV6 donor delivery

The adoptive transfer of engineered T cells for the treatment of cancer, autoimmunity, and infectious disease is a rapidly growing field that has shown great promise in recent clinical trials. Nuclease-driven genome editing provides a method in which to precisely target genetic changes to further enhance T cell function in vivo. We describe the development of a highly efficient method to genome edit both primary human CD8 and CD4 T cells by homology-directed repair at a pre-defined site of the genome. Two different homology donor templates were evaluated, representing both minor gene editing events (restriction site insertion) to mimic gene correction, or the more significant insertion of a larger gene cassette. By combining zinc finger nuclease mRNA delivery with AAV6 delivery of a homologous donor we could gene correct 41% of CCR5 or 55% of PPP1R12C (AAVS1) alleles in CD8+ T cells and gene targeting of a GFP transgene cassette in >40% of CD8+ and CD4+ T cells at both the CCR5 and AAVS1 safe harbor locus, potentially providing a robust genome editing tool for T cell-based immunotherapy.


Supplementary Figure S2
Supplementary Figure S2. CD4 + T cells are efficiently transduced by AAV serotype 6. CD4 + T cells were transduced with increasing doses of GFP-expressing AAV vectors of the indicated serotypes, and GFP expression was determined at 5 days post-transduction by flow cytometry. The vector doses used were 1 x 10 4 , 3 x 10 4 , 1 x 10 5 , 3 x 10 5 , and 1 x 10 6 vector genomes (vg)/cell. (A) Combined results of 3 independent experiments using different CD4 + T cell donors were shown as mean +/-SD. (B) Flow cytometric plots from a representative experiment at 3 x 10 5 vg/cell are shown.

Supplementary Figure S4
Supplementary Figure S4. Combination of ZFN mRNA and AAV6 vectors promotes high levels of transgene addition at the CCR5 locus in CD4 + T cells. (A) CD4 + T cells were treated as described in Supplementary Figure 2A, but using CCR5-GFP donor vectors, with and without CCR5 ZFN mRNA electroporation. Cells were collected at 7 days post-transduction and analyzed by flow cytometry for % GFP + . Results are combined from two experiments using different CD4 + T cell donors, and show mean +/-SD. * p<0.05, 2-tailed t-test to compare %GFP+ between conditions with different doses of AAV6 donor in the presence of ZFN mRNA treatment. (B) Flow cytometry plots from one representative experiment using 1 x 10 6 vg/cell CCR5-GFP donor, at 7 days post-electroporation. (C) Confirmation of targeted integration of GFP expression cassette at the CCR5 locus by semi-quantitative PCR. The % GFP HDR was estimated by comparison to standards.

Supplementary Figure S5
Supplementary Figure S5: Confirmation of high levels of transgene addition at the AAVS1 locus in CD8 + T cells. CD8 + T cells were transduced with AAV6 vectors carrying the AAVS1-GFP donor at indicated doses (vg/cell) for 16 hours and/or electroporated with AAVS1 ZFN mRNA. Cells were analyzed 14 days postelectroporation by an Out-Out PCR as described in Materials And Methods to detect targeted integration (TI) of GFP at the AAVS1 locus by comparison to the HDR standards. The presence of GFP-TI is indicated by the top arrow.

In-Out PCR primers CCR5
in-out GAGGATTGGGAAGACAATAGCAG CCAGCAATAGATGATCCAACTCAAATTCC control GATTTGCACAGCTCATCTGGC CCATCTTGTTCCACCCTGTGC * Only the target-specific portion of the MiSeq adaptor PCR primers is listed.
Reverse primer sequence is preceded by 5'-GACGTGTGCTCTTCCGATCT.   Table S2. CD8 + T cells were transduced with 1 X 10 6 vg/cell AAV6 vectors carrying the CCR5-RFLP donor for 16 hours, then electroporated with CCR5 ZFN mRNA (60 μg/ml). Cells were analyzed 7 days post-electroporation by deep sequencing to measure the efficiency of genome modification (% indels and RFLP HDR). The top 20 sequences from a treated sample were aligned against the wild-type CCR5 sequence. Only the most important portion of the sequences is shown. In sequence alignment, CCR5 ZFN binding sites are underlined. Deleted bases are indicated by "-". Inserted bases are indicated in red color. *One representative sequence for each type of alignment is shown.   Table S3. CD8 + T cells were transduced with 3 X 10 5 vg/cell AAV6 vectors carrying the AAVS1-RFLP donor for 16 hours, then electroporated with AAVS1 ZFN mRNA (40 μg/ml). Cells were analyzed 7 days post-electroporation by deep sequencing to measure the efficiency of genome modification (% indels and RFLP HDR). The top 20 sequences from a treated sample were aligned against the wild-type AAVS1 sequence. Only the most important portion of the sequences is shown. In sequence alignment, AAVS1 ZFN binding sites are underlined. Deleted bases are indicated by "-". Inserted bases are indicated in red color. *One representative sequence for each type of alignment is shown.