Efficient modification of CCR5 in primary human hematopoietic cells using a megaTAL nuclease and AAV donor template

Abstract

Genetic mutations or engineered nucleases that disrupt the HIV co-receptor CCR5 block HIV infection of CD4(+) T cells. These findings have motivated the engineering of CCR5-specific nucleases for application as HIV therapies. The efficacy of this approach relies on efficient biallelic disruption of CCR5, and the ability to efficiently target sequences that confer HIV resistance to the CCR5 locus has the potential to further improve clinical outcomes. We used RNA-based nuclease expression paired with adeno-associated virus (AAV)-mediated delivery of a CCR5-targeting donor template to achieve highly efficient targeted recombination in primary human T cells. This method consistently achieved 8 to 60% rates of homology-directed recombination into the CCR5 locus in T cells, with over 80% of cells modified with an MND-GFP expression cassette exhibiting biallelic modification. MND-GFP-modified T cells maintained a diverse repertoire and engrafted in immune-deficient mice as efficiently as unmodified cells. Using this method, we integrated sequences coding chimeric antigen receptors (CARs) into the CCR5 locus, and the resulting targeted CAR T cells exhibited antitumor or anti-HIV activity. Alternatively, we introduced the C46 HIV fusion inhibitor, generating T cell populations with high rates of biallelic CCR5 disruption paired with potential protection from HIV with CXCR4 co-receptor tropism. Finally, this protocol was applied to adult human mobilized CD34(+) cells, resulting in 15 to 20% homologous gene targeting. Our results demonstrate that high-efficiency targeted integration is feasible in primary human hematopoietic cells and highlight the potential of gene editing to engineer T cell products with myriad functional properties.

Fig. 1. CCR5-TALEN and CCR5-megaTAL activity in T cells and comparison of NHEJ and HDR events in a TLR reporter line

(A) Location of the CCR5-TALEN (red line) and CCR5-megaTAL nuclease (blue highlight) binding sites within the human CCR5 gene. Nuclease architecture at the target site is schematically diagrammed beneath. CCR5 sequence used as homology arms in donor templates in subsequent figures is indicated at top. (B) Percentage of NHEJ events in primary human T cells detected using the T7 and TIDE sequencing 18 to 21 days after transfection with megaTAL, TALEN, or BFP control mRNA (1 μg of each mRNA and 1 μg of RNA per TALEN monomer) (n = 5). (C) Schematic diagram of traffic light reporter (TLR) cassette and representative data showing fluorescent marker expression based on repair pathway. Translational reading frame is indicated in parenthesis for each coding sequence. Fluorescence-activated cell sorting (FACS) plots beneath show green fluorescent protein (GFP) and mCherry expression in representative CCR5 TLR assay. GibberishFP, open reading frame (ORF) encoded by out-of-frame translation of GFP; eGFP, enhanced GFP. (D) HDR versus NHEJ events at CCR5 TLR locus in human embryonic kidney (HEK) 293T cells after treatment with CCR5-megaTAL or CCR5-TALEN mRNA and increasing MOI of AAV.GFP repair template. Bars show the means ± SEM; significance was calculated using the unpaired two-tailed t test; P values <0.05 are in red. CDS, coding sequences; FOK1, Flavobacterium okeanokoites nuclease.

Fig. 2. Efficient HDR in primary T cells leading to introduction of a GFP expression cassette within the CCR5 locus

(A) (Top) Diagram of AAV constructs used as donor templates. (Bottom) Timeline of gene-editing procedure beginning with bead stimulation of CD4⁺ T cells. pA, SV40 poly-adenylation signal; HA, homology arm. (B) Representative flow cytometry plots showing BFP versus GFP expression at days 2 and 16 after gene editing. The percentage of live single lymphocytes within the relevant quadrant is indicated. (C) Cell viability and BFP and GFP expression over time after gene editing (n = 5). Significance was calculated using the unpaired t test with the Holm-Sidak correction for multiple comparisons; significant P values (<0.05) are in red. (D) Diagram of human CCR5 locus before and after HDR with the AAV.CCR5.GFP donor template. Primers binding outside homology arms were used to amplify gDNA from treated cells visualized on agarose gel; expected sizes of bands for unedited and edited alleles are indicated; right chart shows indel % (determined by TIDE sequencing) within the lower–molecular weight (MW) bands (n = 3). P value was calculated using the unpaired two-tailed t test. (E) Nested polymerase chain reaction (PCR) of single cells (31 to 100 cells per condition) edited using megaTAL and AAV.CCR5.GFP donor at indicted MOIs. Graph shows percentages of amplicons with a single higher–molecular weight band (biallelic HDR) and a single lower–molecular weight band (no HDR detected) or both bands (monoallelic HDR).

Fig. 3. Comparison of HDR rates in human primary T cells with CCR5-megaTAL versus those with CCR5-TALENs

(A) Representative FACS plots showing HDR events observed 16 days after treatment with CCR5-megaTAL versus those with CCR5-TALEN at varying MOI of the AAV.CCR5. GFP donor. The percentage of live single lymphocytes within a relevant quadrant is indicated. (B) Time course of cell viability (top), % BFP⁺ cells (middle), and % GFP⁺ cells (bottom) after treatment with control BFP (1 μg), CCR5-megaTAL (MT; 1 μg), or CCR5-TALEN (TAL; 1 μg of each TALEN half-site) mRNA with and without various MOI of the AAV.CCR5.GFP donor (or negative control AAV.BFP lacking CCR5 homology arms). (C) Comparison of % GFP 16 days after gene editing with CCR5-megaTAL versus that with TALEN at various MOI of AAV.CCR5.GFP donor template. (D) Ratio of % GFP⁺ cells after megaTAL- versus that of TALEN-mediated HDR over time and at various MOI of the AAV donor. All bars show means ± SEM of n = 5; significance was calculated using unpaired t test with the Holm-Sidak correction for multiple comparisons; P values (<0.05) are in red.

Fig. 4. Maintenance of TCR diversity and sustained engraftment of gene-edited T cells

(A) Representative TCR spectratypes of Vβ subfamilies 1 to 25 in mock-treated T cells (bottom panel) or T cells 8 to 19 days after treatment with CCR5-megaTAL mRNA with and without AAV.CCR5. GFP donor. (B) Total number and relative percentage of CD4 lymphocytes (upper panels), GFP⁺ CD4 lymphocytes (lower left panel), and CD4⁺ CCR5⁺ lymphocytes (lower right panel) obtained from spleens of NSG mice 4 weeks after transfer of gene-edited T cells; numbers of mice for each condition is shown below x axis of GFP⁺ panel and is the same for all panels. Bars show the means ± SEM; P values were calculated using the unpaired two-tailed t test; P values <0.05 are in red. n = number of mice. (C) % GFP variation versus splenocyte number for each mouse receiving CCR5-megaTAL and AAV.CCR5.GFP donor–edited T cells. (D) gDNA from total splenocytes obtained from two recipient mice for each condition at 4 weeks after T cell transfer was PCR-amplified using primers outside of the donor template homology arms and run on an agarose gel. The predicted size of HDR-modified (4.4 kb) and NHEJ/unmodified alleles (3.2 kb) are indicated. Splenocytes from mice receiving T cells edited with megaTAL and AAV.CCR5.GFP donor co-delivery were flow-sorted before obtaining gDNA to detect HDR and NHEJ events in GFP⁺ and GFP⁻ cells. WT, wild-type.

Fig. 5. Gene editing of mobilized adult CD34⁺ cells using co-delivery of megaTAL and AAV.CCR5.GFP donor

(A) Timeline of CD34⁺ cell gene-editing procedures and analyses. (B) (Left) % NHEJ events detected (by TIDE sequencing) 10 days after treatment with megaTAL mRNA and % of cells separately transfected with BFP mRNA 24 hours after transfection. (Right) representative flow plots (gated on live singlets) showing BFP expression 24 hours after transfection. (C) Representative flow analysis (gated on live singlets) of BFP and GFP expression in CD34⁺ cells transduced with the AAV.CCR5.GFP template with and without CCR5-megaTAL mRNA transfection at 4 and 10 days post-transduction. (D) Viability and % BFP⁺ and GFP⁺ CD34⁺ cells after treatment with the specified gene-editing reagents. (E) DNA gel of PCR-amplified gDNA obtained from gene-edited CD34⁺ cells. Predicted sizes of HDR-modified (4.4 kb) and NHEJ/unmodified alleles (3.2 kb) are indicated. Chart shows the indel % (by TIDE sequencing) within the lower–molecular weight band (n = 3). All P values were calculated using the unpaired two-tailed t test; n = number of independent experiments, performed using cells from two donors.

Fig. 6. Efficient expression of genetic therapies targeted to the CCR5 locus in primary CD3 T cells

(A to C) AAV donor templates used to target C46 (A), HIV-CAR (B), and CD19-CAR (C) to the CCR5 locus, including viral packaging size and MOI. FACS plots show expression 16 days after gene editing. For each, we show negative controls that were mock-transfected or transduced with only the AAV donor and the % of cells in relevant quadrant for transgene expression. HIV-CAR and CD19-CAR populations were column- or sort-purified before analysis at day 16. (D) CD137 expression in engineered T cells (x axis) cultured for 24 hours in the presence (blue or pink bars) or absence (black bars) of target cells expressing their indicated cognate antigen; P values were calculated using the unpaired two-tailed t test, and significant P values (<0.05) are in red; n = 3 independent experiments using T cells from different subjects. (E) Selective loss of GFP⁺ CD19–expressing K562 target cells (T) relative to CD19⁻ iRFP⁺ K562 cells (y axis) 48 hours after treatment with CD19-CAR⁺ T cell effectors (E) (ratio of E/T indicated on the x axis); n = 3. (F) DNA gels showing PCR amplicons of gDNA using primers binding outside of the CCR5 homology arms. gDNA was obtained from T cells edited with the AAV donors diagrammed in (A), after purification by flow cytometry. Predicted sizes of HDR-modified alleles (CD19-CAR. T2A.BFP, 6.4 kb; HIV-CAR.T2A.RQR8, 6.3 kb; C46.T2A.GFP, 6.0 kb) are indicated by arrowheads at the right; open arrow indicates size of unmodified allele (3.2 kb). WPRE, woodchuck hepatitis virus posttranscriptional regulatory element; PE, phycoerythrin.