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Replacement of the endogenous TCR by non-viral genome targeting.
a, Schematic of HDR template used to replace the endogenous TCR. b, Non-viral genome targeting successfully replaced the endogenous TCR with the NY-ESO-1 antigen specific 1G4 TCR. c, Antigen-specific cytokine production and degranulation in CD8+ T cells with the replaced TCR. d, Antigen-specific target cell killing by CD8+ T cells with the replaced TCR. e, Melanoma tumour mouse xenograft model. f, Scalability of non-viral replacement of the endogenous TCR for adoptive cell therapy. g, Preferential in vivo localization of NY-ESO-1 TCR+ T cells to the tumour. h, Tumour growth following adoptive transfer of NY-ESO-1 TCR+ non-virally or lentivirally modified or vehicle alone (saline). One representative donor from n=6 (b) or n=2 (c, d) independent healthy donors with mean and standard deviation of technical triplicates (c, d). n=6 (f) or n=2 (g, h) independent healthy donors in 5 (g) or 7 mice (h) with mean and standard deviation (f-h). **P<0.01, ***P<0.001, ****P<0.0001 (Two Way ANOVA with Holm-Sidak’s multiple comparisons test). See also Extended Data Figs 9, ,1010.
Next, we assessed the tumour antigen-specific function of targeted human T cells. When the targeted T cells were co-cultured with two different NY-ESO-1+ melanoma cell lines, M257 and M407, the modified T cells robustly and specifically produced IFN-ɣ and TNF-α and induced T cell degranulation (measured by CD107a surface expression) (Fig. 4c). Cytokine production and degranulation only occurred when the NY-ESO-1 TCR T cells were exposed to cell lines expressing the appropriate HLA-A*0201 class I MHC allele required to present the cognate NY-ESO-1 peptide. Both the CD8+ and CD4+ T cell response was consistent across healthy donors, and was comparable to the response of T cells from the same healthy donor in which the NY-ESO-1 TCR was transduced by gamma retrovirus and heterologously expressed using a viral promoter (Fig. 4c and Extended Data Fig. 9j). NY-ESO-1 TCR knock-in T cells rapidly killed target M257-HLA-A*0201 cancer cells in vitro at rates similar to the positive control, retrovirally transduced T cells (Fig. 4d). Killing was selective for target cells expressing NY-ESO-1 antigen and the HLA-A*0201 allele, consistent across donors, and depended on the T cells being modified using both the correct gRNA and HDR template (Extended Data Fig. 9n-q).
Finally, we confirmed that non-viral genome targeting could be used to generate NY-ESO-1 TCR cells at scale and that these cells have in vivo anti-tumour function (Fig. 4e and Extended Data Fig. 10a). Given that knock-in efficiency was lower with non-viral targeting than with comparable sized AAV templates4, we first wanted to ensure that we could generate sufficient numbers of NY-ESO-1 positive cells for adoptive cell therapies. We electroporated 100 million T cells from six healthy donors, which after ten days of expansion yielded an average of 385 million NY-ESO-1 TCR T cells per donor (Fig. 4f and Extended Data Fig. 9i-m). NY-ESO-1 TCR knock-in T cells preferentially localized to, persisted at, and proliferated in the tumour rather than the spleen, similar to positive control lentivirally-transduced T cells (Fig. 4g and Extended Data Fig. 10b-f). Adoptive transfer of sorted NY-ESO-1 TCR T cells also reduced the tumour burden in treated animals (Fig. 4h).
Our therapeutic gene editing in human T cells is a process that takes only a short time from target selection to production of the genetically modified T cell product. In approximately one week, novel guide RNAs and DNA repair templates can be designed, synthesized, and the DNA integrated into primary human T cells that remain viable, expandable, and functional. The whole process and all required materials can be easily adapted to good manufacturing practices (GMP) for clinical use. Avoiding the use of viral vectors will accelerate research and clinical applications, reduce the cost of genome targeting, and potentially improve safety.
Looking forward, the technology could be used to “rewire” complex molecular circuits in human T cells. Multiplexed integration of large functional sequences at endogenous loci should allow combinations of coding and non-coding elements to be corrected, inserted, modified, and rearranged. Much work remains to be done to improve our understanding endogenous T cell circuitry if we are going to create synthetic circuits. Rapid and efficient non-viral tagging of endogenous genes in primary human cells will facilitate live-cell imaging and proteomic studies to decode T cell programs. Non-viral genome targeting provides an approach to re-write these programs in cells for the next generation of immunotherapies.
No statistical methods were used to predetermine sample size. For all in vivo experiments experimental conditions were allocated randomly at the time of adoptive transfer, and experimental conditions were mixed among littermates. For in vivo tumour sizing experiments the investigator was blinded to experimental condition. No power analysis was used to determine sample sizes.
All antibodies used in the study for fluorescence activated cell sorting, flow cytometry, and cellular stimulations are listed in Supplementary Table 2.
All guide RNAs used in the study are listed in Supplementary Table 3.
Isolation of human primary T cells for gene targeting
Primary human T cells were isolated from healthy human donors either from fresh whole blood, residuals from leukoreduction chambers after Trima Apheresis (Blood Centers of the Pacific), or leukapheresis products (StemCell). Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood samples by Ficoll centrifugation using SepMate tubes (STEMCELL, per manufacturer’s instructions). T cells were isolated from PBMCs from all cell sources by magnetic negative selection using an EasySep Human T Cell Isolation Kit (STEMCELL, per manufacturer’s instructions). Unless otherwise noted, isolated T cells were stimulated as described below and used directly (fresh). When frozen cells were used, previously isolated T cells that had been frozen in Bambanker freezing medium (Bulldog Bio) per manufacturer’s instructions were thawed, cultured in media without stimulation for 1 day, and then stimulated and handled as described for freshly isolated samples. Fresh blood was taken from healthy human donors under a protocol approved by the UCSF Committee on Human Research (CHR #13-11950). Patient samples used for gene editing were obtained under a protocol approved by the Yale Human Investigation Committee (HIC). Additional leukapheresis products from healthy donors were collected either under UCLA Institutional Review Board (IRB) approval #10-001598 or purchased from AllCells, LLC. All patients and healthy donors provided informed consent.
Primary human T cell culture
Unless otherwise noted, bulk T cells were cultured in XVivo15 medium (STEMCELL) with 5% Fetal Bovine Serum, 50 mM 2-mercaptoethanol, and 10 mM N-Acetyl L-Cystine. Immediately following isolation, T cells were stimulated for 2 days with anti-human CD3/CD28 magnetic dynabeads (ThermoFisher) at a beads to cells concentration of 1:1, along with a cytokine cocktail of IL-2 at 200 U/mL (UCSF Pharmacy), IL-7 at 5 ng/mL (ThermoFisher), and IL-15 at 5 ng/mL (Life Tech). Following electroporation, T cells were cultured in media with IL-2 at 500 U/mL. Throughout the culture period T cells were maintained at an approximate density of 1 million cells per mL of media. Every 2-3 days post-electroporation additional media was added, along with additional fresh IL-2 to bring the final concentration to 500 U/mL, and cells were transferred to larger culture vessels as necessary to maintain a density of 1 million cells/mL.
RNPs were produced by complexing a two-component gRNA to Cas9, as previously described10. Briefly, crRNAs and tracrRNAs were chemically synthesized (Dharmacon, IDT), and recombinant Cas9-NLS, D10A-NLS, or dCas9-NLS were recombinantly produced and purified (QB3 Macrolab). Lyophilized RNA was resuspended in 10 mM Tris-HCL (7.4 pH) with 150 mM KCl at a concentration of 160 μM, and stored in aliquots at −80C. crRNA and tracrRNA aliquots were thawed, mixed 1:1 by volume, and annealed by incubation at 37C for 30 min to form an 80 μNM gRNA solution. Recombinant Cas9 or the D10A Cas9 variant were stored at 40 μM in 20 mM HEPES-KOH pH 7.5, 150 mM KCl, 10% glycerol, 1 mM DTT, were then mixed 1:1 by volume with the 80 μM gRNA (2:1 gRNA to Cas9 molar ratio) at 37C for 15 min to form an RNP at 20 μM. RNPs were electroporated immediately after complexing.
Double stranded DNA HDRT production
Novel HDR sequences were constructed using Gibson Assemblies to insert the HDR template sequence, consisting of the homology arms (commonly synthesized as gBlocks from IDT) and the desired insert (such as GFP) into a cloning vector for sequence confirmation and future propagation. These plasmids were used as templates for high-output PCR amplification (Kapa Hotstart polymerase). PCR amplicons (the dsDNA HDRT) were SPRI purified (1.0X) and eluted into a final volume of 3 μL H2O per 100 μL of PCR reaction input. Concentrations of HDRTs were determined by nanodrop using a 1:20 dilution. The size of the amplified HDRT was confirmed by gel electrophoresis in a 1.0% agarose gel. All homology directed repair template sequences used in the study, both dsDNA and ssDNA, are listed in Supplementary Table 3.
Single stranded DNA HDRT production by exonuclease digestion
To produce long ssDNA as HDR templates, the DNA of interest was amplified via PCR using one regular, non-modified PCR primer and a second phosphorylated PCR primer. The DNA strand that will be amplified using the phosphorylated primer, will be the strand that will be degraded using this method. This makes it possible to prepare either a single-stranded sense or single-stranded antisense DNA using the respective phosphorylated PCR primer. To produce the ssDNA strand of interest, the phosphorylated strand of the PCR product was degraded by treatment with two enzymes, Strandase Mix A and Strandase Mix B, for 5 minutes (per 1kb) at 37C, respectively. Enzymes were deactivated by a 5 minute incubation at 80C. The resulting ssDNA HDR templates were SPRI purified (1.0X) and eluted in H2O. A more detailed protocol for the Guide-it™ Long ssDNA Production System (Takara Bio USA, Inc. #632644) can be found at the manufacturer’s website.
Single stranded DNA HDRT production by reverse synthesis
ssDNA HDR templates were synthesized by reverse transcription of an RNA intermediate followed by hydrolysis of the RNA strand in the resulting RNA:DNA hybrid product, as described21. Briefly, the desired HDR donor was first cloned downstream of a T7 promoter and the T7-HDR donor sequence amplified by PCR. RNA was synthesized by in vitro transcription using HiScribe T7 RNA polymerase (New England Biolabs) and reverse-transcribed using TGIRT-III (InGex). Following reverse transcription, NaOH and EDTA were added to 0.2 M and 0.1 M respectively and RNA hydrolysis carried out at 95C for 10 min. The reaction was quenched with HCl, the final ssDNA product purified using Ampure XP magnetic beads (Beckman Coulter) and eluted in sterile RNAse-free H2O. ssDNA quality was analysed by capillary electrophoresis (Bioanalyzer, Agilent).
Primary T cell electroporation
RNPs and HDR templates were electroporated 2 days following initial T cell stimulation. T cells were harvested from their culture vessels and magnetic anti-CD3/anti-CD28 dynabeads were removed by placing cells on an EasySep cell separation magnet for 2 minutes. Immediately prior to electroporation, de-beaded cells were centrifuged for 10 minutes at 90g, aspirated, and resuspended in the Lonza electroporation buffer P3 using 20 μL buffer per one million cells. For optimal editing, one million T cells were electroporated per well using a Lonza 4D 96-well electroporation system with pulse code EH115. Alternate cell concentrations from 200,000 up to 2 million cells per well resulted in lower transformation efficiencies. Alternate electroporation buffers were used as indicated, but had different optimal pulse settings (EO155 for OMEM buffer). Unless otherwise indicated, 2.5 μL of RNPs (50 pmols total) were electroporated, along with 2 μL of HDR Template at 2 μg/μL (4 μg HDR Template total).
The order of cell, RNP, and HDRT addition appeared to matter (Extended Data Fig. 1). For 96-well experiments, HDRTs were first aliquoted into wells of a 96-well polypropylene V-bottom plate. RNPs were then added to the HDRTs and allowed to incubate together at RT for at least 30 seconds. Finally, cells resuspended in electroporation buffer were added, briefly mixed by pipetting with the HDRT and RNP, and 24 μLs of total volume (cells + RNP + HDRT) was transferred into a 96 well electroporation cuvette plate. Immediately following electroporation, 80 μLs of pre-warmed media (without cytokines) was added to each well, and cells were allowed to rest for 15 minutes at 37C in a cell culture incubator while remaining in the electroporation cuvettes. After 15 minutes, cells were moved to final culture vessels.
Flow cytometry and cell sorting
Flow cytometric analysis was performed on an Attune NxT Acoustic Focusing Cytometer (ThermoFisher) or an LSRII flow cytometer (BD). Fluorescence activated cell sorting was performed on the FACSAria platform (BD). Surface staining for flow cytometry and cell sorting was performed by pelleting cells and resuspending in 25 μL of FACS Buffer (2% FBS in PBS) with antibodies at the indicated concentrations (Supplementary Table 2) for 20 minutes at 4C in the dark. Cells were washed once in FACS buffer before resuspension.
Samples were prepared by drop casting 10 μl of a solution of suspended live T cells onto a 3×1” microscope slide onto which a 25 mm2 coverslip was placed. Imaging was performed on an upright configuration Nikon A1r laser scanning confocal microscope. Excitation was achieved through a 488 nm OBIS laser (Coherent). A long working distance (LWD) 60x Plan Apo 1.20 NA water immersion objective was used with additional digital zoom achieved through the NIS-Elements software. Images were acquired under “Galvano” mirror settings with 2x line averaging enabled and exported as TIFF to be analyzed in FIJI (ImageJ, NIH).
CUT&RUN was performed using epitope-tagged primary human T cells 11 days after electroporation and 4 days after re-stimulation with anti-CD3/anti-CD28 dynabeads (untagged cells were not electroporated). Approximately 20% and 10% of electroporated cells showed GFP-BATF expression as determined by flow cytometry in donor 1 and donor 2 samples, respectively. CUT&RUN was performed as described12, using anti-GFP (ab290), anti-BATF (sc-100974), and rabbit anti-mouse (ab46540) antibodies. Briefly, 6 million cells (30 million cells for anti-GFP CUT&RUN in GFP-BATF-containing cells) were collected and washed. Nuclei were isolated and incubated rotating with primary antibody (GFP or BATF) for 2 hours at 4C. BATF CUT&RUN samples were incubated an additional hour with rabbit anti-mouse antibody. Next, nuclei were incubated with proteinA-micrococcal nuclease (kindly provided by the Henikoff lab) for one hour at 4C. Nuclei were equilibrated to 0C and MNase digestion was allowed to proceed for 30 minutes. Solubilized chromatin CUT&RUN fragments were isolated and purified. Paired-end sequencing libraries were prepared and analysed on Illumina Nextseq machines and sequencing data was processed as described12. For peak calling and heatmap generation, reads mapping to centromeres were filtered out.
TLA sequencing and analysis
TLA sequencing was performed by Cergentis as previously described16. Similarly, data analysis of integration sites and transgene fusions was performed by Cergentis as previously described16. TLA sequencing was performed in two healthy donors, each edited at the RAB11A locus with either a dsDNA or ssDNA HDR template to integrate a GFP fusion (Fig. 1b). Sequencing reads showing evidence of primer dimers or primer bias (i.e. greater than 99% of observed reads came from single primer set) were removed.
In vitro Treg suppression assay
CD4+ T cells were enriched using the EasySep Human CD4+ T cell enrichment kit (STEMCELL Technologies). CD3+CD4+CD127loCD45RO+TIGIT+ enriched Treg-like cells from IL2RA-deficient subjects and HD as well as CD3+CD4+IL2RAhiCD127lo Tregs from IL2RA+/− individuals were sorted by flow cytometry. CD3+CD4+IL2RA-CD127+ responder T cells (Tresps) were labeled with CellTrace CFSE (Invitrogen) at 5 μM. Tregs and HD Tresps were co-cultured at a 1:1 ratio in the presence of beads loaded with anti-CD2, anti-CD3 and anti-CD28 (Treg Suppression Inspector; Miltenyi Biotec) at a 1 bead: 1 cell ratio. On days 3.5 to 4.5, co-cultures were analyzed by FACS for CFSE dilution. % inhibition is calculated using the following formula: 1 - (% proliferation with Tregs / % proliferation of stimulated Tresps without Tregs).
Sorting and TSDR analysis of corrected Tregs
Ex-vivo expanded Tregs and T effector cells from a healthy control and a patient with IL2RA compound heterozygous mutations (D6) were thawed and stained. Live cells were sorted based on expression of CD25 and CD62L markers directly into ZymoResearch M-digestion Buffer (2x) (cat# D5021-9) supplemented with proteinase K. The lysate was incubated at 65°C for greater than 2 hours and then frozen. Bisulfite conversion and pyrosequencing of the samples was performed by EpigenDx (assay ID ADS783-FS2) to interrogate the methylation status of 9 CpG sites intron 1 of the FOXP3 gene, spanning −2330 to −2263 from ATG.
Generation of retrovirally and lentivirally transduced control T cells
For retroviral infections, clinical grade MSGV-1-1G4 (NY-ESO-1 TCR transgene) retroviral vector (IUVPC, Indianapolis, IN) was used. For lentiviral production, HEK 293 cells were plated at 18 million cells in 15 cm dishes the night before transfection. Cells were transfected using the lipofectamine 3000 reagent following the manufacturer’s protocol (L3000001). Transfection media was changed the following day to fresh HEK 293 media (DMEM + 5% FBS + 1% pen/strep) with viral boost reagent per the manufacturer’s protocol at 500x (Alstem viral boost reagent #VB100). 48 hours after transfection the viral supernatant was collected, filtered, and the Alstem precipitation solution was added, mixed, and refrigerated at four degrees for four hours, concentrated by centrifugation, and the viral pellet was then resuspended at 100x in cold PBS following the manufacturer’s protocol (lentivirus precipitation solution #VC100).
T cells for viral infection were activated similarly to non-virally edited cells. Both retroviral and lentiviral transductions occurred 48 hours after TCR/cytokine stimulus, followed by expansion in IL-2 similarly to non-virally edited cells. For retroviral transduction, T cells were infected by spinoculation in retronectin (Clontech, Mountain View, CA) coated plates. Control mock-transduced T cells were also generated. For lentiviral transduction, viral concentrate was added to 1X final concentration.
Antigen specific TCR expression analysis
The expression of the NY-ESO-1 TCR was assessed in virally and non-virally modified cells with an NY-ESO-1 specific (SLLMWITQC) dextramer-PE (Immundex, Copenhagen, Denmark) according to the manufacturer’s protocol. Negative dextramer (Immudex, Copenhagen, Denmark) was used as a negative control.
T cell activation and cytokine production analysis
Melanoma cell lines were established from the biopsies of melanoma patients under the UCLA IRB approval #11-003254. Cell lines were periodically screened for mycoplasma contamination as well as authenticated using GenePrint® 10 System (Promega, Madison, WI), and were matched with the earliest passage cell lines. M257 (NY-ESO-1+ HLA-A*0201-), M257-A2 (NY-ESO-1+ HLA-A*0201+) and M407 (NY-ESO-1+ HLA-A*0201+) were cocultured 1:1 with the modified PBMCs in cytokine free media. The recommended amount per test of CD107a-APC-H7 (Supplementary Table 2) antibody was added to the coculture. After 1 hour, half the recommended amount of BD Golgi Plug and BD Golgi Stop (BD bioscience, San Jose, CA) was added to the coculture. After 6 hours, surface staining was performed followed by cell permeabilization using BD cytofix/cytoperm (BD bioscience, San Jose, CA) and intracellular staining according to manufacturer instructions (Supplementary Table 2). Negative dextramer and Fluorescence minus one (FMOs) staining were used as controls.
T Cell in vitro killing assay
M202-nRFP (NY-ESO-1-, HLA-A*0201+), M257-nRFP (NY-ESO-1+ HLA-A*0201-), M257-A2-nRFP (NY-ESO-1+ HLA-A*0201+), M407-nRFP (NY-ESO-1+ HLA-A*0201+), and A375-nRFP (NY-ESO-1+ HLA-A*0201+) melanoma cell lines stably transduced to express nuclear RFP (Zaretsky 2016 NEJM) were seeded approximately 16 hours before starting the coculture (~1500 cells seeded per well). Modified T cells were added at the indicated E:T ratios. All experiments were performed in cytokine free media. Cell proliferation and cell death was measured by nRFP real time imaging using an IncuCyte ZOOM (Essen, Ann Arbor, MI) for 5 days.
In vivo mouse solid tumour model
All mouse experiments were completed under a UCSF Institutional Animal Care and Use Committee protocol. We used 8 to 12 week old NOD/SCID/IL-2Rɣ-null (NSG) male mice (Jackson Laboratory) for all experiments. Mice were seeded with tumours by subcutaneous injection into a shaved right flank of 1×106 A375 human melanoma cells (ATCC CRL-1619). At seven days post tumour seeding, tumour size was assessed and mice with tumour volumes between 15-30 mm3 were randomly assigned to experimental and control treatment groups. Indicated numbers of T cells were resuspended in 100 μl of serum-free RPMI and injected retro-orbitally. For tumour sizing experiments, the length and width of the tumour was measured using electronic calipers and volume was calculated as v = 1/6 * π * length * width * (length + width) / 2. The investigator was blinded to experimental treatment group during sizing measurements. A bulk edited T cell population (5×106) or a sorted NY-ESO-1 TCR+ population (3×106) was transferred as indicated in figures and legends. For bulk edited T cell transfers, lentivirally edited cells generally had a higher percentage of NY-ESO-1 positive cells, so mock-infected cells were added to normalize the percentage of total T cells NY-ESO-1+ to equal that of the bulk population of non-virally edited T cells (~10% NY-ESO-1+). For sorted T cell transfers, NY-ESO-1+ T cells were FACS sorted eight days following electroporation, expanded for two additional days, and frozen (Bambanker freezing medium, Bulldog Bio). Non-virally or lentivirally modified human T cells were then thawed and rested in media overnight prior to adoptive transfer. For flow cytometric analysis of adoptively transferred T cells, single-cell suspensions from tumours and spleens were produced by mechanical dissociation of the tissue through a 70 μm filter. All animal experiments were performed in compliance with relevant ethical regulations per an approved IACUC protocol (UCSF), including a tumor size limit of 2.0 cm in any dimension.
Data and reagent availability
CUT&RUN data has been deposited in GEO as record GSE108600. TLA and amplicon sequencing data is available upon request. Source data for animal experiments (Fig. 4g, h and Extended Data Fig. 10) is provided. Plasmids containing the HDR template sequences used in the study are available through AddGene.
Extended Data Figure 1: