Reprogramming human T cell function and specificity with non-viral genome targeting

Reprogramming human T cell function and specificity with non-viral genome targeting


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Decades of work have aimed to genetically reprogram T cells for therapeutic purposes1 using recombinant viral vectors, which do not target transgenes to specific genomic sites2,3. In addition, the need for viral vectors has slowed down research and clinical use as their manufacturing and testing is lengthy and expensive. Genome editing brought the promise of specific and efficient insertion of large transgenes into target cells through homology-directed repair (HDR)4,5. Here, we developed a CRISPR-Cas9 genome targeting system that does not require viral vectors, allowing rapid and efficient insertion of large DNA sequences (> 1kb) at specific sites in the genomes of primary human T cells, while preserving cell viability and function. This permits individual or multiplexed modification of endogenous genes. First, we apply this strategy to correct a pathogenic IL2RA mutation in cells from patients with monogenic autoimmune disease, demonstrating improved signaling function. Second, we replace the endogenous T cell receptor (TCR) locus with a new TCR redirecting T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognize tumour antigen and mount productive anti-tumour cell responses in vitro and in vivo. Taken together, these studies provide preclinical evidence that non-viral genome targeting can enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells.

The major barrier to effective non-viral T cell genome targeting of large DNA sequences has been the toxicity of the DNA6. While the introduction of short single-stranded oligodeoxynucleotide (ssODN) HDR templates does not cause significant T cell toxicity, it has been shown that larger linear double stranded (dsDNA) templates are toxic at high concentrations7,8. Contrary to expectations, we found that co-electroporation of human primary T cells with CRISPR-Cas9 ribonucleoprotein (Cas9 RNP9,10) complexes and long (>1kb) linear dsDNA templates reduced the toxicity associated with the dsDNA template (Extended Data Fig 1a-e). Cas9 RNPs were co-electroporated with a dsDNA HDR template designed to introduce an N-terminal GFP-fusion in the housekeeping gene RAB11A (Fig. 1a). Both cell viability and the efficiency of this approach were optimized by systematic exploration (Fig. 1b and Extended Data Fig. 1f-h) resulting in GFP expression in ~50% of both primary human CD4+ and CD8+ T cells. The method was reproducibly efficient with high cell viability (Fig. 1c, d, e). The system is also compatible with current manufacturing protocols for cell therapies. The method can be used with fresh or cryopreserved cells, bulk T cells or FACS-sorted sub-populations, and cells from whole blood or leukapheresis (Extended Data Fig. 2a-d).

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Figure 1:

Efficient non-viral genome targeting in primary human T cells.

a, HDR mediated integration of a GFP fusion tag to the housekeeping gene Rab11A. b, Development and optimization of non-viral genome targeting for both cell viability and HDR efficiency. c, Insertion of a GFP fusion into the endogenous RAB11A gene using non-viral targeting in primary human CD4+ and CD8+ T cells. d, Average efficiency with the RAB11A-GFP HDR template was 33.7% and 40.3% in CD4+ and CD8+ cells respectively. e, Viability (number of live cells relative to non-electroporated control) after non-viral genome targeting averaged 68.6%. Efficiency and viability were measured 4 days following electroporation. Mean of n=12 independent healthy donors displayed (d-e). See also Extended Data Fig 1.

We next confirmed that the system could be applied broadly by targeting sequences in different locations throughout the genome. We efficiently engineered primary T cells by generating GFP fusions with different genes (Fig. 2a and Extended Data Fig. 2e-g). Live-cell imaging with confocal microscopy confirmed the specificity of gene targeting, revealing the distinct sub-cellular locations of each of the resulting GFP-fusion proteins11 (Fig. 2b). Appropriate chromatin binding of a transcription factor GFP-fusion protein was confirmed by performing genome-wide CUT & RUN12 analysis with an anti-GFP antibody (Fig. 2c and Extended Data Fig. 2h). Finally, we showed that gene targeting preserved the regulation of the modified endogenous gene. Consistent with correct cell-type specific expression, a CD4-GFP fusion was selectively expressed in the CD4+ population of T cells (Fig. 2d). Using HDR templates encoding multiple fluorescent proteins, we demonstrated that we could generate cells with bi-allelic gene targeting (Fig. 2e and Extended Data Fig. 3a-d) or multiplex modification of two (Fig. 2f and Extended Data Fig. 3e-h) or even three (Fig. 2g and Extended Data Fig. 3i) different genes13,14. These results show that multiple endogenous genes can be directly engineered without virus in T cells, and that gene and protein regulation are preserved.

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Figure 2:

Individual and multiplexed modification of endogenous T cell genes.

a, Non-viral genome targeting with GFP-fusion constructs into multiple endogenous genes. b, Confocal microscopy of live human T cells electroporated with the indicated HDR templates confirmed fusion-protein localization. Scale = 5 μm. c, GFP fused to the endogenous transcription factor BATF enabled genome-wide binding analysis (CUT&RUN) using anti-GFP or anti-BATF antibodies. d, RAB11A-fusions produced GFP positive CD4+ and CD8+ cells, whereas the CD4-fusions were selectively expressed in CD4+ cells. e, Bi-allelic non-viral genome targeting of two distinct fluorescent proteins into the same locus. f, Multiplexed non-viral genome targeting of HDR templates into two separate genomic loci. g, Simultaneous targeting of three distinct genomic loci. Cells positive for one (Q-II, Q-III) or two integrations (Q-IV), were highly enriched for a third HDR integration. One representative donor displayed from n=6 (a), n=4 (b, d-g), or n=2 (c) independent healthy donors. See also Extended Data Figs 2​,33.

For therapeutic use of genetically modified T cells, integrated sequences should be introduced specifically without unintended disruption of other critical genome sites15. We performed targeted locus amplification (TLA) sequencing16 and found no evidence of off-target integrations above the assay’s limit of detection (~1% of alleles) (Extended Data Fig. 4a-b). We further assessed potential off-target integrations at the single cell level by quantifying GFP+ cells generated using a Cas9 RNP that cuts outside the homology site. Similar to what has been described with viral HDR templates4,17, we found evidence to suggest that double-stranded templates could integrate independent of target homology18,19, albeit at low rates (Extended Data Fig. 4c-i). These rare events could be reduced almost completely by using single-stranded DNA templates20,21 (Extended Data Fig. 5a-d). As an additional safeguard that could be important for some applications, we demonstrated that efficient non-viral T cell genome targeting also could be achieved using either a single-stranded or double-stranded template with a Cas9 “nickase” engineered to reduce potential off-target double-stranded cuts22,23 (Extended Data Fig. 5e-h).

Having optimized this non-viral genome engineering approach in primary human T cells, we demonstrated its utility it in two different clinically relevant settings where targeted replacement of a gene would provide proof-of-principle that the method can be used to create therapeutically relevant gene modifications. Specifically, we tested the ability to rapidly and efficiently correct an inherited genetic alteration in T cells and we also tested the targeted insertion of the two chains of a TCR to redirect the specificity of T cells to recognize cancer cells.

We identified a family with monogenic primary immune deficiency with autoimmune disease caused by recessive loss-of-function mutations in the gene encoding the IL-2 alpha receptor (IL2RA)24 (Supplementary Table 4), which is essential for healthy regulatory T cells (Tregs)25 (Extended Data Fig. 6a-h). Whole exome sequencing revealed that the IL2RA-deficient children harboured compound heterozygous mutations in IL2RA (Fig. 3a and Extended Data Fig. 6i). One mutation, c.530A>G, creates a premature stop codon. With non-viral genome targeting, we were able to correct the mutation and observed IL2RA expression on the surface of corrected T cells from the patient (Fig. 3b). Long dsDNA templates led to efficient correction of the mutations. Because only two base pair changes were necessary (one to correct the mutation and one to silently remove the gRNA’s PAM sequence), a short single-stranded DNA (~120 bps) could also be used to make the correction. These single-stranded DNAs were able to correct the mutation at high frequencies, although here the efficiency of correction was lower than with the longer dsDNA template (Extended Data Fig. 7a​,8a).8a). Correction was successful in T cells from all three siblings, but lower rates of IL2RA expression were seen in compound het 3, which could be due to altered cell-state associated with the patient’s disease or the fact she was the only sibling treated with immunosuppressive therapy (Supplementary Table 4 and Extended Data Fig. 8f). The second mutation identified, c.800delA, causes a frameshift in the reading frame of the final IL2RA exon. This frameshift mutation could be corrected both by HDR as well as by RNP cutting alone, presumably due to some of the small indels restoring the reading frame (Extended Data Fig. 8). Taken together, these data show that distinct mutations can be corrected in patient T cells using HDR template-dependent and non-HDR template-dependent mechanisms.

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Non-viral genome targeting is consistent across T cell types and reproducible across target loci.

a, Efficient genome targeting was accomplished with a variety of T cell processing and handling conditions that are used with current manufacturing protocols for cell therapies. Non-viral genome targeting of a RAB11A-GFP fusion protein using a linear dsDNA HDR template was performed in bulk CD3+ T cells isolated from either whole blood draws or by leukapheresis. b, Targeting was similar either using bulk CD3+ T cells fresh after isolation or after cryopreservation (stored in liquid nitrogen and thawed prior to initial activation). c, CD4+ T cells isolated by fluorescent activated cell sorting (FACS) showed detectable GFP+ cells indicative of efficient editing, albeit at lower rates than targeting in CD4+ cells isolated by negative selection (potentially due to the added cellular stress of sorting). d, Using the same optimized non-viral genome targeting protocol (Methods), a variety of T cell types could be successfully edited, including peripheral blood mononuclear cells, without any selection (T cell culture conditions cause preferential growth of T cells from PBMCs). Sorted T cell subsets CD8+, CD4+, and CD4+IL2RA+CD127lo regulatory T cells (Tregs) could be successfully targeted with GFP integration. PBMCs were cultured for two days identically to primary T cells (Methods). Bulk CD3+ T cells were isolated by negative enrichment. The electroporations in panel d used only 2 μg of dsDNA HDR template, a concentration that was later found to be less efficient than the final 4 μg (contributing to the lower efficiencies seen compared to Fig. 1d). RAB11A-GFP template was used with on-target gRNA was used in a-de, Four days after electroporation of different GFP templates along with a corresponding RNP into primary CD3+ T cells from six healthy donors, GFP expression was observed across both templates and donors. f, High viability post-electroporation was similarly seen across target loci. g, The fusion tagged proteins produced by integrating GFP into specific genes localized to the subcellular location of their target protein (Fig. 2b), and were also expressed under the endogenous gene regulation, allowing protein expression levels to be observed in living primary human T cells. Note how GFP tags of the highly expressed cytoskeletal proteins TUBA1B (beta tubulin) and ACTB (beta actin) show consistently higher levels of expression compared to the other loci targeted across six donors. GFP mean fluorescent intensity (MFI) was calculated for the GFP+ cells in each condition/donor, and normalized as a percentage of the maximum GFP MFI observed in the experiment. h, Gene fusions not only permitted the imaging and analysis of expression of endogenous proteins in live cells, but also could be used for biochemical targeting of specific proteins. For example, ChIP-Seq, and more recently CUT & RUN, have been widely used to map transcription factor binding sites; however, these assays are often limited by the availability of effective and specific antibodies. As a proof-of-principle we used anti-GFP antibodies to perform CUT & RUN in primary T cells where the endogenous gene encoding BATF, a critical transcription factor, had been targeted to generate a GFP-fusion. Binding sites identified with anti-GFP CUT & RUN closely matched the sites identified with an anti-BATF antibody. Anti-BATF, anti-GFP, and no antibody heatmaps of CUT&RUN data obtained from primary human T cell populations electroporated with GFP-BATF fusion HDR template (untagged cells were not electroporated). Aligned CUT&RUN binding profiles for each sample were centered on BATF CUT&RUN peaks in untagged cells and ordered by BATF peak intensity in untagged cells. Experiment (h) was performed in two independent healthy donors.

Extended Data Figure 3:

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HDR and non-HDR mediated correction of IL2RA c.800delA frameshift loss-of-function mutation.

a,Histograms of IL2RA surface expression in CD3+ T cells in all children from a family carrying two loss-of-function IL2RA mutations, including three compound heterozygotes that express minimal amounts of IL2RA on the surface of the T cells (No electroporation, Grey). Two days following electroporation of an RNP containing a gRNA for the site of one of the two mutations, a one base pair deletion in the final exon of IL2RA (c.800delA) causing a run-on past the normal stop codon, CD3+ T cells from a healthy donor and single hets (c.800 Het 2 and 3) showed slight increases in IL2RA- cells (RNP only, Blue). This modest reduction is potentially due to the gRNA targeting the C-terminus of the protein where small indels may cause less pronounced loss of surface protein expression. Surprisingly, the RNP alone resulted in IL2RA surface expression in almost 50% of edited T cells in all three compound heterozygotes. In cells from two of the compound heterozygous children, increases in the percent of cells with IL2RA correction compared to RNP only could be achieved by inclusion of an ssODN HDR template sequence with the mutation correction (RNP+ssODN, Green), and further increased at this site when using a longer dsDNA HDR template to correct the mutation (RNP + dsDNA HDRT, Yellow) (Extended Data Fig. 6i)b, Amplicon sequencing was performed in select targeted patient cells. c, Stat5 phosphorylation (pStat5) in response to high dose IL-2 stimulation (200 U/mL) in targeted CD3+ T cells following 7 days of expansion post-electroporation. Increased numbers of pStat5+ cells correlated with increased IL2RA surface expression (a)d, Following 9 days of expansion post-electroporation, intracellular FoxP3 staining revealed an increased proportion of IL2RA+ FoxP3+ cells in CD3+ T cells compared to no electroporation controls. Electroporations were performed according to optimized non-viral genome targeting protocol (Methods). For ssODN electroporations, 100 pmols in 1 μL water were electroporated. e, Flow cytometric analysis of GFP expression 6 days following electroporation of a positive HDR control RAB11A-GFP dsDNA HDR template into CD3+ T cells from the indicated patients revealed lower GFP expression in the three compound heterozygotes compared to their two c.800 heterozygote siblings. Compared to a cohort of twelve healthy donors similarly edited (Fig. 1d), both c.800 heterozygotes as well as compound het 1 and 2 were within the general range observed across healthy donors, whereas compound het 3 had lower GFP expression than any healthy donor analysed. Of note, in compound het 3 HDR mediated correction at the c.530 mutation was substantially lower than the other two compound heterozygotes (Fig. 3b). IL2RA surface expression after electroporation of the c.800delA targeting RNP alone was similar though. Compared to HDR-mediated repair, NHEJ mediated frameshift correction at c.800delA may be less dependent on cell proliferation, consistent with compound het 3 being the only compound heterozygous patient on active immunosuppressants at the time of blood draw and T cell isolation (Supplementary Note 3)f, Altered cell-state associated with the patient’s disease could also contribute to diminished HDR rates. TIGIT and CTLA4 expression levels in non-edited, isolated CD4+ T cells from each indicated patient was measured by flow cytometry. Consistent with altered cell states and or/ cell populations, cells from compound het 3 had a distinct phenotype, with increased TIGIT and CTLA4 expression compared both to healthy donors, the single heterozygous family members, as well the other two compound heterozygous siblings.

Extended Data Figure 9:

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Endogenous TCR replacement strategy and functional characterization.

a-d, Schematic description of HDR template for endogenous TCR replacement by in-frame integration of a new TCR-β chain and a new variable region of a TCR-α chain at the TCR-α locus and the new TCR’s subsequent transcription and translation. e, HDR template for endogenous TCR replacement at the TCR-β locus. f, Multiplexed integration of a new TCR-α at the TCR-α locus and a new TCR-β at the TCR-β locus. Detailed description of TCR replacement strategy in Supplementary Note 4. g, TCR mispair analysis after retroviral delivery or non-viral TCR replacement of an NY-ESO-1 specific TCR in gated CD4+ or CD8+ T cells. With viral introduction of the new TCR, an infected cell will potentially express at least four different TCRs (new TCR-α + new TCR-β; new TCR-α + endogenous TCR-β; endogenous TCR-α and new TCR-β; endogenous TCR-α + endogenous TCR-β). Staining for the specific beta chain in the new introduced TCR (VB13.1) along with MHC-peptide multimer (NYESO) can provide a rough estimate of TCR mispairing by distinguishing between cells that predominantly expressed the introduced TCR (VB13.1+ NYESO+; new TCR-α + new TCR-β) vs those that expressed predominantly one of the potential mispaired TCRs (VB13.1+ NYESO-; endogenous TCR-α + new TCR-β). h, i, TCR replacement by targeting an entire new TCR into TRAC (a-d, also possible with a multiplexed knockout of TCR-β), an entire new TCR into TRBC1/2 (f), or multiplexed replacement with a new TCR-α into TRAC and a new TCR-β into TRBC1/2j, Functional cytokine production was observed selectively following antigen exposure in gated CD4+ T cells, similarly to gated CD8+ T cells (Fig. 4c). k, Non-viral TCR replacement was consistently observed at four days post electroporation in both CD8+ and CD4+ T cells across a cohort of six healthy blood donors. l, In a second cohort of six additional healthy blood donors, 100 million T cells from each donor were electroporated with the NY-ESO-1 TCR replacement HDR template and on-target gRNA/Cas9 (Fig. 4f). The percentage of CD4+ and CD8+ T cells that were NY-ESO-1 TCR+ was consistent over ten days of expansion following electroporation. m, Over 10 days of expansion following non-viral genome targeting, CD8+ T cells showed a slight proliferative advantage over CD4+ T cells. n, The indicated melanoma cell lines were co-incubated with the indicated sorted T cell populations at a ratio of 1:5 T cells to cancer cells. At 72 hours post co-incubation the percent cancer cell confluency was recorded with by automated microscopy (where nuclear RFP marks the cancer cells). T Cells expressing the NY-ESO-1 antigen specific TCR, either by retroviral transduction (Black) or by non-viral knock-in endogenous TCR replacement (Red) both showed robust target cell killing only in the target cancer cell lines expressing both NY-ESO-1 and the HLA-A*0201 class I MHC allele. o, To ensure that target cell killing by non-viral TCR replacement T cells (Red) was not due to the either the gRNA or the HDR template used for TCR replacement alone, a matrix of on/off target gRNAs and on/off target HDR templates was assayed for target cell killing of the NY-ESO-1+ HLA-A*0201+ A375 cancer cell line (off-target gRNA and HDRT were specific for RAB11A-GFP fusion protein knock-in). Only cells with both the on-target gRNA as well as the on-target HDR template demonstrated target cell killing. p, Sorted NY-ESO-1+ TCR+ cells from a bulk T cell edited population (on-target gRNA, on-target HDR template) showed a strong dose-response effect for target cancer cell killing. Within 48 hours T cell to cancer cell ratios of 2:1 and greater showed almost complete killing of the target cancer cells. By 144 hours, T cell to cancer cell ratios of less than 1:16 showed evidence of robust target cell killing. q, Target cell killing by non-viral TCR replacement T cells was due specifically to the NY-ESO-1-recognizing TCR+ cell population observed by flow cytometry after non-viral TCR replacement (Fig. 4b). Starting with the bulk edited T cell population (all of which had been electroporated with the on-target gRNA and HDR template), we separately sorted three populations of cells: the NY-ESO-1+TCR+ cells (non-virally replaced TCR) (red), the NY-ESO-1-TCR- cells (TCR knockout) (grey), and the NY-ESO-1-TCR+ cells (those that retained their native TCR but did not have the NY-ESO specific knock-in TCR) (orange). Only the sorted NY-ESO-1+ TCR+ population demonstrated target cell killing (4:1 T cell to cancer cell ratio). One representative donor from n=2 (g, j) or n=3 (h, i) independent healthy donors with mean and standard deviation of technical triplicates (j). Mean and standard deviations of n=6 independent healthy donors (l, m) or of four technical replicates for n=2 independent healthy donors (o-q) are shown. Mean and individual values for n=2 independent healthy donors (n).

Extended Data Figure 10:

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In vivo functionality of T cells with non-viral TCR replacement.

a,Diagram of in vivo human antigen specific tumour xenograft model. 8 to 12 week old NSG mice were seeded with 1×106 A375 cells (human melanoma cell line; NY-ESO-1 antigen+ and HLA-A*0201+) subcutaneously in a shaved flank. Primary human T cells edited to express an NY-ESO-1 antigen specific TCR were generated (either through lentiviral transduction or non-viral TCR replacement), expanded for 10 days following transduction or electroporation, and frozen. Either a bulk edited population was used (b,c) or a NY-ESO-1 TCR+ sorted population (d-f) was used. At seven days post tumour seeding, T cells were thawed and adoptively transferred via retro-orbital injection. b, Two days following transfer of 5×106 bulk non-virally targeted T cells (~10% TCR+ NYESO-1+ (Red), ~10% TCR+ NYESO-1- (Orange), and ~80% TCR- NYESO-1- (Green), see Fig 4b), NY-ESO-1+ non-virally edited T cells preferentially accumulated in the tumour vs. the spleen. n=5 mice for each of four human T cell donors. c, Ten days following transfer of 5×106 bulk non-virally targeted CFSE labeled T cells, NYESO-1 TCR+ cells showed greater proliferation than TCR- or TCR+NYESO-1- T cells, and showed greater proliferation (CFSE Low) in the tumour than in the spleen. At ten days post transfer TCR- and TCR+NYESO- T cells were difficult to find in the tumour (Fig 4g). d, Individual longitudinal tumour volume tracks for data summarized in Fig 4h. 3×106 sorted NY-ESO-1 TCR+ T cells generated either by lentiviral transduction (Black) or non-viral TCR replacement (Red) were transferred on day 7 post tumour seeding and compared to vehicle only injections until 24 days post tumour seeding. Note that the same data for vehicle control data are shown for each donor in comparison to lentiviral delivery (above) and non-viral TCR replacement (below). e,f, In these experiments, seventeen days following T cell transfer (d), non-virally TCR replaced cells appeared to show greater NY-ESO-1 TCR expression and lower expression of exhaustion markers. Transfer of both lentivirally transduced and non-viral TCR replaced cells showed significant reductions in tumour burden on day 24. In this experimental model, non-viral TCR replacement showed further reductions compared to the lentiviral transduction (Fig. 4h), potentially due to knockout of the endogenous TCR, endogenous regulation of the new TCR’s expression, some difference in the cell populations amenable to non-viral vs lentiviral editing, or confounding variables in cell handling between lentiviral transduction and non-viral genome targeting. n=4 (b), n=2 (d-f), or n=1 (c) independent healthy donors in 5 (b, c) or 7 mice (d-f) per donor with mean (b, e, f) and standard deviation (b).

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We thank members of the Marson Lab, Chris Jeans (QB3 MacroLab), Kyle Marchuk (UCSF BIDC), Jeffrey Bluestone and Qizhi Tang (UCSF Regulatory T Cell Therapy Program), Ryan Wagner (Parnassus CAT), the UCSF Flow Cytometry Core (NIH P30 DK063720 and 1S10OD021822-01), Lonza, Jacob Corn and Sarah Pyle for suggestions and assistance. This research was supported by NIH grants DP3DK111914-01 (A.M.), P50GM082250 (A.M.), R35 CA197633 (A.R.), K23 DK094866 (S.W.G.), T32GM007618 (T.L.R, J.H.), T32 DK007418 (T.L.R.), and P30 DK020595 (S.W.G.), the NIH NCI Intramural Program (A.L.F., S.H.H.), grants from the Keck Foundation (A.M.), National Multiple Sclerosis Society (A.M.; CA 1074-A-21), gifts from Jake Aronov, Galen Hoskin, the Jeffrey Modell Foundation (A.M), and awards from the Burroughs Wellcome Fund (A.M.) and the Ressler Family Fund (C.P.S., J.S., A.R.). A.M. is a Chan Zuckerberg Biohub investigator. A.R. is a Parker Institute for Cancer Immunotherapy member.

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Competing Financial Interests

A.M. is a co-founder of Spotlight Therapeutics. A.M. serves as an advisor to Juno Therapeutics and PACT Pharma and the Marson laboratory has received sponsored research support (Juno Therapeutics, Epinomics, Sanofi) and a gift from Gilead. T.L.R., C.P.S., E.S., A.R., and A.M. are inventors on new patent applications related to this manuscript.


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Jawahar Raina

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