WO2017070429A1 - Procédés consistant à éditer des polynucléotides codant pour un récepteur de lymphocytes t - Google Patents

Procédés consistant à éditer des polynucléotides codant pour un récepteur de lymphocytes t Download PDF

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WO2017070429A1
WO2017070429A1 PCT/US2016/058045 US2016058045W WO2017070429A1 WO 2017070429 A1 WO2017070429 A1 WO 2017070429A1 US 2016058045 W US2016058045 W US 2016058045W WO 2017070429 A1 WO2017070429 A1 WO 2017070429A1
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nuclease
cells
cell
genome
cas9
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Mark John OSBORN
Jakub Tolar
Joseph A. Zasadzinski
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Regents Of The University Of Minnesota
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]

Definitions

  • This disclosure describes, in one aspect, a method for disrupting T cell receptor (TCR) expression.
  • the method includes introducing into a T cell a genome-editing nuclease designed to edit the TRAC coding region that encodes TCRa, and culturing the T cell under conditions for the genome-editing nuclease to modify the TRAC coding region to either produce a modified TCRa that does not pair with TCRP or to inhibit expression of TCRa.
  • introducing the genome-editing nuclease into the T cell includes introducing into the T cell a polynucleotide that encodes the genome-editing nuclease.
  • introducing the genome-editing nuclease into the T cell includes introducing into the T cell a Cas9 polypeptide.
  • the genome-editing nuclease includes a TALEN nuclease, a CRISPR/Cas9 nuclease, or a megaTAL nuclease.
  • the CRISPR/Cas9 nuclease is derived from either Streptococcus pyogenes or Staphylococcus aureus.
  • the CRISPR/Cas9 nuclease includes a nuclease-resistant gRNA such as, for example, at least one 2'-OMe-phosphorothioate modified base, at least one 2'-0-methyl modified base, or at least one 2'-0-methyl 3' thioPACE modified base.
  • the TALEN nuclease or the megaTAL nuclease is encoded by an RNA that has an exogenous polyadenylation signal.
  • the method further includes culturing the T cell under conditions effective for expanding the population of genome-modified T cells.
  • this disclosure describes a method of treating a subject having a tumor.
  • the method includes collecting allogenic T cells; introducing into at least a portion of the allogenic T cells a JR ⁇ C-targeted nuclease; culturing the modified allogenic T cells under conditions effective for the ⁇ 4 C-targeted nuclease to disrupt expression of T cell receptor a (TCRa) by the modified T cells; and administering the allogenic T cells to the subject.
  • TCRa T cell receptor a
  • introducing the JR ⁇ C-targeted nuclease into the allogenic T cell includes introducing into the allogenic T cell a polynucleotide that encodes the JR ⁇ C-targeted nuclease.
  • the JR ⁇ C-targeted nuclease includes a TALEN nuclease, a
  • CRISPR/Cas9 nuclease or a megaTAL nuclease.
  • disrupting expression of TCRa further disrupts assembly of TCRa and TCRp. In some embodiments, disrupting expression of TCRa further disrupts formation of a complex between TCR and CD3. In some embodiments, disrupting expression of TCRa involves further disrupting assembly of TCRa and TCRp.
  • FIG. 1 TRAC gene targeting and nuclease architecture.
  • A Exon 1 of the TRAC locus with the positions of the nuclease target sites shown in relation to one another.
  • B TALE recognition code and megaTAL (MT) architecture.
  • TALE repeat variable diresidue:DNA base recognition code is represented by colored bars. The amino acid sequences HD recognize DNA base C, NN binds G, NI interacts with A, and NG binds T. Eleven TALE repeat regions are fused to the meganuclease domain by a peptide linker and the hybrid protein is termed a megaTAL.
  • the central four bases in SEQ ID NO:27 that are common to the parental I-Onul homing endonuclease from which the MT is derived are underlined.
  • C The dimeric TALEN proteins contain a deletion of 152 amino acids at the N-terminus and maintenance of 63 TAL amino acids at the C-terminus.
  • the individual RVDs each bind a single base of DNA (SEQ ID NO:28, SEQ ID NO:29) and each half array is joined to a sub-unit of the Fokl heterodimeric nuclease.
  • D CRISPR/Cas9 architecture. A chimeric gRNA is shown.
  • the constant portion of the molecule interacts extensively with the Cas9 protein and the gene-specific component (SEQ ID NO:50) is shown.
  • the gRNA contacts a target sequence (SEQ ID NO:48, SEQ ID NO:49) in the context of a-NGG protospacer adjacent motif.
  • the Cas9 protein contains two domains (HNH and RuvC) each responsible for the cleavage of a single strand of DNA.
  • E Expression platforms.
  • MT, Cas9, and TALEN mRNA was generated with either a T3 or T7 RNA polymerase promoter and the '+/-" refers to the presence or absence of a polyadenylation signal added in vitro.
  • gRNA was produced as an RNA transcript, a circular plasmid with a human U6 polIII promoter, or a linear fragment generated by PCR containing the U6 promoter and full length guide RNA sequences.
  • FIG. 2 Nuclease comparison in Jurkat T-ALL cell line. Nucleic acids were delivered by electroporation into Jurkats and seven days later the amount of CD3 loss from the cell surface was determined by flow cytometry.
  • the first lane is the GFP transfection control with 95+% CD3 expression levels. Lane two shows the MT disruption rates. Lanes three and four are TALEN optimization conditions. TALEN mRNA was generated from a T3 polymerase promoter without (T3 alone) or with (T3 + pA) an exogenous polyadenylation signal.
  • B Cas9 mRNA and protein with varying platforms of gRNA editing rates.
  • the first lane is GFP followed by a linear DNA fragment encoding the gRNA (Cas9, linear), or circularized gRNA plasmid (Cas9, plasmid) borne expression systems.
  • gRNA transcript Cas9, RNA
  • 2'0-Methyl (2'-OMe) bases 2'0-Methyl (2'-OMe) bases that were delivered at doses of 5 ⁇ g or 10 ⁇ g by either complexing with Cas9 protein (RNP) or with Cas9 mRNA.
  • RNP Cas9 protein
  • Cas9 mRNA complexing with Cas9 protein
  • **** represent p values (Student' s t-test) of ⁇ 0.05
  • FIG. 3 Nuclease activity in primary T-lymphocytes.
  • A Experimental schema. T-cells were isolated from peripheral blood, cultured at a 3 : 1 CD3/CD28 beadxell ratio followed by bead removal and electroporation with the indicated dose of nuclease. Cells were cultured transiently for 24 hours at 30°C. At day seven, gene knockout efficiencies and cellular viability were assessed.
  • B CD3 disruption rates using TALEN mRNA.
  • C MT mRNA doses of 1 ⁇ g, 2 ⁇ g, 4 ⁇ g, or 8 ⁇ g.
  • D Cas9 RNP or mRNA with nuclease protected gRNA at 5 ⁇ g or 10 ⁇ g. Experiments were done using at least three unrelated donors in quadruplicate. Average CD3 disruption rate with SEM are shown.
  • Dashed lines indicate the demarcation of CD3 disruption rates on the left portion of the graph and the cellular viability on the right side.
  • FIG. 4 Expansion and Scaling of CD3 negative cells.
  • A Experimental Schema. T-cells were harvested, activated, and at 48 hours electroporated, and transiently cold shocked. During the first nine days the cells were grown in the presence of IL-2, IL-7, and IL-15. Following CD3 cell depletion the cells were maintained in IL-7 and IL-15 until day 15 when they were enumerated and, if indicated, electroporated with TRAC mRNA and re-stimulated with
  • CD3/CD28 beads in the presence of IL-2 (B) CD3 negative selection. Post-nuclease treated cells were depleted of CD3 positive cells by completion of one (left) or two (right) treatments with the EASYSEP (StemCell Technologies, Inc., Vancouver, Canada) procedure. (C) Re-introduction of TRAC mRNA. At day zero, 200,000 cells were treated with MT and at day 15 post sorted, CD 3 negative cells were cultured in IL-7 and IL-15 alone (labeled no TRAC no stim) or with a 3 : 1 CD3/CD28 bead: cell ration (labeled no TRAC + stim).
  • a third group received 1 ⁇ g of TRAC mRNA via electroporation followed CD3/CD28 bead stimulation (labeled + TRAC + stim).
  • Cell counts were performed at 2 days , 4 days, 6 days, and 8 days post gene transfer.
  • (D) 500,000 cells were treated with 1 ⁇ g of Cas9 mRNA and 5 ⁇ g of modified gRNA or
  • (E) 2 x 10 6 cells were treated with 10 ⁇ g of MT. Cells were grown in bulk to day 9 when they were enumerated. Negative depletion was performed, the CD3 null cells were re-plated in media with IL-7 and IL- 15 and cultured to day 15 when they counted.
  • Experiments are from three donors and are the total from three experiments with four experimental replicates with averages and standard error of the mean shown. Arrow indicates CD3 depletion step with subsequent plating of CD3 null cells.
  • FIG. 5 T-cell phenotyping.
  • FIG. 6 CAR transduction and anti-tumor properties of gene modified cells.
  • CD 19 CAR lentiviral transduction was performed on day 0 with a self-inactivating (SIN) lentiviral construct encoding the CD19R single-chain variable fragment with the CD28 transmembrane domain (CD28 tm), the 4-1BB costimulatory domain (41BB), the CD3-zeta co- stimulatory signaling domain ( ⁇ ), a self-cleaving T2A picornaviral peptide sequence (T2A) and a non-ligand binding truncated epidermal growth factor receptor (tEGFR).
  • SI self-inactivating
  • CD3 negative depletion was performed on day nine with culture overnight followed by incubation of T-cells with K562 or CD 19 transgenic K562 cells.
  • B Anti -tumor activity of engineered T-cells. Equal numbers of T-cells and K562 transgenic cells expressing human CD 19 (CD 19 Tg K562) or CD 19 null K562 were incubated and analyzed for degranulation. Shown is a representative FACS analysis on cells from two donors for CD 107a with gating on CD4 and CD8 subsets.
  • FIG. 7. Off target genome mapping.
  • A Experimental schema for IDLV gene trapping. Nuclease mRNA for MT and TALEN and Cas9 mRNA and gRNA plasmid were introduced into Jurkats followed by transduction with an IDLV expressing GFP and puromycin. The IDLV is integrated into loci where a DNA break has occurred. LAM and nRLAM PCR experimental schema. LTR priming results in linear fragments that are converted to double stranded DNA products that are barcoded, deep sequenced, and interrogated against the genome for off target sites.
  • B TRAC IDLV gene trapping confirmation at on target TRAC locus. A PCR using LTR and TRAC specific primers (arrows shown in A) revealed presence of the IDLV cargo at the TRAC locus for all of the nuclease platforms visualized by agarose gel.
  • FIG. 8. Off target assessment in primary T-cells. Target loci were amplified from 100% CD3 null cells and analyzed by the Surveyor assay for evidence of nuclease activity.
  • A KAT2B Surveyor cleavage products image and genomic sequence (SEQ ID NO:30) alignment compared to TRAC (SEQ ID NO:27). Cleavage products indicated by arrows and quantitative gel analysis indicating is shown at bottom of gel.
  • B GBP5 Surveyor cleavage products image and genomic sequence (SEQ ID NO:31) in relation to TRAC (SEQ ID NO:27).
  • C PDE11A, DR1, HIAT1, KIAA1217, and EXOC2 Surveyor analysis that did not reveal MT OT cleavage.
  • KAT2B K(Lysine) acetyltransferase 2B;
  • G J Si 5 5 guanylate binding protein 5;
  • FIG. 9 Individual nuclease target sequences in the TRAC gene.
  • the double stranded DNA sequence of the TRAC locus is shown (SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:51) with a gap represented by the double dash separating SEQ ID NO:42 and SEQ ID NO:51.
  • Nuclease sequences (SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:45) are identified and the MT (SEQ ID NO:34), CRISPR/Cas9 (SEQ ID NO:38), and TALEN (SEQ ID NO:40 and SEQ ID NO:44) sequences used in this study are identified.
  • the MT that recognizes a 39 bp sequence
  • the CRISPR/Cas9 gRNA recognizes a 23 bp sequence on the anti-sense strand
  • the TALEN binds 55 bp.
  • the TALEN sites previously described (Poirot et al., 2015.
  • FIG. 10 Representative FACS plots for data used in FIG. 2. Note: The Cas9 linear DNA treatment group and the groups at bottom were not co-transfected with GFP as were the others.
  • FIG. 11 Representative FACS plots for data in primary T cells used in FIG. 3. Dose for MT and TALEN was 1 ⁇ of mRNA. At bottom are the Cas9 treatment groups treated with two doses of modified gRNA (5 ⁇ g or 10 ⁇ g). Boxes below the FACS plot indicate the platform for Cas9: either protein (RNP) or mRNA each at 1 ⁇ g.
  • FIG. 12 KAT2B indel and cDNA analysis in primary T-cells.
  • the KAT2B locus is shown from exons 2-17 with introns shown as solid black line.
  • A Indel analysis. Limiting cycle PCR (25 cycles) was used with primers indicated by blue arrows to amplify the region containing the KAT2B OT site and amplicons were sequenced and aligned to reference sequence.
  • B cDNA analysis. Using the primers in exons 2 and 7 (outer arrows) KAT2B cDNA was amplified and sequenced by the Sanger method. The splice junction between adjacent exons is highlighted in blue and the first and last two base pairs of each exon is shown in relation.
  • FIG. 13 (A) Cas9 ribonucleoprotein. (B) Representative FACS data for primary T cells either unmodified (TCR+) or treated with the Cas9 ribonucleoprotein shown in (A).
  • CAR T cells chimeric antigen receptor T cells
  • CAR approaches have so far been implemented using autologous patient T cells, rendering them cumbersome to generate for widespread or urgent use, and potentially leading to variable clinical outcomes due to differential functional properties of each patient's starting T cell populations.
  • Potential approaches to address the variability of autologous approaches include the use of allogeneic T cells from healthy donors whose functional properties can be carefully defined prior to administration to a patient.
  • TCR endogenous T cell receptor
  • TCRa The TCR a chain (TCRa) is encoded by a single TRAC gene and pairs with the TCR ⁇ chain encoded by two TCRB genes. Since the TCR ⁇ / ⁇ dimer produces a fully functioning TCR complex, disrupting TCRa function is a simple approach to reduce (even eliminate) TCR expression and undesired TCR-driven off tumor recognition.
  • Zinc finger nucleases are heterodimeric arrays that co-localize at a target DNA site. ZFNs include individual finger subunits that bind DNA and are tethered to the Fokl nuclease domain that cleaves DNA.
  • Transcription activator-like effector nucleases include repeating units that bind DNA by virtue of a hypervariable two amino acid sequence (repeat variable diresidue; RVD) that governs DNA base recognition. Similar to ZFNS, TALENs function as dimeric proteins that are fused to the Fokl endonuclease domain for DSB generation.
  • MN Meganucleases
  • CRISPR clustered regularly interspaced short palindromic repeats
  • gRNA small guide RNA
  • CRISPR/Cas9 have been employed for disrupting the TCR complex by targeting either TRAC or TRBC of the TCR.
  • ZFNs delivered as mRNA results in gene disruption rates of 27-37% for TRAC and 4-15% for TRBC.
  • Delivering this same pair in either integrase deficient lentiviral (IDLV) or adenoviral delivery vehicles resulted in TRAC disruption rates of 10% with IDLV and 50% with adenovirus, and -5% with IDLV and -40% with adenovirus for TRBC.
  • TALEN mRNA delivery, at maximal rates, has resulted in -60% TRAC and -40% TRBC gene disruption.
  • a fusion protein of a meganuclease to TAL repeats (termed a megaTAL; MT) can achieve editing rates for TRAC of >60% using a first generation enzyme co-delivered with the Trex2 gene product, an exonuclease that increases gene disruption rates.
  • Nucleases from three different platforms were generated, each targeted to overlapping sequences within exon 1 of the TRAC coding region (FIG. 1 A and FIG. 9).
  • the MT site was nearly identical to the Streptococcus pyogenes (Sp) CRISPR/Cas9 (located on the opposite strand) and the TALEN site covered the same genomic locus (FIG. la and FIG. 9) as the previously reported ZFN (Torikawa et al., 2012. Blood 119:5697-5705).
  • Sp Streptococcus pyogenes
  • the hybrid MT protein contains the I-Onul LAGLIDADG homing endonuclease architecture that maintains its 'central 4' bp recognition domain, but has been repurposed for TRAC gene binding and is fused to 11 TAL repeat regions (FIG. IB).
  • the TALEN candidate target site contained a 5' T nucleotide and 22 bp and 18 bp contacting RVDs, respectively, with an architecture containing an N-terminal deletion of 152 residues and 63 wild type TAL sequences at the C-terminus (FIG. 1C).
  • the Sp CRISPR/Cas9 platform employs a guide RNA that contacts a target locus possessing a GN 20 GG sequence motif that serves to recruit the Cas9 protein to the target site where it induces a double-stranded break (FIG. ID).
  • the TALENs were assembled using the Golden Gate methodology into the RCIscript Goldy TALEN backbone that allows for in vitro mRNA production using T3 polymerase (Carlson, et al., 2012. Proc Natl Acad Sci USA 109: 17382-17387).
  • Cas9 can be delivered as a protein or, like the MT, can be in vitro transcribed using a T7 promoter.
  • the gRNA was evaluated as DNA (plasmid or linear DNA expression fragment) or RNA for efficiency of gene editing (FIG. IE). These RNA species were either transcribed in vitro locally or acquired from a commercial vendor as an unmodified RNA or one whose first and last three bases contained a 2'- O-methyl (2'-OMe) phosphorothioate modification.
  • the megaTAL (MT) candidate was tested for activity in Jurkat cells by electroporating 1 ⁇ g of mRNA generated following the standard protocol for T7 RNA polymerase that includes adding an exogenous polyadenylation signal by the E. coli poly (A) polymerase enzyme.
  • Flow cytometry was employed to quantify TRAC gene disruption rates using an anti-CD3 antibody that recognizes the intact TCR complex.
  • CD3 disruption rates as evidenced by the lack of cell surface CD3 expression, were -80% in MT -treated cells, while GFP treated cells with rates of gene transfer of 95% showed >95% CD3 (FIG. 2a).
  • the TALEN RCIscript Goldy backbone contains 5' and 3' UTR sequences derived from the Xenopus ⁇ -globin gene, included to increase translation efficiency.
  • T3 polymerase-based system to generate mRNA resulted in TRAC gene disruption rates of approximately 30% (FIG. 2A).
  • the T3-based procedure does not include a polyadenylation step and, therefore, may increase the stability and expression of TALEN mRNA.
  • T3 mRNA with an added polyA exhibited a knockout rate increased to -60% (FIG. 2A).
  • gRNA transcripts may not reach the nucleus at sufficient rates and/or are degraded by intracellular nucleases prior to complexing with the Cas9 protein.
  • Advances in synthetic RNA allows one to obtain high doses of the gRNA as an unmodified or nuclease protected species by virtue of 2'-OMe modified bases.
  • the unmodified and modified gRNAs were employed at a dose of 5 ⁇ g using either Cas9 protein or Cas9 mRNA.
  • Use of the nuclease-protected modified gRNA resulted in higher editing rates than unmodified gRNA using both the complexed ribonucleoprotein (RNP) format as well as an all RNA delivery platform (FIG. 2B).
  • FIG. 10 A representative FACS plot of FIG. 2 is shown as FIG. 10 and, collectively, the results suggest effective delivery vehicles for each class of nuclease.
  • MT functioned at high levels with in vitro transcribed mRNA and TALEN activity could be enhanced with the addition of an exogenous polyadenylation signal.
  • CRISPR/Cas9 the delivery of the gRNA as a modified gRNA species yielded maximum editing rates.
  • the nucleases were assessed in primary human T-lymphocytes for TRAC gene modification.
  • TALEN T3 mRNA with a poly A employs an initial cellular activation step followed by electroporation-based gene transfer.
  • the delivery platform identified as described above for each candidate was used in this study: MT T7 polymerase generated mRNA, TALEN T3 mRNA with a poly A, and Cas9 mRNA or protein with a modified gRNA. Nuclease-treated cells were incubated at 30°C for 24 hours post gene transfer. The TALEN showed a significant decrease compared to their profile in Jurkat cells; moreover, significant toxicity was associated with TALEN expression in primary T-cells (FIG. 3b).
  • a pool of cells lacking the TCR can be generated using a manufacturing process for CD3 negative cells that involves electroporating T cells at a low, mid, and high scale with either MT or CRISPR/Cas9 reagents.
  • the experimental schema is shown in FIG. 4a and employs the use of IL2, IL-7, and IL-15 and 9 days of culture followed by negative selection using the column free EASYSEP system (StemCell Technologies, Inc., Vancouver, Canada) that resulted in a purity of 85%) that was improved to 99% by repeating the procedure (FIG. 4B). TRAC gene edited cells were then maintained in IL-7 and IL-15 alone.
  • the TRAC gene mRNA was re-introduced by electroporation at day 15 and then re-stimulated cells with CD3/CD28 activation beads.
  • Adding the CD3/CD28 beads to cells that did not receive TRAC mRNA resulted in a slight proliferative increase, perhaps due to CD28 stimulation or activation of the ⁇ 1% of CD3+ cells that remain after negative depletion (FIG. 4B and 4C, no TRAC+stim).
  • transient recapitulation of the CD3 complex by TRAC mRNA electroporation and stimulation with CD3/28 activation beads allowed for a further five-fold expansion (FIG. 4C, TRAC+stim), thus providing for the rapid and efficient generation of TCR-deficient T cells from a small starting population.
  • T cell transduction was evaluated at higher order scaling.
  • the CRISPR/Cas9 system required 5 ⁇ g of modified gRNA, a dose that may not be conducive for large-scale application.
  • the input cell number was increased to 500,000 cells.
  • Nuclease treatment, negative selection, and culture of CD3 null cells in homeostatic cytokines allowed for the recovery of ten times the input number at day 15 of the procedure (FIG. 4D).
  • ⁇ 10 8 TRAC null cells were obtained at day 15 (FIG. 4E).
  • FIG. 5 comprehensive flow cytometry -based phenotyping panel.
  • the majority of the cells were CD4+ (FIG. 5 A) that retained effector function (FIG. 5B) (e.g., secretion of IL-2, IFNy, GzmA, GzmB). Further, the in vitro culture period needed for TRAC ablation did not affect
  • FIG. 5C shows that the schema shown in FIG. 6A was used to perform CD19-CAR lentiviral transduction followed by gene editing.
  • CD3 negative CD4+ or CD8+ CAR transduced cells showed the ability to form lytic granules in response to K562 cells expressing the CD 19 antigen (FIG. 6B).
  • IDLV integrase-deficient lentivirus
  • LAM-PCR linear amplification mediated PCR
  • nrLAM-PCR nrLAM-PCR methodology (Gabriel et al., 2011. Nature Biotechnol 29:816-823; Paruzynski et al., 2010. Nature Protoc 5: 1379-1395) were used in Jurkat cells (FIG. 7A). Inserting the IDLV vector into the genome can occur in the presence of double-stranded breaks, permanently marking the locus and enabling mapping by LAM and nrLAM PCR (FIG. 7A). DNA breaks can occur naturally in the absence of engineered nucleases due to normal cellular physiology or genomic fragile spots and these non-nuclease associated events, in the parlance of the assay nomenclature, are termed integration sites (IS). IDLVs delivered in tandem with a nuclease results in multiple insertions at either on-target or off-target (OT) sites and are termed clustered integration sites (CLIS).
  • IS integration sites
  • the designed TALEN pair overlapped the previously reported ZFN site and when the TALEN dimers were delivered as mRNA gave similar, albeit slightly lower, rates of editing compared to ZFNs (FIG. 3).
  • the Torikai 3 study using ZFNs showed TRAC disruption rates of -30% using 2.5 ⁇ g of mRNA, while a rate of -10% was observed using 1 ⁇ g of TALEN. Higher doses for the TALEN were not pursued since toxicity associated with TALEN expression was observed (FIG. 3B).
  • the TALEN disruption rates were increased by adding a polyadenylation signal (FIG. 2).
  • the CRISPR/Cas9 system is a highly flexible and user-friendly system the produced effective levels of TRAC gene disruption (FIGS. 2 and 3). Poor editing rates were observed when employing gRNA transcripts produced by in vitro transcription. Using commercially synthesized gRNA at a dose of 5 ⁇ g/ ⁇ l, however, resulted in ⁇ 60+% CD3 disruption rates using Cas9 mRNA (FIG. 2B and FIG. 3D).
  • gRNA with Cas9 protein can be an alternative effective delivery strategy and efficient editing rates in Jurkat and primary T cells were observed using the RNP approach (FIG. 2B and FIG. 3D).
  • Guide RNAs containing modified RNA bases that protect from nuclease degradation resulted in high gene knockout rates in Jurkat and primary T cells using nuclease protected gRNA (FIGS. 2-4).
  • a challenge associate with this strategy is the high dose, associated cost, and specialized manufacturing process required to generate and modify RNA oligos of >100 bp in length.
  • a solution to this challenge can involve a strategy whereby low or midrange numbers of cells could be employed for CD3 disruption and expansion (FIG. 4).
  • TCR/CD3 negative cells were isolated with >99% purity (FIG. 4B)— an extremely high level of purity that may allow for clinical translation, as even a very small number of residual TCR- expressing cells may be capable of causing significant TCR-driven inflammation and tissue damage.
  • the purified cells maintained a high degree of viability (95+%) after incubation with homeostatic cytokines IL-7 and IL-15 that promote maintenance of CD3 null cells, but they did not robustly expand (FIG. 4C).
  • the TRAC gene mRNA were re-introduced into the cells via electroporation and the cells were subsequently stimulated with CD3/CD28 beads, achieving a consistent and rapid 5-fold further expansion (FIG. 4C).
  • CAR T cells have an ability to expand in the presence of antigen that is provided via artificial antigen presenting and retain their ability to lyse cells. The studies reported herein complement this approach and represent a manner in which cells can be generated without artificial antigen presenting cells.
  • This disclosure describes a genome-level methodology to assess nuclease off-target effects for MT, TALEN, and CRISPR/Cas9 head-to-head for a clinically relevant gene (FIG. 7). Moreover, the MT -treated and CRISPR/Cas9-treated primary T cells showed high level TRAC gene disruption with correspondingly high viability and expansion (FIG. 3 and FIG. 4). This indicates that the KAT2B OT site that represented 62.5% of the OT CLIS is well tolerated in T cells. Additionally, by design, the CAR construct described herein also co-expresses a truncated, non-ligand binding form of the epidermal growth factor gene (tEGFR) (FIG.
  • tEGFR epidermal growth factor gene
  • This disclosure further describes gene disruption using TALENs, MT, or CRISPR/Cas9.
  • the MT and CRISPR/Cas9 reagents showed robust TRAC knockout rates.
  • the differential targeting sites and their associated accessibility to nucleases with regard to epigenetic factors can factor in nuclease design and application for the most complete targeting strategy.
  • a component of this is the ability to assess off-target events and to engineer, maintain, and expand the cells in a manner that is clinically viable.
  • This disclosure describes a genome-wide off-target screen comparing candidates from three classes of nucleases.
  • CRISPR/Cas9 is a user-friendly platform that yielded high TRAC disruption rates and did not exhibit off-target activity; however, large doses of synthetic RNA were required that for many laboratories will require commercial acquisition.
  • This challenge can be addressed, at least in part, by either expanding gene edited cells by, for example, transiently re-expressing TRAC and re-stimulating the cells or by controlling the initial number of cells as part of a scalable process. Under either condition, one can successfully obtain adequate numbers of cells such that putative cell or dosing hurdles can be surmounted, resulting in a cell population whose dose is within the range of those being used for T cell therapy.
  • CRISPR/Cas9 platform is delivered to a cell by delivering a polynucleotide that encodes is the required polypeptides, the methods described herein may be performed by, for example, introducing the Cas9 protein directly into the cell. Delivery can be accomplished through electroporation, inclusion/conjugation of Cas9 with cell penetrating peptides or ligands for cellular surface receptors, complexing with lipids, liposomes, and conjugated alone or in tandem with a gRNA to nanoparticles and hollow gold nanoparticles.
  • the Cas9 can also be a photo- activatable version or an induced dimerization version using drug (e.g., rapamycin, giberelin, etc.) administration to achieve activity or inhibit activity with agents such as rapalogs.
  • drug e.g., rapamycin, giberelin, etc.
  • Cas9 may also exist as a functionally inert form with respect to nuclease activity that relies on dimerization and nucleolytic activity through alternate nuclease domains (e.g., FokT).
  • CRISPR/Cas9 is derived from Staphylococcus aureus or Streptococcus pyogenes, the methods described herein can be performed using CRISPR/Cas from any suitable prokaryotic source.
  • Exemplary alternative sources of CRISPR/Cas9 include, for example, Francisella novicida, Campylobacter jejuni, Neisseria meningitides, Francisella tularensis, Pasteurella multocida, Streptococcus thermophilus, Campylobacter lari, Mycoplasma gallisepticum, Nitratifractor salsuginis, Parvibaculum lavamentivorans, Roseburia intestinalis, and/or nuclease components derived from the class II Cpfl Cas9 and single component C2C2 variants, as well as other Cas derived sequences with nuclease activity: Cas3, Cas8a, Cas8b, Casl Od, Csel, C
  • the methods described herein can be performed using other gRNAs.
  • the gRNA can have an alternative modification that confers nuclease resistance such as, for example, at least one 2'-0-methyl modified base or at least one 2'-0-methyl 3' thioPACE modified base.
  • the gRNA can be a standard gRNA— i.e., a gRNA that does not include a base modification to make the gRNA nuclease resistant.
  • the gRNA can be delivered in a manner that either decreases susceptibility to intracellular nucleases and/or in a manner that confers stability to unmodified guides.
  • exemplary delivery strategies include, for example, minicircle DNA, microsponge RNA, lipid particles, nanoparticles, and/or hollow gold nanoparticles that are activated by near infrared light.
  • Another exemplary delivery strategy involves Cas9 conjugated to a cell-penetrating peptide that allows for transit of the plasma membrane, transit of the nuclear membrane, and/or endosomal release.
  • the cell-penetrating peptide may be from, or be derived from, a natural (e.g., transferrin, LDL, LDL receptor, cell surface receptors, polyclonal antibody or monoclonal antibody), viral (e.g., HIV TAT), or bacterial source.
  • a natural source e.g., transferrin, LDL, LDL receptor, cell surface receptors, polyclonal antibody or monoclonal antibody
  • viral e.g., HIV TAT
  • bacterial source e.g., HIV TAT
  • the term "derived from" a natural source simply allows for the cell-penetrating peptide to be, for example, a truncated version of a naturally-occurring larger cell-penetrating peptide and/or include, for example, conservative amino acid substitutions compared to the naturally-occurring version of the peptide.
  • a cell-penetrating peptide "derived from” a natural-occurring cell-penetrating peptide also can include one or more amino acid residues not found in the naturally-occurring peptide such as, for example, a short "linking" peptide sequence that links the cell-penetrating peptide to the Cas9 polypeptide.
  • a gRNA can include non-classical/synthetic nucleic acid bases (e.g., iso-C, iso-G, xanthosine, d5SICS, dNaM, 6-amino-5-nitro-3-(l '-P-D-2'-deoxyribofuranosyl)-2(lH)-pyridone, and 2-amino-8-( -P-d-2'-deoxyribofuranosyl)-imidazo[l,2-a]-l,3,5-triazin-4(8H)-one)).
  • non-classical/synthetic nucleic acid bases e.g., iso-C, iso-G, xanthosine, d5SICS, dNaM, 6-amino-5-nitro-3-(l '-P-D-2'-deoxyribofuranosyl)-2(lH)-pyridone, and 2-amino-8-( -P-d-2'-
  • the gRNA can be conjugated with and/or fused to a polynucleotide sequence with stability bearing properties such as those derived from Kunjin and Dengue virus.
  • exemplary polynucleotides that confer stability bearing properties include, for example, SEQ ID NO:52-55.
  • the term “and/or” means one or all of the listed elements or a combination of any two or more of the listed elements; the terms “comprises,” “comprising,” and variations thereof are to be construed as open ended— i.e., additional elements or steps are optional and may or may not be present; unless otherwise specified, "a,” “an,” “the,” and “at least one” are used interchangeably and mean one or more than one; and the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).
  • the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.
  • the MT was constructed as previously described (Boissel et al., 2014. Nucleic Acids Res
  • the TALEN site was selected using the TAL effector Nucleotide Targeter 2.0 (Doyle et al., 2012. Nucleic Acids Res 40:W117-122) and were assembled using a previously described methodology (Cermak et al., 2011. Nucleic Acids Res 39(12):e82) and cloned into the RCIscript Goldy backbone (Addgene #38142, Addgene, Cambridge, MA; Carlson et al., 2012. Proc Natl Acad Sci USA 109: 17382-17387). CRISPR/Cas9 target site selection and in silico predictive off target assessment were accomplished using the CRISPR Design Tool
  • Cas9 plasmid was obtained from Addgene (Mali et al., 2013. Science 339(6121):823-826).
  • RNA Production The MT plasmid was linearized with Swal, TALEN plasmid linearization was accomplished with Sad, and Cas9 was linearized with Nhel.
  • TRAC NC_000014.9
  • cDNA was a linear PCR fragment.
  • T3 TALEN
  • T7 MT, Cas9, TRAC
  • promoter generated mRNA in vitro transcription using the mMESSAGE mMACHINE T3 or mMESSAGE mMACHINE T7 Ultra kits (Thermo Fisher Scientific,
  • RNA transcript was performed by amplifying a single stranded oligonucleotide (target site underlined): ⁇ TRAC):
  • AAAAAAAGCACCGACTCGGTGCC (SEQ ID NO:3) with Q5 Hot Start High-Fidelity 2X Master Mix (New England BioLabs, Ipswich, MA).
  • mRNA and gRNA transcripts were DNase treated and then purified using the RNeasy MinElute Cleanup Kit (Qiagen, Valencia, CA) with elution in water.
  • RNeasy MinElute Cleanup Kit Qiagen, Valencia, CA
  • commercially synthesized gRNAs were obtained from TriLink Biotechnologies, LLC (San Diego, CA) and the sequences are:
  • T cell isolation and negative selection Eight mL of whole blood was obtained by phlebotomy and heparinized with 15 USP of heparin and T-cells were isolated using the ROSETTESEP Human T cell enrichment cocktail (StemCell Technologies, Inc., Vancouver, Canada). Negative selection was performed by subjecting the T-cells to either one or two rounds of depletion using the EASYSEP
  • T cells were cultured in X-VIVO 20 media (Lonza, Basel, Switzerland) supplemented with 20% AB human serum (Thermo Fisher Scientific, Waltham, MA). For activation the cells were further supplemented with recombinant IL-2 (Chiron Corp., Emeryville, CA) at a concentration of 300 IU/mL. Anti-CD3/CD28 DYNABEADS (Thermo Fisher Scientific, Waltham, MA) were added at a 3: 1 beadxell ratio and cells were cultured at 37°C and 5% C0 2 at a concentration of 500,000/mL. Where indicated, recombinant human IL-7 or IL-15 (PeproTech, Rocky Hill, NJ) was included at 5 ng/mL (Cieri et al., 2013. Blood 121 :573-84).
  • CD3/CD28 beads Forty-eight hours after activation the CD3/CD28 beads were magnetically removed and the cells were cultured in the absence of beads for 6-12 hours. 200,000 cells were electroporated with the indicated amounts of nucleic acid.
  • RNP For the RNP conditions either 5 ⁇ g or 10 ⁇ g of gRNA was incubated with 1 ⁇ g of Cas9 protein (Thermo Fisher Scientific, Waltham, MA) in a total volume of 5 ⁇ for 15 minutes at room temperature. The 10 ⁇ or 100 ⁇ tip of the NEON Transfection System (Thermo Fisher Scientific, Waltham, MA) was used for gene transfer with the following conditions: primary T-cells (in Buffer T): 1400V, 10 ms, three pulses.
  • Jurkats (In Buffer R): 1325V, 10 ms, three pulses. Cells were plated in 200 ⁇ of antibiotic free media and cultured at 30°C for 24 hours. Cells were maintained at 500,000/mL in media containing antibiotics after the initial plating in antibiotic free media.
  • Genomic DNA was isolated using the PURELINK genomic DNA mini kit (Thermo Fisher Scientific, Waltham, MA) and amplified with the primers listed below using the PURELINK genomic DNA mini kit (Thermo Fisher Scientific, Waltham, MA) and amplified with the primers listed below using the PURELINK genomic DNA mini kit (Thermo Fisher Scientific, Waltham, MA) and amplified with the primers listed below using the PURELINK genomic DNA mini kit (Thermo Fisher Scientific, Waltham, MA) and amplified with the primers listed below using the primers listed below using the primers listed below using the primers listed below using the primers listed below using the primers listed below using the primers listed below using the primers listed below using the primers listed below using the primers listed below using the primers listed below using the primers listed below using the primers listed below using the primers listed below using the primers listed below using the primers listed below using the primers listed below using the primers listed below using the primers listed below using the primers listed below using the primers listed below using
  • ACCUPRIME Ta DNA polymerase high fidelity (Thermo Fisher Scientific, Waltham, MA) follows: 95°C for two minutes, and 40 cycles of 95°C for 40 seconds, 59°C for 40 seconds, and 68°C for one minute.
  • Nine microliters of the PCR product was denatured/renatured with IX PCR buffer and then incubated for 20 minutes with the Enhancer and Surveyor enzyme (Integrated DNA Technologies, Inc., Coralville, IA; Guschin et al., 2010. Methods Mol Biol 649:247-256). Products were then resolved on a 10% TBE PAGE gel (Thermo Fisher Scientific, Waltham, MA) at 200V and stained with ethidium bromide.
  • TRAC F ATCACGAGCAGCTGGTTTCT (SEQ ID NO:6)
  • TRAC R CCCGTGTCATTCTCTGGACT (SEQIDNO:7)
  • DPT F AAGCAAGGCCAGTTTCTCA (SEQ ID NO:8)
  • DPT R TTAGGATGTGCCCAGGTTATG (SEQIDNO:9)
  • KAT2B F AGC C TAGGACGAT GAGGGA. (SEQ ID NO: 10)
  • KAT2b R CCTATCCCACCAGCATCCAA (SEQ ID NO: 11)
  • HIAT1 R TATCCCCAGCACCCAGTAAG (SEQIDNO:15)
  • DR1 F TCCATTGTGTAATGAAGATGATTTG (SEQIDNO:16)
  • KIAA1217R AGACCTAGAATTGCCAAAACA (SEQIDNO:19)
  • GBP5 F TGGTCAAGTGTCGAGTTTGT (SEQ ID NO:20)
  • GBP5 R AT C C AG T C AC C T T C C AC C AG (SEQ ID NO:21)
  • EXOC2 F AGGCCATAGTCACCCAAACA (SEQIDNO:22)
  • EXOC2 R T TGGGT TCT TGGTCACGAAG (SEQ ID NO:23)
  • TCR disruption rate assessment Jurkats and primary T-cells were cultured for seven or nine days post electroporation respectively, and analyzed for the presence of CD3 by flow cytometry. Similarly, phenotypic analysis was performed on day 9 T-cells using the antibodies indicated in FIG. 5.
  • FACS FACS, cells were washed with PBS and stained at 1 : 100 dilution with the appropriate antibody (all antibodies obtained from eBiosciences, San Diego, CA) for 60 minutes at 4°C in FACS buffer consisting of PBS+1% FBS+lmM EDTA. Cells were then washed three times in FACS buffer and re-suspended in FACS buffer containing Sytox Blue
  • VSV-G pseudotyped CD19 CAR-tEGFR lentivirus was produced in 293T cells and 50 mL of supernatant was concentrated with Lenti-X concentrator (Clontech Laboratories, Inc., Mountain View, CA) was added and the viral pellet resuspended in 2 mL of T cell culture media. 500 ⁇ of viral supernatant was added to a retronectin coated plate and spun for three hours at 1200 rpm. 1 ⁇ 10 6 T cells were added with CD3/CD28 beads and cultured for 48 hours and then treated with the appropriate nuclease.
  • Cas9 protein at a concentration of 10 ⁇ g (Aldevron, Fargo, ND) was complexed with unmodified TRAC gRNA (5 ⁇ g) in 5 ⁇ of NEON Transfection Buffer T (ThermoFisher, Waltham, MA, USA) for 10 minutes at room temperature.
  • the ribonucleotide complex (shown in FIG. 13 A) was electroporated into primary T cells using the NEON Transfection System (ThermoFisher, Waltham, MA, USA) with the following conditions: 1400V, 10ms, three pulses. Cells were plated in 200 ⁇ of antibiotic free media (X-VIVO 20 media (Lonza, Basel,
  • FACS analysis was performed on day nine and the cells were washed with PBS and stained at 1 : 100 dilution with the anti-human CD3 APC-eFluor 780 antibody (Affymetrix, Inc., Santa Clara, CA) for 60 minutes at 4°C in FACS buffer consisting of PBS+1% FBS+1 mM EDTA. Cells were then washed three times in FACS buffer and re-suspended in FACS buffer containing Sytox Blue (ThermoFisher, Waltham, MA) for dead cell exclusion. Acquisition was performed on a BD FACSCanto (BD Biosciences, San Jose, CA) and data was subsequently analyzed using FlowJo (Tree Star Inc., Ashland OR).
  • BD FACSCanto BD Biosciences, San Jose, CA

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Abstract

La présente invention concerne des procédés qui consistent à éditer le génome d'un lymphocyte T pour interrompre l'expression normale d'un récepteur de lymphocytes T (TCR). De manière générale, les procédés consistent à introduire à l'intérieur d'un lymphocyte T une nucléase d'édition de génome conçue pour éditer la région codant le TRAC qui code le TCRα, et à cultiver le lymphocyte T dans des conditions permettant à la nucléase d'édition du génome de modifier le TRAC de sorte à interrompre la formation du complexe TCR/CD3. Selon certains aspects, les procédés peuvent consister à administrer à un sujet lesdits lymphocytes T dont le génome a été édité en tant qu'immunothérapie adoptive.
PCT/US2016/058045 2015-10-22 2016-10-21 Procédés consistant à éditer des polynucléotides codant pour un récepteur de lymphocytes t WO2017070429A1 (fr)

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