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Sequential gene editing in primary immune cells — Cellectis (US11674155B2)

Cellectis · Google Patents

Google Patents · Patents · License: Open Access

patent, google patents, intellectual property, US11674155B2, Cellectis, Jean-Pierre Cabaniols, en, 2023

ABSTRACT

Abstract

The invention pertains to the field of adaptive cell immunotherapy. It aims at reducing the occurrence of translocations and cell deaths when several specific endonuclease reagents are used altogether to genetically modify primary immune cells at different genetic loci. The method of the invention allows to yield safer immune primary cells harboring several genetic modifications, such as triple or quadruple gene inactivated cells, from populations or sub-populations of cells originating from a single donor or patient, for their subsequent use in therapeutic treatments.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of pending U.S. application Ser. No. 16/314,697, filed on Jan. 2, 2019, which is a U.S. National Stage Entry under 35 U.S.C. Â§ 371 of PCT/EP2017/066355, filed on Jun. 30, 2017, which itself claims the benefit under 35 U.S.C. 119(e) of Denmark Patent Application No. PA201670503, filed on Jul. 6, 2016, the contents of each of which are incorporated herein by reference in their entireties for all purposes.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on Aug. 5, 2022, is named 116983-5073-US01sequencelisting.xml and is 26,563 bytes in size.

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

The potential of gene editing in various therapies has long been envisioned by the applicant (WO2004067753), especially in the field of cell therapy, where immune cells can be genetically modified ex-vivo and then reintroduced into patients, as already described, for instance, in U.S. Pat. No. 8,921,332.

Since the emergence of the first programmable sequence-specific reagents by the turn of the century, initially referred to as Meganucleases [Smith et al. (2006) A combinatorial approach to create artificial homing endonucleases cleaving chosen sequences. Nucl. Acids Res. 34 (22):e149.], endonucleases reagents have rapidly evolved, offering improved specificity, safety and reliability. In particular, TALE-nucleases (WO2011072246), which are fusions of a TALE binding domain with a cleavage catalytic domain have been successfully applied to primary immune cells, in particular T-cells from peripheral blood mononuclear cells (PBMC). Such TALE-nucleases, marketed under the name TALENÂ®, are currently used to simultaneously inactivate gene sequences in T-cells originating from donors, in particular to produce allogeneic therapeutic T-Cells, in which the genes encoding TCR (T-cell receptor) and CD52 are disrupted. These cells can be endowed with chimeric antigen receptors (CAR) or recombinant TCR for treating cancer patients (US2013/0315884). TALE-nucleases are very specific reagents because they need to bind DNA by pairs under obligatory heterodimeric form to obtain dimerization of the cleavage domain Fok-1. Left and right heterodimer members each recognizes a different nucleic sequences of about 14 to 20 bp, together spanning target sequences of 30 to 50 bp overall specificity.

More recently, further endonucleases reagents have been developed based on the components of the type II prokaryotic CRISPR (Clustered Regularly Interspaced Short palindromic Repeats) adaptive immune system of the bacteria S. pyogenes . This multi-component system referred to as RNA-guided nuclease system [Gasiunas, Barrangou et al. (2012) Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria; PNAS 109(39): E2579-E2586]; Doudna, J. Charpentier E. (2014) The new frontier of genome engineering with CRISPR-Cas9 Science 346 (6213):1258096], involves members of Cas9 or Cpf1 [Zetsche et al. (2015). Cpf1 is a single RNA-guided endonuclease that provides immunity in bacteria and can be adapted for genome editing in mammalian cells. Cell 163:759-771] endonuclease families coupled with a guide RNA molecules that have the ability to drive said nuclease to some specific genome sequences. Such programmable RNA-guided endonucleases are easy to produce because the cleavage specificity is determined by the sequence of the RNA guide, which can be easily designed and cheaply produced. The specificity of CRISPR/Cas9 although stands on shorter sequences than TAL-nucleases of about 10 pb, which must be located near a particular motif (PAM) in the targeted genetic sequence.

Other endonuclease systems derived from homing endonucleases (ex: I-Onul, or I-Crel), combined or not with TAL-nuclease (ex: MegaTAL) or zing-finger nucleases have also proven specificity, but with less efficiency so far.

Various proofs of concept of the efficiency and safety of the above specific endonuclease reagents have been reported in human cells in-vitro or ex-vivo, but the co-delivery into the same cells of sequence specific reagents acting on different loci has still to be carefully considered as a potential factor of off-site mutations, large genomic deletions and translocations inherent to the DNA repair mechanisms (Poirot et al. (2015) Multiplex Genome-Edited T-cell Manufacturing Platform for âOff-the-Shelfâ Adoptive T-cell Immunotherapies Cancer Res. 75: 3853-64).

In parallel, novel specificities have been conferred to immune cells through the genetic transfer of transgenic T-cell receptors or so-called chimeric antigen receptors (CARs) (Jena et al. (2010) Redirecting T-cell specificity by introducing a tumor-specific chimeric antigen receptor. Blood. 116:1035-1044). CARs are recombinant receptors comprising a targeting moiety that is associated with one or more signaling domains in a single fusion molecule. In general, the binding moiety of a CAR consists of an antigen-binding domain of a single-chain antibody (scFv), comprising the light and heavy variable fragments of a monoclonal antibody joined by a flexible linker. Binding moieties based on receptor or ligand domains have also been used successfully. The signaling domains for first generation CARs are derived from the cytoplasmic region of the CD3zeta or the Fc receptor gamma chains. First generation CARs have been shown to successfully redirect T cell cytotoxicity, however, they failed to provide prolonged expansion and anti-tumor activity in vivo. Signaling domains from co-stimulatory molecules including CD28, OX-40 (CD134), ICOS and 4-1BB (CD137) have been added alone (second generation) or in combination (third generation) to enhance survival and increase proliferation of CAR modified T cells. CARs, as well as the expression of recombinant TCRs, have successfully allowed T cells to be redirected against antigens expressed by tumor cells from various malignancies including lymphomas and solid tumors.

Recently engineered T-cells disrupted in their T-cell receptor (TCR) using TALE-nucleases, endowed with chimeric antigen receptor (CAR) targeting CD19 malignant antigen, referred to as âUCART19â product, have shown therapeutic potential in at least two infants who had refractory leukemia (Leukaemia success heralds wave of gene-editing therapies (2015) Nature 527:146-147). To obtain such UCART19 cells, the TALE-nuclease was transiently expressed into the cells upon electroporation of capped mRNA to operate TCR gene disruption, whereas a cassette encoding the chimeric antigen receptor (CAR CD19) was introduced randomly into the genome using a retroviral vector.

In this later approach, the steps of gene inactivation and of expressing the chimeric antigen receptor are independently performed after inducing activation of the T-Cell âex-vivoâ.

However, engineering primary immune cells is not without any consequences on the growth/physiology of such cells. In particular one major challenge is to ovoid cells exhaustion/anergy that significantly reduces their immune reaction and life span. This is more likely to happen when the cells are artificially activated ahead of their infusion into the patient. It is also the case when a cell is endowed with a CAR that is too reactive.

The introduction of the polynucleotides expressing recombinant receptors into those cells, through an independent step of viral transduction, also has an impact on the overall production process.

The inventors have explored safer means for ex-vivo delivery of endonucleases reagents into primary cells with the requirements that said cells (1) are modified at different genetic loci, (2) not bearing too many translocations, (3) produced in sufficient number to enable treating at least a hundred patients, and (4) produced in a limited time frame of less than 30 days to avoid exhaustion of the cells. They came up with the invention as described herein, where sequential gene editing is performed instead of multiplexing gene editing. Surprisingly, this occurred to be less destructive to the cells, resulting into higher quality primary immune cells.

This invention paves the way to standard and affordable adoptive immune cell therapy treatments.

SUMMARY OF THE INVENTION

The present invention is drawn to a method of sequential gene editing aiming to improve the genetic modification of primary human cells, especially immune cells originating from individual donors or patients.

Primary immune cells, in particular T-cells or NK cells, have a limited life span, and although they can be expanded and activated ex-vivo by methods known in the art [Rasmussen A. M. et al. (2010) Ex-vivo expansion protocol for human tumor specific T cells for adoptive T cell therapy. Journal of Immunological Methods 355:52-60], their immune reactivity tends to reduce over time. They can also get exhausted from the moment they are collected from a donor to the moment they are reintroduced in-vivo to track down and eliminate malignant or infected cells into the patient.

Gene editing techniques using nucleotide sequence-specific reagents, such as rare-cutting endonucleases, have become the state of the art for the introduction of genetic modifications into primary cells. However, when such endonucleases are used to cleave target sequences simultaneously at different loci, the risk of inter- or intra-chromosomal translocations increases significantly, which concur to a higher risk of unwanted genetic recombination or off-site mutations.

To overcome this drawback and minimize adverse genome effects, the inventors have applied a safer approach, where gene editing is sequentially applied, in particular through several rounds of electroporation. To their surprise, sequential gene editing resulted into cells of higher quality and even into an increase of the yield of the engineered cells modified at different loci, as compared to multiplexing gene editing (i.e. gene editing performed simultaneously at different loci).

The present invention thus primarily concerns a method comprising one or several steps of:

Providing at least one primary immune cell from a culture or a blood sample, such as from peripheral blood mononuclear cells (PBMCs); Subjecting said cell to a step of gene editing, where a first set of sequence-specific reagent(s) is introduced into said cell; Cultivating said cell to enable said first sequence-specific reagent to stably modifying its genome at a first locus, Subjecting said cell to at least a second gene editing step to introduce at least a second set of sequence-specific reagent(s) into said cell, and optionally Cultivating said cell to enable said second sequence-specific reagent to stably modifying its genome at said second locus.

According to a preferred embodiment, the first, second and any subsequent sequence-specific reagents are introduced into said cell by electroporation, so that the method of the invention comprises:

a) Subjecting the immune cells to a first electroporation to introduce at least a first sequence-specific reagent into said immune cell; b) cultivating said immune cell to enable said first sequence-specific reagent to modify its genome at a first locus, c) submitting said cell to at least a second electroporation to introduce at least a second sequence-specific reagent into said cell, and optionally d) cultivating said immune cell to enable said second sequence-specific reagent to modify its genome at said second locus.

Several strategies may be applied when applying the sequential gene editing of the present invention to obtain immune cells with higher recovery, better activation, persistence or therapeutic efficiency. As an example, the first electroporation step can be performed in view of editing or modifying a gene, in such a way that the cell will better expand or get more permissive to the subsequent modification steps.

As another example, the first step of gene editing can be performed on a receptor or surface protein, such as TCR. The TCR negative cells obtained by this first step can be purified by removal of the cells remaining TCR positive, such that said TCR negative cells can be cultivated and then subjected to a second step of gene editing, for instance to make them resistant to a chemotherapy drug. The resulting population of cells in which the second gene editing has been achieved, can then be enriched in TCR negative drug resistant cells by culture in a medium containing said chemotherapy drug.

Several examples are developed herein showing that, although deemed more destructive, the successive gene editing steps surprisingly contribute to improve the yield and therapeutic potential of the engineered immune cells.

The invention is drawn to the methods, but also to the new gene edited cells obtainable by these methods, especially new triple and quadruple gene inactivated immune cells, as well as the population of cells resulting thereof, which are useful for the preparation of therapeutic compositions.

The present invention may be further summarized by the following items:

1) A method for introducing genetic modifications at different loci of a primary immune cell, comprising the sequential steps of:

a) subjecting said primary immune cell to a first electroporation step to introduce at least a first sequence-specific reagent into said immune cell; b) cultivating said primary immune cell thereby enabling said first sequence-specific reagent to modify its genome at a first locus, c) subjecting said primary immune to at least a second electroporation step to introduce at least a second sequence-specific reagent into said cell, d) cultivating and expanding said primary immune thereby enabling said second sequence-specific reagent to modify its genome at said second locus.

CROSS-REFERENCE TO RELATED APPLICATIONS

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

In this later approach, the steps of gene inactivation and of expressing the chimeric antigen receptor are independently performed after inducing activation of the T-Cell âex-vivoâ.

The introduction of the polynucleotides expressing recombinant receptors into those cells, through an independent step of viral transduction, also has an impact on the overall production process.

This invention paves the way to standard and affordable adoptive immune cell therapy treatments.

SUMMARY OF THE INVENTION

The present invention thus primarily concerns a method comprising one or several steps of:

According to a preferred embodiment, the first, second and any subsequent sequence-specific reagents are introduced into said cell by electroporation, so that the method of the invention comprises:

The present invention may be further summarized by the following items:

1) A method for introducing genetic modifications at different loci of a primary immune cell, comprising the sequential steps of:

2) The method according to item 1, wherein the primary immune cell is cultivated in step b) from 12 to 72 hours, preferably from 24 to 48 hours. 3) The method according to item 1, wherein a purification step is performed between step b) and c) relying on a product resulting from the expression or the deletion of the gene that is modified at least at said first locus. 4) The method according to any one of items 1 to 3, wherein steps a) to d) are performed within 240 hours, preferably within 120 hours, more preferably within 96 hours, even more preferably within 72 hours. 5) The method according to any one of items 1 to 4, wherein said method comprises at least one further step of submitting said primary immune cell to a third electroporation step to introduce at least a third sequence-specific reagent into said cell. 6) The method according to item 1, wherein said first and/or second sequence-specific reagent is a polynucleotide or polypeptide encoding a rare-cutting endonuclease, a subunit thereof, or a conjugate of both a polynucleotide and a polypeptide. 7) The method according to item 2, wherein said first and/or second sequence-specific reagent is a polynucleotide or polypeptide encoding a rare-cutting endonuclease selected from programmable RNA or DNA guided endonuclease, TALEN, ZFN or a homing endonuclease. 8) The method according to item 3, wherein said first and/or second sequence-specific reagent is a conjugate of RNA guide and a Cas9 or Cpf1 polypeptide. 9) The method according to item 1, wherein said first and/or second sequence-specific reagent is an interference RNA (RNAi) or a polynucleotide encoding same. 10) The method according to item 1, wherein a transduction step is introduced between b) and c) with a retroviral or lentiviral vector. 11) The method according to item 10, wherein said transduction step involves an integrative lentiviral or retroviral vector for stable expression of a transgene. 12) The method according to item 11, wherein said transgene encodes a Chimeric Antigen Receptor (CAR). 13) The method according to item 10, wherein said transduction step involves a non-integrative viral vector. 14) The method according to item 13, wherein said non integrative viral vector is used as a template for homologous recombination or NHEJ integration of said transgene into the immune cell's genome. 15) The method according to item 10, wherein said first sequence-specific reagent is acting on a genomic sequence that facilitates the transduction step. 16) The method according to any one of items 1 to 15, wherein said first sequence-specific reagent is acting on a genomic sequence that facilitates the genetic modification of step d). 17) The method according to any one of items 1 to 16, wherein step b) is performed below about 35Â° C., preferably at about 30Â° C. 18) The method according to any one of items 1 to 17, wherein said immune cell is a T-cell. 19) The method according to item 18, comprising a preliminary step of activating the primary T-cell by signal transduction. 20) The method according to item 18 or 19, wherein said first sequence-specific reagent permanently reduces or prevents expression of TCR by the primary T-cell. 21) The method according to any one of items 1 to 20, wherein said first or second sequence-specific reagent permanently reduces or prevents expression of at least one gene encoding an immune checkpoint. 22) The method according to item 21, wherein said at least one gene encoding an immune checkpoint is selected from PD1, CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, LAG3, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3. 23) The method according to any one of items 1 to 20, wherein said first or second sequence-specific reagent permanently confers resistance of said primary immune cell against drugs or immune depleting agents. 24) The method according to item 23, wherein said resistance is conferred by inactivating a gene expressing CD52, dCK, GGH or HPRT. 25) The method according to any one of items 1 to 24, wherein a final step of purification is performed relying on a at least one product resulting from the expression or the deletion of one gene that is modified at said first and/or second and/or third locus. 26) A population of primary TCR negative T-cells resulting from a single donor obtainable according to the method according to any one of items 1 to 25, comprising at least two subpopulations of T-cells selected from:

TCR negative and PD1 negative, TCR negative and CD52 negative, TCR negative and CTLA4 negative, TCR negative and dCK negative, TCR negative and GGH negative, TCR negative and HPRT negative, and TCR negative and Î²2m negative.

27) A population of primary TCR negative T-cells originating from a single donor, wherein at least 20%, preferably 30%, more preferably 50% of the cells in said population have been modified using sequence-specific reagents in at least three different loci. 28) A pharmaceutical composition comprising a population of primary T cells according to any one of items 26 or 27.

BRIEF DESCRIPTION OF THE FIGURES AND TABLES

FIG. 1 : Examples of genes and cell functions which can be sequentially modified by gene editing according to the method of the present invention to produce engineered allogeneic primary immune T-cells. Arrows within the cell represents the various genetic loci that can be inactivated by the sequence specific nuclease reagent introduced into the T-cells.

FIG. 2 : Rationale for the sequential gene editing according to the present invention versus the multiplexing gene editing from the prior art in terms of yielding primary immune cells stacking mutations at three different loci, such as genes encoding TCR, PD1 and DCK. The method of the present invention ends up with a population of immune cells where at least 80% of the cells are triple mutants. By contrast, with the same reagent efficiency, simultaneous gene editing amounts about 50% of triple mutants. Upon cell expansion, this proportion should not much increase in the population, if not be decreasing.

FIG. 3 : Schematic representation of one embodiment of the method according to the invention, where a viral transduction step is performed between two electroporation gene editing steps. The viral transduction is preferably performed after a cell sorting step where the cells modified by the first gene editing modifications are purified. This cell sorting step reduces the overall number of cells, thereby reducing the number of viral particles to be used for the viral transduction. This can be advantageous, for instance, when TCR is first inactivated, to follow-up with stable viral transduction and expression of the CAR. Subsequent gene editing step can then be carried out to make the cells resistant to a drug. In such a situation, culturing or expanding the cells in a medium containing the drug allows the selection of the cells that are both TCR negative and drug resistant in view of their therapeutic use.

FIG. 4 : Schematic representation of one embodiment of the method of the invention, wherein a cell sorting step is performed between the two electroporation gene editing steps.

FIG. 5 : Schematic representation of one embodiment of the method of the invention, wherein a cell sorting step plus a viral transduction is performed between the two electroporation gene editing steps.

FIG. 6 : Schematic representation of one embodiment of the method according to the invention, where a culture step in a selective medium is performed between two electroporation gene editing steps, to select the cells that have been made resistant to a compound as per the first gene modification. This culture step increases the number of cells that are eventually modified at multiple loci after the second gene editing step. This approach can be applied to produce drug resistant CAR positive T-cells, that are further gene edited to be more active (inactivation of a locus inhibiting T-cell activation/cytotoxicity, such as PD1 and/or CTLA4), especially in a context of an autologous treatment where the T-cells (eg: Tumor Infiltrating Lymphocytes (TIL)) are collected from a patient, engineered and re-infused to said patient.

FIG. 7 : Schematic representation of one embodiment of the method according to the invention, where the first gene editing step is performed in a gene coding or regulating the expression of a surface antigen and the second gene editing step is performed in a gene coding or regulating the expression of a product that is not a surface antigen. A cell separation step is performed between the two gene editing steps to enrich the cells allowed to pass to the second gene editing step. Optionally, the second gene editing step is followed by a culture step in a selective medium to favor/select expansion of the cells bearing the second gene editing.

FIG. 8 : Schematic representation of one embodiment wherein a cell sorting based on immune cells subtypes, for instance CD4+ and CD8+ cells is performed after the first gene editing step (ex: TCR inactivation) and before the second gene editing step. As per this embodiment, a different gene editing can be applied to the different subtypes respectively, such as for instance the inactivation of FOXP3 in CD4+ cells and the inactivation of PD1 in CD8+ cells. The gene edited cells from the separate batches (ex: CD8+ and CD4+) can be respectively mixed at a predetermined ratio (ex: 1 to 1) to produce more active therapeutic compositions.

FIG. 9 : Diagrams displaying the % number of cells TCR negative, CD52 negative and both TCR and CD52 negative after simultaneous gene editing (TALEN CD52 and TCR together) (see Example 1). Controls are untransfected cells, which results are represented the left columns of the diagrams

FIG. 10 : T-cell growth curve observed after the different electroporation strategies: simultaneous and sequential according to the invention (6, 20 and 40 h interval between the two CD52 and TCR gene editing stepsâsee example 1).

FIG. 11 : Frequency of In-deletions (Indels) for 5 off-site targets (OFS1, OFS2, OFS3, OFS4 and OFS5) and TRAC and PD-1 on-site targets after simultaneous or sequential electroporation of TRAC and PD-1 TALENÂ®. Red stars highlight the abrogation of off-site 3 (OFS3) target cleavage by the method according to the invention (see example 2).

FIG. 12 : Diagrams showing the growth of the engineered cells populations over time from Day 5 (D5) post thawing to Day 15 (D15) with respect to the different gene editing strategies detailed in Example 3 and Table 2.

FIG. 13 : Schematic representation of workflow for the generation of triple KO CAR T cells, as carried out in example 4.

FIG. 14 : Diagram displaying the triple KO efficacy (% number TCR/B2M and PD1 negative cells), in two different donors using indicated doses of mRNA enconding TRAC, Î²2m and PD-1 TALENÂ®s that were electroporated either simultaneously (Sim.) or sequentially (Seq.), resulting into [TCR] neg [Î²2m] neg [PD1] neg therapeutically effective number of cells originating from donors, as explained in Example 4.

FIG. 15 : Diagrams showing cytotoxic activity on Raji cells at different effector to target ratios (E:T) of CD22 CAR-T cells that were either untransfected with TALENÂ® reagents (WT:black) or TALENÂ® transfected sequentially (dark grey) or TALENÂ® transfected simultaneously (light grey).

Table 1: List of genes involved into immune cells inhibitory pathways

Table 2: gene editing efficiency of various sequential gene editing strategies according to the invention as presented in Example 3 (percentage of gene edited cells based on numbers of CD38, TCR and/or CD52 negative cells; D4, D5 and D6 are the number of days after thawing frozen primary cells).

Table 3: Sequence of TALENÂ® used in the examples.

Table 4: Selection of antigen markers of various cancers found to be expressed on the surface of T-cells. The inactivation of the genes encoding these antigen markers is proposed as part of one of one of the gene editing steps according to the invention, especially when the engineered immune cells are endowed with chimeric antigen receptors targeting these very antigens.

DETAILED DESCRIPTION OF THE INVENTION

Unless specifically defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by a skilled artisan in the fields of gene therapy, biochemistry, genetics, and molecular biology.

All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, with suitable methods and materials being described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will prevail. Further, the materials, methods, and examples are illustrative only and are not intended to be limiting, unless otherwise specified.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and son Inc, Library of Congress, USA); Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al, 2001, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Harries & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the series, Methods In ENZYMOLOGY (J. Abelson and M. Simon, eds.-in-chief, Academic Press, Inc., New York), specifically, Vols. 154 and 155 (Wu et al. eds.) and Vol. 185, âGene Expression Technologyâ (D. Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

In a general aspect, the present invention relates to methods to perform genome modification in multiple loci in primary cells through sequential electroporation steps, spaced by cell culture, sorting and/or expansion phase(s).

In particular, these methods comprise the steps of:

a) subjecting the primary immune cell to a first electroporation to introduce at least a first sequence-specific reagent into said immune cell;

b) cultivating said primary immune cell thereby enabling said first sequence-specific reagent to modify its genome at a first locus,

c) subjecting said primary immune to at least a second electroporation to introduce at least a second sequence-specific reagent into said cell,

d) cultivating and expanding said primary immune thereby enabling said second sequence-specific reagent to modify its genome at said second locus.

By âprimary cellâ or âprimary cellsâ are intended cells taken directly from living tissue (e.g. biopsy material) and established for growth in vitro for a limited amount of time, meaning that they can undergo a limited number of population doublings. Primary cells are opposed to continuous tumorigenic or artificially immortalized cell lines. Non-limiting examples of such cell lines are CHO-K1 cells; HEK293 cells; Caco2 cells; U2-OS cells; NIH 3T3 cells; NSO cells; SP2 cells; CHO-S cells; DG44 cells; K-562 cells, U-937 cells; MRC5 cells; IMR90 cells; Jurkat cells; HepG2 cells; HeLa cells; HT-1080 cells; HCT-116 cells; Hu-h7 cells; Huvec cells; Molt 4 cells. Primary cells are generally used in cell therapy as they are deemed more functional and less tumorigenic.

In general, primary immune cells are provided from donors or patients through a variety of methods known in the art, as for instance by leukapheresis techniques as reviewed by Schwartz J. et al. (Guidelines on the use of therapeutic apheresis in clinical practice-evidence-based approach from the Writing Committee of the American Society for Apheresis: the sixth special issue (2013) J Clin Apher. 28(3):145-284). The primary immune cells according to the present invention can also be differentiated from stem cells, such as cord blood stem cells, progenitor cells, bone marrow stem cells, hematopoietic stem cells (HSC) and induced pluripotent stem cells (iPS).

By âimmune cellâ is meant a cell of hematopoietic origin functionally involved in the initiation and/or execution of innate and/or adaptative immune response, such as typically CD3 or CD4 positive cells. The immune cell according to the present invention can be a dendritic cell, killer dendritic cell, a mast cell, a NK-cell, a B-cell or a T-cell selected from the group consisting of inflammatory T-lymphocytes, cytotoxic T-lymphocytes, regulatory T-lymphocytes or helper T-lymphocytes. Cells can be obtained from a number of non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and from tumors, such as tumor infiltrating lymphocytes. In some embodiments, said immune cell can be derived from a healthy donor, from a patient diagnosed with cancer or from a patient diagnosed with an infection. In another embodiment, said cell is part of a mixed population of immune cells which present different phenotypic characteristics, such as comprising CD4, CD8 and CD56 positive cells.

By âendonuclease reagentâ is meant a nucleic acid molecule that contributes to an endonuclease catalytic reaction in the target cell, itself or as a subunit of a complex, preferably leading to the cleavage of a nucleic acid sequence target. The endonuclease reagents of the invention are generally sequence-specific reagents, meaning that they can induce DNA cleavage in the cells at predetermined loci, referred to by extension as âgene targetsâ. The nucleic acid sequence which is recognized by the sequence specific reagents is referred to as âtarget sequenceâ. Said target sequence is usually selected to be rare or unique in the cell's genome, and more extensively in the human genome, as can be determined using software and data available from human genome databases, such as www.ensembl.org/index.html.

âRare-cutting endonucleasesâ are sequence-specific endonuclease reagents of choice, insofar as their recognition sequences generally range from 10 to 50 successive base pairs, preferably from 12 to 30 bp, and more preferably from 14 to 20 bp.

According to a preferred aspect of the invention, the endonuclease reagent is transiently expressed into the cells, meaning that said reagent is not supposed to integrate into the genome or persist over a long period of time, such as be the case of RNA, more particularly mRNA, proteins or complexes mixing proteins and nucleic acids (eg: Ribonucleoproteins). In general, 80% the endonuclease reagent is degraded by 30 hours, preferably by 24, more preferably by 20 hours after transfection.

According to a preferred aspect of the invention, said endonuclease reagent is a nucleic acid encoding an âengineeredâ or âprogrammableâ rare-cutting endonuclease, such as a homing endonuclease as described for instance by Arnould S., et al. (WO2004067736), a zing finger nuclease (ZFN) as described, for instance, by Urnov F., et al. (Highly efficient endogenous human gene correction using designed zinc-finger nucleases (2005) Nature 435:646-651), a TALE-Nuclease as described, for instance, by Mussolino et al. (A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity (2011) Nucl. Acids Res. 39(21):9283-9293), or a MegaTAL nuclease as described, for instance by Boissel et al. (MegaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering (2013) Nucleic Acids Research 42 (4):2591-2601).

According to the invention, the endonuclease reagent is preferentially under RNA form to allow transient endonuclease activity of said reagent into the target cell and make the entire capsule biodegradable in-vivo. Even more preferably, the endonuclease reagent is under the form of a mRNA for the expression of the rare cutting endonuclease into the cells. The endonuclease under mRNA form is preferably synthetized with a cap to enhance its stability according to techniques well known in the art, as described, for instance, by Kore A. L., et al. (Locked nucleic acid (LNA)-modified dinucleotide mRNA cap analogue: synthesis, enzymatic incorporation, and utilization (2009) J Am Chem Soc. 131(18):6364-5).

Due to their higher specificity, TALE-nuclease have proven to be particularly appropriate for therapeutic applications, especially under heterodimeric formsâi.e. working by pairs with a ârightâ monomer (also referred to as â5â²â or âforwardâ) and âleftâ monomer (also referred to as â3â²â or âreverseâ) as reported for instance by Mussolino et al. (TALENÂ® facilitate targeted genome editing in human cells with high specificity and low cytotoxicity (2014) Nucl. Acids Res. 42(10): 6762-6773).

According to another embodiment, the endonuclease reagent is a RNA-guide to be used in conjunction with a RNA guided endonuclease, such as Cas9 or Cpf1, as per, inter alia, the teaching by Doudna, J., and Chapentier, E., (The new frontier of genome engineering with CRISPR-Cas9 (2014) Science 346 (6213):1077), which is incorporated herein by reference.

However, because engineered rare-cutting endonuclease are sequence specific unique reagents, it cannot be excluded that, in some instances, they can promote or induce some chromosomal rearrangements, especially when several gene loci need to be cleaved in the same cell.

Rearrangements are more prompt to happen, for instance, when multiple cleavage sites get simultaneously cut on the same chromosome or when pseudo cleavage sites appear from the unexpected combinations of heterodimers not initially designed to work together.

The inventors have more particularly sought for lowering this risk without reducing the yield of the engineered cells when treated with any of the above endonuclease reagents (i.e. maintaining high gene KO efficacy and high cellular viability).

Sequential Steps to Produce Batches of Engineered Primary Immune Cells of Therapeutic Grade

The present invention thus provides a method allowing stacking gene editing in mammalian cells, while preventing undesirable genome deletions or translocations.

By âgene editingâ is meant, throughout the present specification, any methods by which a genomic sequence is modified by insertion, deletion or replacement at a selected locus by using at least an enzyme that cleaves phosphodiester bond within a polynucleotide chain.

The method of the present invention can be associated with other methods involving physical of genetic transformations, such as a viral transduction or transfection using nanoparticles, in particular for transient expression of exogenous genetic sequences.

The invention can be applied to human immune cells, in particular activated T-cells by alternating transfection (preferably by electroporation) and culture steps. Examples of such cycles as delineated below as examples:

(TNxâhâTNx)Ãn (TNx/TNxâhâTNx)Ãn (TNxâhâTNx/TNx)Ãn (TNx/TNxâhâTNx/TNx)Ãn

where TNx is a Transfection (T) of a specific endonuclease reagent N, (xâ¥1)

h an interval of time in hours, and (hâ¥1)

n is the number of subsequent transfection (nâ¥1)

The above cycle may be combined with each other depending on the number of the different endonuclease reagents to be used sequentially.

Preferably, each gene editing step targets one locus at a time.

On another hand, the use of sequence-specific endonuclease reagents can be combined with other types of cell transformation not involving sequence specific endonuclease reagents, such as a retroviral transduction. This is of particular interest, for instance, for the production of primary immune cells expressing a recombinant receptors, such as a chimeric antigen receptor (CAR) or a recombinant TCR. Such chimeric antigen receptors are generally encoded by exogenous sequences which are introduced into cells by means of viral vectors, in particular lentiviral vectors. It is therefore advantageous to combine the sequential gene editing steps of the present invention with viral transduction steps, such as illustrated in FIGS. 3 , 5 , 6 and 7 .

Example of combinations of sequential gene editing steps and transduction steps are delineated below:

Transductionâhâ(TNxâhâTNx)Ãn; Transductionâhâ(TNx/TNxâhâTNx)Ãn; Transductionâhâ(TNxâhâTNx/TNx)Ãn; Transductionâhâ(TNx/TNxâhâTNx/TNx)Ãn; (TNxâhâTNx)ÃnâhâTransduction)Ãn; (TNx/TNxâhâTNx)ÃnâhâTransduction)Ãn; (TNxâhâTNx/TNx)ÃnâhâTransduction; (TNx/TNxâhâTNx/TNx)ÃnâhâTransduction; (TNxâhâTransductionâhâTNx)Ãn; (TNx/TNxâhâTransductionâhâTNx)Ãn; (TNxâhâTransductionâhâTNx/TNx)Ãn; (TNx/TNxâhâTransductionâhâTNx/TNx)Ãn;

The transduction step may also take place prior to a gene editing step, when, for instance, vector introduces a template DNA into the primary cell to be integrated at a locus during the subsequent gene editing step. The sequence-specific endonuclease reagent is then introduced into the cell to promote the integration of said exogenous DNA at said locus. One preferred aspect is a method of the present invention comprising a first gene editing step, followed by a transduction step involving an AAV vector comprising an exogenous sequence to be integrated at a predetermined locus, prior to a second gene editing step wherein the exogenous DNA comprised in the AAV vector is integrated at said predetermined locus.

According to one aspect of the invention, the sequential gene editing method may combine single gene editing steps (TNx) with multiplexing gene editing steps (TNx/TNx). During the multiplexing gene editing step, different endonuclease reagents may be used at different/multiple loci together. Endonucleases have different types of cleavage signatures: some of them can create âblunt endsâ, such as CRISPR, 5â² âcohesive endsâ such as zing Finger nucleases or TALE-nucleases or 3â² âcohesive endsâ by using homing endonucleases. Combinations of endonuclease reagents can be made based on the type of cleavage sought. For instance, cohesive ends are better suited for integration of exogenous DNA than blunt ends for instance. One preferred aspect of the invention is the combined use of endonuclease reagents creating different types of cleavage signatures to reduce the deletions or translocations occurring at/or between the different gene-edited loci.

According to a preferred embodiment of the invention, the primary immune cells are cultivated for an interval of time (h) as referred to above that is more than 10 hours, preferably from 12 to 72 hours and more preferably from 12 to 48 hours.

According to a particular embodiment of the invention, a purification step can be performed between general step b) and c), as also illustrated in FIGS. 4 , 7 and 13 .

This purification step can be performed for the sake of purity by any standard method known in the art. In the present case, the purification step can help to select the cells which have undergone the gene editing achieved in step a). The purification can thus rely on the product resulting from the first gene editing reaction, such as a product resulting from the modification or insertion of a genetic sequence at said first locus, or the absence of a gene product in case of a deletion of such genetic sequence. In a preferred embodiment, the first gene editing step may help the expression of receptors or membrane proteins, which make the immune cells more receptive to the second gene editing step. The cells can also become more receptive to viral vectors transduction if genes involved into viral transformation are modified prior to the second gene editing.

The genes that can be targeted as part of the first gene editing step, can be genes the modification of which will facilitate viral transduction or the realization of the second gene editing step or of any subsequent transduction or transfection step. Such genes can be, for instance, genes encoding cell restriction factors, such as TRIM5a (Uniprot Q9C035), APOBEC protein family (apolipoprotein B mRNA editing enzyme), and SAMHD1 (Uniprot Q9Y3Z3). By âcell restriction factorsâ is meant molecules that directly and dominantly cause a significant decrease in viral infectivity. TRIM5a, for instance, is a protein known to mediate/inhibit lentiviral, such as HIV, entry into immune cells (Stremlau M, et al. âSpecific recognition and accelerated uncoating of retroviral capsids by the TRIM5alpha restriction factorâ (2006) PNAS 103(14): 5514-9). The inactivation of TRIM5a, SAMHD1 or proteins of APOBEC family as part of one of the gene editing steps of the present invention can increase the infectivity of primary cells to viral vectors during subsequent transduction steps.

According to preferred embodiments, the invention provides that steps a) to d) of the general method are performed within 240 hours, preferably within 120 hours, more preferably within 96 hours, even more preferably within 72 hours. This limited period of time allows better recovery of the primary immune cells and limits their exhaustion. Limited exhaustion can be controlled at different steps during the process by one skilled in the art by using specific exhaustion markers such as reviewed by Wherry, J. A. (T cell exhaustion (2011) Nature Immunology 12:492-499).

According to a preferred embodiment of the invention, the transfection step T, such as steps a) and c) in the general method previously described, as well as any further steps TN (where N is the number of gene editing steps) are performed by electroporation.

Surprisingly, successive electroporation steps were found to be less destructive and/or less genotoxic for the primary cells than any other methods performing multiplex gene editing where various endonuclease reagents are transfected in one shot.

The method according to the present invention can comprise at least one further step of submitting the primary immune cell to a third electroporation to introduce at least a third sequence-specific reagent into said cell.

Such electroporation steps are typically performed in closed chambers comprising parallel plate electrodes producing a pulse electric field between said parallel plate electrodes greater than 100 volts/cm and less than 5,000 volts/cm, substantially uniform throughout the treatment volume such as described in WO/2004/083379, which is incorporated by reference, especially from page 23, line 25 to page 29, line 11. One such electroporation chamber preferably has a geometric factor (cm â1 ) defined by the quotient of the electrode gap squared (cm2) divided by the chamber volume (cm 3 ), wherein the geometric factor is less than or equal to 0.1

CLAIMS

Claims ( 17 )

We claim:

1. A pair of PD-1-targeting TALE nuclease (TALEN) monomers comprising:

1) A TALEN monomer comprising the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 9; and

2) A TALEN monomer comprising the amino acid sequence of SEQ ID NO: 6.

2. The pair of PD-1-targeting TALEN monomers of claim 1 , comprising:

1) A TALEN monomer comprising the amino acid sequence of SEQ ID NO: 5; and

2) a TALEN monomer comprising the amino acid sequence of SEQ ID NO: 6.

3. The pair of PD-1-targeting TALEN monomers of claim 1 , comprising:

1) A TALEN monomer comprising the amino acid sequence of SEQ ID NO: 9; and

2) a TALEN monomer comprising the amino acid sequence of SEQ ID NO: 6.

4. A method of gene-editing primary immune cells by transiently expressing a rare-cutting sequence-specific endonuclease reagent that is a PD-1-targeting TALEN in the primary immune cells, wherein the PD-1-targeting TALEN comprises the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 9.

5. The method of claim 4 , wherein the PD-1-targeting TALEN comprises the amino acid sequence of SEQ ID NO: 5.

6. The method of claim 4 , wherein the PD-1-targeting TALEN comprises the amino acid sequence of SEQ ID NO: 6.

7. The method of claim 4 , wherein the PD-1-targeting TALEN comprises the amino acid sequence of SEQ ID NO: 9.

8. The method of claim 4 , comprising transiently expressing a pair of PD-1-targeting TALEN monomers in the primary immune cells.

9. The method of claim 8 , wherein the pair of PD-1-targeting TALEN monomers comprise a TALEN monomer having the amino acid sequence of SEQ ID NO: 5 and a TALEN monomer having the amino acid sequence of SEQ ID NO: 6.

10. The method of claim 8 , wherein the pair of PD-1-targeting TALEN monomers comprise a TALEN monomer having the amino acid sequence of SEQ ID NO: 9 and a TALEN monomer having the amino acid sequence of SEQ ID NO: 6.

11. The method of claim 8 , wherein the pair of PD-1-targeting TALEN monomers are transiently expressed in the primary immune cells by electroporation of the primary immune cells with mRNA encoding the TALEN monomers.

12. The method of claim 4 , wherein the primary immune cells are T cells.

13. The method of claim 4 , wherein the primary immune cells are NK cells.

14. The method of claim 4 , wherein the primary immune cells are B cells.

15. The method of claim 4 , wherein the primary immune cells are obtained from a tumor.

16. The method of claim 15 , wherein the primary immune cells are tumor infiltrating lymphocytes (TILs).

17. The method of claim 4 , further comprising expanding the gene-edited primary immune cells.

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2018-01-11

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EP3481861A1

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2019-05-15

US20230287454A1

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AU2017291851A1

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2019-02-07

CA3029761A1

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2020-07-02

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2023-03-23

JP2019520076A

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2019-07-18

Sequential gene editing in primary immune cells — Cellectis (US11674155B2)

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