In vitro validation methods for cd19-targeting cell therapies

Abstract

The invention includes improved methods and compositions for detecting specific insertion of a chimeric antigen receptor (CAR) sequence in the desired locus in the genome of a cell. Methods are useful as quality control for CAR-T cell or CAR-NK cell preparations.

Description

IN VITRO VALIDATION METHODS FOR CD19-TARGETING CELL THERAPIESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. provisional application serial no. 63 / 515,757 filed on 26 July 2023.STATEMENT REGARDING GOVERNMENT-SPONSORED RESEARCH

[0002] None.SEQUENCE LISTING

[0003] The 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 July 17, 2024, is named “CBI052.30.xml” and is 9,858 bytes in size The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety.FIELD OF THE INVENTION

[0004] The invention related to the field of cell therapy. More specifically, the invention related to a method of validating genetically engineered immune cells used in cell therapies by detecting and quantifying chimeric antigen receptor (CAR) gene insertion in the cells.BACKGROUND OF THE INVENTION

[0005] The promise of adoptive cell therapy is to deliver engineered immune cells that will repeatedly seek and destroy tumor cells or other harmful or diseased cells in the patient’s body. The innate cytotoxic potential persists through the life of the cell and its progeny, making adoptive immune cells a “living medicine.”

[0006] The state-of-the-art adoptive immune cells are armed with chimeric antigen receptors (CARs) inserted into the cellular genome. First attempts at inserting CARS used randomly integrating vectors such as retroviral vectors. Much more precise method of genome editing utilized the CRISPR / Cas system. Gene editing with CRISPR nucleases is more efficient and moreprecise making a much safer choice. Nevertheless, the efficiency of CRISPR / Cas endonucleases in human cells is less than 100%. Many cells in the edited population lack one or more of the desired genome edits. A recent study has reported that using CRISPR / Cas9 to insert an anti-CD19 CAR into the T cell receptor alpha (TRAC) locus resulted in 70% efficiency of the gene knockout and 40% efficiency of the gene knock-in, so that just over 50% of the cells have acquired the desired double modification. (Eyquem el al., (2017), Targeting CAR to the TRAC locus with CRTSPR / Cas9 enhances tumour rejection, Nature 543(7643): 113.)

[0007] There is a need for reproducible methods of validating engineered immune cell preparations to ensure safety and potency of the preparations prior to them being administered to the patients.SUMMARY OF THE INVENTION

[0008] In one embodiment, the invention is a method of detecting the presence of a chimeric antigen receptor (CAR)-expressing nucleic acid construct at an integration site in a genome of an engineered cell, the method comprising: contacting a sample comprising nucleic acids from one or more engineered cells with amplification primers, wherein the first amplification primer is capable of hybridizing to a sequence present in the genome of wild type cells adjacent to the integration site, and the second amplification primer is capable of hybridizing to a sequence in the CAR-expressing nucleic acid construct, and a polymerase, and performing an amplification by the polymerase chain reaction (PCR) to produce an amplicon, wherein the presence or absence of the amplicon is indicative of the presence or absence of the integrated CAR-expressing nucleic acid construct at the integration site. In some embodiments, the presence or absence of the amplicon is detected by detecting fluorescence of a labeled probe. In some embodiments, the CAR-expressing nucleic acid construct comprises in the following order, a promoter, a CAR-encoding sequence, and a polyadenylation signal. In some embodiments, the integration site is in the T cell receptor alpha constant (TRAC) gene. In some embodiments, the CAR is an anti-CD19 CAR. In some embodiments, the first amplification primer consists essentially of SEQ ID NO: 2, the second amplification primer consists essentially of SEQ ID NO: 1, and the probe consists essentially of SEQ ID NO: 3. In some embodiments, the first amplification primer consists essentially of SEQ ID NO: 5, the second amplification primer consists essentially of SEQ ID NO: 4, and the probe consists essentially of SEQ ID NO: 6. In some embodiments, the method further comprisesamplifying a control nucleic acid sequence present in the genome of wild type cells and in the genome of engineered cells and detecting the presence of the control nucleic acid with a control probe. In some embodiments, the method further comprises comparing the fluorescence of the probe detecting the CAR-expressing nucleic acid and the fluorescence of the probe detecting the control nucleic acid, thereby determining copy number of the CAR-expressing nucleic acid in the engineered cell. In some embodiments, the CAR-expressing nucleic acid has been introduced into the engineered cell with the use of an AAV vector, the method further comprising amplifying a nucleic acid sequence present in the AAV vector and detecting the presence of the AAV vector. In some embodiments, the nucleic acid sequence present in the AAV vector is amplified with a first primer capable of hybridizing to the inverted terminal repeat (ITR) of the AAV, and a second primer, and the presence of the AAV is detected with a fluorescently labeled probe. In some embodiments, the first primer consists essentially of SEQ ID NO: 7 and the second primer is selected from the sequence consisting essentially of SEQ ID NO: 1 and the sequence consisting essentially of SEQ ID NO: 4. In some embodiments, the method further comprises comparing the fluorescence of the probe detecting the nucleic acid sequence present in the AAV vector and the fluorescence of the probe detecting the control nucleic acid, thereby determining copy number of the AAV in the sample. In some embodiments, the PCR is digital PCR.

[0009] In one embodiment, the invention is a reaction mixture for detecting the presence of chimeric antigen receptor (CAR)-expressing nucleic acid construct at an integration site in a genome of an engineered cell, the reaction mixture comprising: nucleic acids from one or more engineered cells, a first amplification primer capable of hybridizing to a sequence present in the genome of wild type cells adjacent to the integration site, and a second amplification primer capable of hybridizing to a sequence in the CAR-expressing nucleic acid construct. In some embodiments, the first amplification primer consists essentially of SEQ ID NO: 2 and the second amplification primer consists essentially of SEQ ID NO: 1. In some embodiments, the first amplification primer consists essentially of SEQ ID NO: 5 and the second amplification primer consists essentially of SEQ ID NO: 4. In some embodiments, the reaction mixture further comprises one or more labeled probe selected from a sequence consisting essentially of SEQ ID NO: 3 and a sequence consisting essentially of SEQ ID NO: 6. In some embodiments, the reaction mixture further comprises a first and second control amplification primers capable of amplifying a control nucleic acid sequence present in the genome of wild type cells and in the genome ofengineered cells, and a control probe capable detecting amplification of the control nucleic acid. In some embodiments, the reaction mixture further comprises a primer capable of amplifying an AAV sequence, the primer consisting essentially of SEQ ID NO: 7.

[0010] In one embodiment, the invention is a kit for detecting the presence of chimeric antigen receptor (CAR)-expressing nucleic acid construct at an integration site in a genome of an engineered cell, the reaction mixture comprising: a first amplification primer capable of hybridizing to a sequence present in the genome of wild type cells adjacent to the integration site, and a second amplification primer capable of hybridizing to a sequence in the CAR-expressing nucleic acid construct. In some embodiments, the first amplification primer consists essentially of SEQ ID NO: 2 and the second amplification primer consists essentially of SEQ ID NO: 1. In some embodiments, the first amplification primer consists essentially of SEQ ID NO: 5 and the second amplification primer consists essentially of SEQ ID NO: 4. In some embodiments, the kit further comprises one or more labeled probe selected from a sequence consisting essentially of SEQ ID NO: 3 and a sequence consisting essentially of SEQ ID NO: 6. In some embodiments, the kit further comprises a first and second control amplification primers capable of amplifying a control nucleic acid sequence present in the genome of wild type cells and in the genome of engineered cells, and a control probe capable detecting amplification of the control nucleic acid. In some embodiments, the kit further comprises a primer capable of amplifying an AAV sequence, the primer consisting essentially of SEQ ID NO: 7. In some embodiments, the kit further comprises nucleoside triphosphates, a thermostable nucleic acid polymerase and buffers and cofactors necessary to sustain enzymatic activity of the polymerase. In some embodiments, the kit further comprises reagents for preparing sequencing libraries and sequencing nucleic acids.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIGURE 1 shows design of the assay for detecting insertion of the anti-CD19 CAR into the TRAC locus.

[0012] FIGURE 2 shows design of the assay for detecting residual viral vector constructs in the cell preparation.DETAILED DESCRIPTION OF THE INVENITON

[0013] Definitions

[0014] The following definitions aid in understanding of this disclosure.

[0015] The term “adoptive cell” refers to a cell that can be genetically modified for use in a cell therapy treatment. Examples of adoptive cells include T-cells, macrophages, and natural killer (NK) cells.

[0016] The term “cell therapy” refers to the treatment of a disease or disorder that utilizes genetically modified cells. The term “adoptive cell therapy (ACT)” refers to a therapy that uses genetically modified adoptive cells. Examples of ACT include T-cell therapies, CAR-T cell therapies, natural killer (NK) cell therapies and CAR-NK cell therapies.

[0017] The term “lymphocyte” refers to a leukocyte that is part of the vertebrate immune system. Lymphocytes include T-cells such as CD4+or CD8+T-cells, alpha / beta T-cells, gamma / delta T-cells, and regulatory T-cells. Lymphocytes also include natural killer (NK) cells, natural killer T (NKT) cells, cytokine induced killer (CIK) cells, and antigen presenting cells (APCs), such as dendritic cells. Lymphocytes also include tumor infiltrating lymphocytes (TILs).

[0018] The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologues, SNPs, and complementary sequences as well as the sequence explicitly indicated.

[0019] The term “modified nucleotide” is used herein to describe a nucleotide in DNA with a base other than the four conventional DNA bases consisting of adenosine, guanosine, thymidine and cytosine. Deoxyuracil (dU) and deoxyinosine (di) are modified nucleotides in DNA. Modified nucleotides may comprise modifications of the sugar moiety. In DNA, modified nucleotides comprise a sugar moiety other that deoxyribose. For example, ribonucleotides are considered “modified nucleotides” in DNA. Modified nucleotides may comprise modified base moieties. C5- methyl-dC, a C5-ethyl-dC, a C5-methyl-dU, a C5-ethyl-dU, a 2,6-diaminopurine, a C5-propynyl- dC, a C5-propynyl-dU, a C7-propynyl-dA, a C7-propynyl-dG, a C5-propargylamino-dC, a C5-propargylamino-dU, a C7-propargylamino-dA, a C7-propargylamino-dG, a 7-deaza-2- deoxyxanthosine, a pyrazolopyrimidine analog, a pseudo-dU, a nitro pyrrole, a nitro indole, 2'-0- methyl Ribo-U, 2'-0-methyl Ribo-C, an N4-ethyl-dC, an N6-methyl-dA, N2 benzyl purines and the like are useful modified nucleotides in amplification primers that reduce non-specific nucleic acid modification and formation of primer dimers, see U.S. Pat. No. 6,001,611.

[0020] The term “primer” refers to an oligonucleotide which binds to a specific region of a single-stranded template nucleic acid molecule and initiates nucleic acid synthesis via a polymerase-mediated enzymatic reaction. Typically, a primer comprises fewer than about 100 nucleotides and preferably comprises fewer than about 30 nucleotides. A target-specific primer specifically hybridizes to a target polynucleotide under hybridization conditions. Such hybridization conditions can include, but are not limited to, hybridization in isothermal amplification buffer (20 mM Tris-HCl, 10 mM (NH4)2SO4), 50 mM KC1, 2 mM MgSO4, 0.1% TWEEN® 20, pH 8.8 at 25 °C) at a temperature of about 40 °C to about 70 °C. Depending on the length of the primer, perfect complementarity is not required for the primer to specifically bind to the target sequence under hybridization conditions. The term “substantially complementary refers to the degree of complementarity sufficient to achieve specific hybridization under hybridization conditions. In addition to the target-binding region, a primer may have one or more additional regions, typically in the 5’-poriton. The additional regions may include a universal primer binding site or a barcode. The presence of the additional regions in the 5 ’-portion does not negatively affect the ability of the primer to hybridize to its intended target under hybridization conditions.

[0021] The term “amplification conditions” refers to conditions in a nucleic acid amplification reaction (e.g., PCR amplification) that allow for hybridization and template-dependent extension of the primers. The terms “amplicon” and “amplification product” refer to a nucleic acid molecule that contains all or a fragment of the target nucleic acid sequence and that is formed as the product of in vitro amplification by any suitable amplification method.

[0022] The term “universal primer” refers to a primer that can hybridize to a universal primer binding site. Universal primer binding sites can be natural or artificial sequences typically added to a target sequence in a non-target-specific manner.

[0023] The term “barcode” refers to a nucleic acid sequence that can be detected and identified. Barcodes can generally be 2 or more and up to about 50 nucleotides long. Barcodes are designed to have at least a minimum number of differences from other barcodes in a population. Barcodescan be unique to each molecule in a sample or unique to the sample and be shared by multiple molecules in the sample. The term “multiplex identifier,” “MID” or “sample barcode” refer to a barcode that identifies a sample or a source of the sample. As such, all or substantially all, MID barcoded polynucleotides from a single source or sample will share an MID of the same sequence; while all, or substantially all (e.g., at least 90% or 99%), MID barcoded polynucleotides from different sources or samples will have a different MID barcode sequence. Polynucleotides from different sources having different MIDs can be mixed and sequenced in parallel while maintaining the sample information encoded in the MID barcode. The term “unique molecular identifier” or “UID,” refer to a barcode that identifies a polynucleotide to which it is attached. Typically, all, or substantially all e.g., at least 90% or 99%), UID barcodes in a mixture of UID barcoded polynucleotides are unique.

[0024] The invention involves detecting specific genetic modifications in the genome of a cell. More precisely, the invention is a method of assessing successful genome engineering of an immune cell such as a T cell or an NK cell into a CAR-T cell or a CAR-NK cell. A CAR-T cell and a CAR-NK cell include a chimeric antigen receptor (CAR) expression construct stably inserted into the genome of the cell. In some embodiments, the CAR expression construct is inserted into a gene whose function is thereby abolished. Such a genome editing strategy comprises a gene knock-out followed by (or simultaneous with) a gene knock-in and therefore constitutes double genome editing.

[0025] The innate diversity of DNA repair pathways in mammalian cells results in less than 100% efficiency of any genome editing. Genome editing with CRSIPR / Cas9 nucleases yields less than 100% of perfectly edited cells in any edited cell population. The DNA repair pathway mostly involved in repair of double strand break (DSB) (such as the ones introduced by the CRISPR-Cas nucleases during genome editing) is highly accurate homologous recombination (HR). HR results in precise insertion of the exogenous sequence at the desired site in the cellular genome. However, less accurate pathways of non-homologous end joining (NHEJ) and micro-homology-mediated end joining (MME J) are also involved (see Xue and Greene, (2021) DNA repair pathway choices in CRISPR-Cas9 mediated genome editing, Trends Genet. 37:639.). These error-prone pathways result in deletions or imperfect integrations of donor sequences.

[0026] Because of innate unpredictability of DNA repair in living cells, engineered immune cell preparations (such as CAR-T cells or CAR-NK cell preparations) require quality controlprocedures before they can be used in the clinic. Indirect ways of assessing success of genome engineering include protein-specific assays that confirm expression and activity of the exogenous protein in the engineered cell. Such assays include flow cytometry and functional assays. Such assays are time consuming and require the sacrifice of a large number of engineered cells. To address this problem, the inventors have devised a method of using a small aliquot of the cell preparation to rapidly detect not only the presence of the exogenous chimeric antigen receptor (CAR) expression construct but also its location to the desired locus in the cellular genome. In some embodiments, the invention is a method of detecting the presence of the chimeric antigen receptor (CAR) construct nucleic acid inserted in the T cell receptor alpha constant (TRAC) locus in the genome of a mammalian cell. The method can be applied as a validation or a quality control measure for a preparation of engineered cells (e.g., CAR-T cells or CAR-NK cells) prior to administering the cells to a patient. The method can also be applied to engineered cells (e.g., CAR- T cells or CAR-NK cells) isolated from a recipient patient as part of monitoring the patient’s response to therapy with the engineered CAR-T cells or CAR-NK cells. The method is applicable to autologous CAR-T or CAR-NK cell populations (immune cells derived from a patient and upon editing, reinfused into the patient). The method is also applicable to allogeneic CAR-T or CAR- NK cell populations (off-the-shelf preparations of immune cells derived from healthy donors and made ready for infusion into any patient).

[0027] In broad terms, the method comprises the steps of detecting the presence of the CAR expression construct nucleic acid in the TRAC locus of the edited cells. More specifically, the method comprises the use of primers and probes specific to the sequences in the TRAC gene and to the terminal regions of the inserted CAR expression construct nucleic acid, such as the polyadenylation signal or the promoter region of the expression construct. In some embodiments, the method further comprises quantifying the number of the insert-specific nucleic acid sequences in each cell. In some embodiments, the quantifying is in reference to one or more reference (endogenous) sequences in the cellular genome. In some embodiments, the method further comprises a step of comparing the number of endogenous sequences to the number of insertionspecific sequences to determine the number of times the exogenous sequence has been inserted into the cellular genome. In some embodiments, the method further comprises detection and quantification residual vector construct, such as viral construct (comprising the viral vector and the CAR expression construct) present in the preparation of engineered cells.

[0028] The invention comprises the use of human or mammalian cells such as immune cells that can be useful in immunotherapy. The immune cells of the instant invention include T cells, CAR-T cells, natural killer (NK) cells, induced natural killer (iNK) cells, macrophages and their engineered derivatives such as CAR-NK cells, CAR- NK cells and the like.

[0029] In some embodiments, the cells analyzed by the method of the invention are present in culture, i.e., maintained in a growth medium outside of the human body.

[0030] In some embodiments, the cells are present in a therapeutic composition that also comprises a suitable excipient comprising one or more of carbohydrates, inorganic salts, antimicrobial agents, antioxidants, surfactants, buffers, acids, bases, water, alcohols, polyols, glycerin, vegetable oils, phospholipids, surfactants, sugars, and derivatized sugars.

[0031] In some embodiments, the cells are retrieved from a patient who has previously received an infusion of engineered immune cells as part of the treatment regimen. In some embodiments, the cells are present among lymphocytes that are isolated from blood, including peripheral blood and cord blood, or from lymphoid organs such as the thymus, bone marrow, lymph nodes, and mucosal-associated lymphoid tissues (MALT). Techniques for isolating lymphocytes from such tissues are well known in the art, see, e.g., Smith, J.W. (1997) Apheresis techniques and cellular immunomodulation, Ther. Apher. 1 :203-206.

[0032] In some embodiments, the isolated lymphocytes are characterized in terms of specificity, frequency, and function. In some embodiments, the isolated lymphocyte population is enriched for specific subsets of cells, such as T cells or NK cells.

[0033] In some embodiments, the isolated lymphocyte population is enriched for specific subsets of T cells, such as CD4+, CD8+, CD25+, or CD62L+. See, e.g., Wang et al., Mol. Therapy - Oncolytics (2016) 3: 16015. In some embodiments, the isolated lymphocyte population is enriched for CD56+phenotype representing NK cells.

[0034] In some embodiments the CAR-T cells or CAR-NK cells may be isolated from a patient’s solid tissue, or a solid tumor. A solid tumor sample may be obtained by biopsy. The bodily fluids other than blood may also comprise the CAR-T cells, CAR-NK cells or nucleic acids (cell-free DNA) derived from such cells, e.g., urine, sputum, blood serum, lymph, saliva, sputum, sweat, tear, cerebrospinal fluid, amniotic fluid, synovial fluid, pericardial fluid, peritoneal fluid, pleural fluid, cystic fluid, bile, gastric fluid, intestinal fluid, and fecal samples.

[0035] In some embodiments, the method of the invention is applied to cell -free DNA obtained from a patient who has been treated with engineered CAR-T and CAR-NK cells and whose blood may comprise cell-free DNA derived from the CAR-T or CAR-NK cells and is therefore informative on the genetic characteristic of such cells.

[0036] The invention involves manipulating isolated nucleic acids that have been isolated or extracted from a sample. Methods of nucleic acid extraction are well known in the art. See J. Sambrook et al., Molecular Cloning: A Laboratory Manual , 1989, 2nd Ed., Cold Spring Harbor Laboratory Press: New York, N.Y.). A variety of kits are commercially available for extracting nucleic acids (DNA or RNA) from biological samples (e.g., KAPA Express Extract (Roche Sequencing Solutions, Pleasanton, Cal ), BD Biosciences (Palo Alto, Cal ), Epicentre Technologies (Madison, Wise.); Gentra Systems, (Minneapolis, Minn.); Qiagen (Valencia, Cal.), Ambion (Austin, Tex.); BioRad Laboratories (Hercules, Cal.), and more.

[0037] In some embodiments, the specific genetic modification of a cellular genome to be detected by the method described herein involves inserting an exogenous protein expression construct into a precise location within the cellular genome. In some embodiments, the exogenous expression construct encodes a chimeric antigen receptor (CAR) described herein. The CAR expression construct is introduced into a cell such as lymphocyte including a T-cell, or a T-cell precursor, or an NK cell or an NK cell precursor. In some embodiments, “naked” nucleic acids comprising exogenous protein expression construct are introduced into the cell by electroporation as described e.g., in U.S. Patent No. 6,410,319.

[0038] In some embodiments, the CAR-encoding nucleic acid is introduced into the cell via electroporation. In some embodiments, the CAR-encoding nucleic acid is introduced into the cell via electroporation of naked DNA. In some embodiments, the CAR-encoding nucleic is introduced into the cell via a vector. In some embodiments, the vector is a viral vector derived from a virus selected from the group consisting of an adenovirus type 2 and an adenovirus type 5, a retrovirus, a lentivirus, an adeno-associated virus (AAV), a simian virus 40 (SV-40), vaccinia virus, Sendai virus, Epstein-Barr virus (EBV), and herpes simplex virus (HSV). In some embodiments, the virus is an AAV virus and the AAV virus incorporates the CAR nucleic acid sequence having all the elements necessary for transcription and translation of the CAR expression construct flanked by homology arms homologous to the sequence of the desired insertion site in the cellular genome. In some embodiments, each homology arm comprises 1000-100 base pairs or 100-500 base pairsor about 500 base pairs of sequence cabaple of hybridizing to the sequence in the cellular genome where insertion of the CAR gene is desired.

[0039] In some embodiments, one or more of the expression constructs described herein are inserted into the genome of the cell with the aid of a sequence-specific endonuclease.

[0040] In some embodiments, the endonuclease is a nucleic acid-guided endonuclease encoded by the CRISPR locus. The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) genomic locus is found many prokaryotic genomes and provides resistance to invasion of foreign nucleic acids. Structure, nomenclature and classification of CRISPR loci are reviewed in Makarova et al., Evolution and classification of the CRISPR-Cas systems. Nature Reviews Microbiology. 2011 June; 9(6): 467-477.

[0041] Briefly, a typical CRISPR locus includes a number of short repeats regularly interspaced with spacers. The CRISPR locus also includes coding sequences for CRISPR- associated (Cas) genes. A spacer-repeat sequence unit encodes a CRISPR RNA (crRNA). In vivo, a mature crRNAs are processed from a polycistronic transcript referred to as pre-crRNA or pre- crRNA array. The repeats in the pre-crRNA array are recognized by Cas-encoded proteins that bind to and cleave the repeats liberating mature crRNAs. CRISPR systems perform cleavage of a target nucleic acid wherein Cas proteins and crRNA form a CRISPR ribonucleoproteins (crRNP). The crRNA molecule guides the crRNP to the target nucleic acid (e.g., a foreign nucleic acid invading a bacterial cell) and the Cas nuclease proteins cleave the target nucleic acid.

[0042] Type I CRISPR systems include means for processing the pre-crRNA array that include a multi-protein complex called CASCADE (CRISPR-associated complex for antiviral defense) comprised of subunits CasA, B, C, D and E. The Cascade-crRNA complex recognizes the target nucleic acid through hybridization of the target nucleic acid with crRNA. The bound nucleoprotein complex recruits the Cas3 helicase / nuclease to facilitate cleavage of target nucleic acid.

[0043] Type II CRISPR systems include a trans-activating CRISPR RNA (tracrRNA). The tracrRNA hybridizes to a crRNA repeat in the pre-crRNA array and recruits endogenous RNaselll to cleave the pre-crRNA array. The tracrRNA / crRNA complex can associate with a nuclease, e.g., Cas9. The crRNA-tracrRNA-Cas9 complex recognizes the target nucleic acid through hybridization of the target nucleic acid with crRNA. Hybridization of the crRNA to the target nucleic acid activates the Cas9 nuclease, for target nucleic acid cleavage.

[0044] Type III CRISPR systems include the RAMP superfamily of endoribonucleases (e.g., Cas6) that cleave the pre-crRNA array with the help of one or more CRISPR polymerase-like proteins.

[0045] Type VI CRISPR systems comprise a different set of Cas-like genes, including Csfl, Csf2, Csf3 and Csf4 which are distant homologues of Cas genes in Type I-III CRISPR systems.

[0046] Type V CRISPR systems are classified into several different subtypes, including, e.g., V-A, V-B, V-C, V-D, V-E, V-F, V-G, V-H, V-I, V-J, V-K and V-U. See, e.g., Makarova et al.Nat. Rev. Microbiol., 2020, 18:67-83) and Pausch etal. (Science, 2020, 369(6501):333-337). The V-A subtype encodes the Casl2a protein (formerly known as Cpfl). Casl2a has a RuvC-like nuclease domain that is homologous to the respective domain of Cas9 but lacks the HNH nuclease domain that is present in Cas9 proteins. Type V systems can comprise a single crRNA sufficient for targeting of the Cas 12 to a target site, or a crRNA-tracrRNA guide pair for targeting of the Cas 12 to a target site.

[0047] CRISPR endonucleases require a nucleic acid targeting nucleic acid (NATNA) also known as guide RNAs. The endonuclease is capable of forming a ribonucleoprotein complex (RNP) with one or more guide RNAs. In some embodiments, the endonuclease is a Type II CRISPR endonuclease and NATNA comprises tracrRNA and crRNA.

[0048] In some embodiments, NATNA is selected from the embodiments described in U.S. Patent No. 9,260,752. Briefly, a NATNA can comprise, in the order of 5' to 3', a spacer extension, a spacer, a minimum CRISPR repeat, a single guide connector, a minimum tracrRNA, a 3' tracrRNA sequence, and a tracrRNA extension. In some instances, a nucleic acid-targeting nucleic acid can comprise, a tracrRNA extension, a 3' tracrRNA sequence, a minimum tracrRNA, a single guide connector, a minimum CRISPR repeat, a spacer, and a spacer extension in any order.

[0049] In some embodiments, the guide nucleic acid-targeting nucleic acid can comprise a single guide NATNA. The NATNA comprises a spacer sequence which can be engineered to hybridize to the target nucleic acid sequence. The NATNA further comprises a CRISPR repeat comprising a sequence that can hybridize to a tracrRNA sequence. Optionally, NATNA can have a spacer extension and a tracrRNA extension. These elements can include elements that can contribute to stability of NATNA. The CRISPR repeat and the tracrRNA sequence can interact, to form a base-paired, double-stranded structure. The structure can facilitate binding of the endonuclease to the NATNA.

[0050] In some embodiments, the single guide NATNA comprises a spacer sequence located 5' of a first duplex which comprises a region of hybridization between a minimum CRISPR repeat and minimum tracrRNA sequence. The first duplex can be interrupted by a bulge. The bulge facilitates recruitment of the endonuclease to the NATNA. The bulge can be followed by a first stem comprising a linker connecting the minimum CRISPR repeat and the minimum tracrRNA sequence. The last paired nucleotide at the 3' end of the first duplex can be connected to a second linker connecting the first duplex to a mid-tracrRNA. The mid-tracrRNA can comprise one or more additional hairpins.

[0051] In some embodiments, the NATNA can comprise a double guide nucleic acid structure. The double guide NATNA comprises a spacer extension, a spacer, a minimum CRISPR repeat, a minimum tracrRNA sequence, a 3' tracrRNA sequence, and a tracrRNA extension. The double guide NATNA does not include the single guide connector. Instead, the minimum CRISPR repeat sequence comprises a 3' CRISPR repeat sequence and the minimum tracrRNA sequence comprises a 5' tracrRNA sequence and the double guide NATNAs can hybridize via the minimum CRISPR repeat and the minimum tracrRNA sequence.

[0052] In some embodiments, NATNA is an engineered guide RNA comprising one or more DNA residues (CRISPR hybrid RNA-DNA or chRDNA). In some embodiments, NATNA is selected from the embodiments described in U.S. Patent No. 9,650,617. Briefly, some chRDNA for use with a Type II CRISPR system may be composed of two strands forming a secondary structure that includes an activating region composed of an upper duplex region, a lower duplex region, a bulge, a targeting region, a nexus, and one or more hairpins. A nucleotide sequence immediately downstream of a targeting region may comprise various proportions of DNA and RNA. Other chRDNA may be a single guide D(R)NA for use with a Type II CRISPR system comprising a targeting region, and an activating region composed of and a lower duplex region, an upper duplex region, a fusion region, a bulge, a nexus, and one or more hairpins. A nucleotide sequence immediately downstream of a targeting region may comprise various proportions of DNA and RNA. For example, the targeting region may comprise DNA or a mixture of DNA and RNA, and an activating region may comprise RNA or a mixture of DNA and RNA.

[0053] In some embodiments, the endonuclease used to insert an exogenous protein expression construct into the genome of a cell is a restriction endonuclease, e.g., a Type II restriction endonuclease.

[0054] In some embodiments, the endonuclease used to insert an exogenous protein expression construct into the genome of a cell is a catalytically inactive CRISPR endonuclease (e.g., catalytically inactive Cas9 or Casl2a) conjugated to the cleavage domain of the restriction endonuclease Fok I. (see e.g., Guilinger, J. P., et al., (2014). Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification, Nature biotechnology, 32(6), 577-582.

[0055] In some embodiments the endonuclease used to insert an exogenous protein expression construct sequence into the genome of a cell is a zinc finger nuclease (ZFN), or a ZFN-Fok I fusion. In such embodiments, the target sequence is about 22-52 bases long and comprises a pair of ZFN recognition sequences, each 9-18 nucleotides long, separated by a spacer, which is 4-18 nucleotides long. (See e.g.., Kim Y.G., et al., (1996). Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain, Proc Natl Acad Sci USA. 93(3): 1156-1160.

[0056] In some embodiments, the endonuclease used to insert an exogenous protein expression construct into the genome of a cell is a transcription activator-like effector nuclease (TALEN), or a TALEN-Fok 1 fusion. In such embodiments, the target sequence is about 48-85 nucleotides long and comprises a pair of TALEN recognition sequences, each 18-30 bases long, separated by a spacer, which is 12-25 bases long. (See e.g., Christian M. et al., (2010) Targeting DNA doublestrand breaks with TAL effector nucleases, Genetics. 186 (2): 757-61 .

[0057] The instant invention is a method of detecting an exogenous chimeric antigen receptor (CAR) gene (or CAR-coding sequence) inserted into the genome of a cell as part of an expression construct. A typical CAR comprises an extracellular domain comprising an antigen binding region, a transmembrane domain and one or more intracellular domains such as6 activation domains and co- stimulatory domains. In some embodiments, the CAR also comprises a hinge domain. In some embodiments, the CAR also comprises a leader peptide directing the CAR to the cell membrane.

[0058] An exemplary CAR is shown in Figure 1. In Figure 1, “EFl” is the EFla promoter, “CD8a” is the CD8A leader sequence, “FMC63” is the anti-CD19 scFv, “CD8H” is a hinge domain, “CD8T” is a transmembrane domain, “4- IBB” is a co-stimulation domain, “CD3z” is the CD3zeta activation domain, “BGH pA” is a polyadenylation signal. The CAR expressing construct is flanked by sequences of exon 3 of the TRAC gene.

[0059] The CAR disclosed herein comprises an extracellular domain comprising an antigen binding region targeting CD 19. In some embodiments, the antigen-binding region comprises a well-studied single-chain variable region (scFv) FMC63 (Nicholson, et al., (1997) Construction and characterization of a functional CD 19 specific single chain Fv fragment for immunotherapy of B lineage leukaemia and lymphoma, Mol. Immunol., 34:1157).

[0060] In some embodiments, the CAR further comprises a hinge domain and the hinge domain is derived from CD8.

[0061] In some embodiments, the CAR further comprises a signal peptide (a signal sequence) that enables trafficking of the CAR to the cell membrane. In some embodiments, the signal sequence comprises a CD28 signal sequence. In some embodiments, the signal sequence consists essentially of a CD28 signal sequence.

[0062] In some embodiments, the CAR further comprises a transmembrane domain derived from a membrane-bound or transmembrane protein. In some embodiments, the transmembrane domain is the CD8ot transmembrane domain. In some embodiments, the transmembrane domain is the CD28 transmembrane domain.

[0063] In some embodiments, the CAR further comprises a cytoplasmic or intracellular signaling domain comprising one or more co-stimulatory domains or activation domains responsible for intracellular signaling leading to activation of one or more effector functions of the immune cell expressing the CAR. In some embodiments, the cytoplasmic domain of the CAR co.mprises a 4-1BB domains and a CD3(^ chain.

[0064] In some embodiments, the CAR-encoding nucleic acid comprises non-coding elements that facilitate mRNA transcription, mRNA maturation and mRNA translation resulting in synthesis of the CAR polypeptide. In some embodiments, such additional elements comprise a promoter, and a polyadenylation signal. In some embodiments, the promoter is an EFla promoter, and the polyadenylation signal is the BGH polyadenylation signal.

[0065] In some embodiments, the CAR-expression construct is inserted into the cellular genome into the endogenous T-cell receptor alpha chain (TRAC) gene. In some embodiments, the CAR is inserted into the TRAC locus on chromosome 14. In some embodiments, the CAR is inserted into exon 3 of the TRAC gene. In some embodiments, the CAR is inserted into approximately between nucleotides 22550579 and 22550601 (hg38). In some embodiments, the CAR is inserted into exon 3 of the TRAC gene on chromosome 14 approximately between nucleotides 22550579 and 22550601 (hg38).

[0066] In some embodiments, the CAR expression construct is introduced into the genome of the cell flanked by homology arms having sequence homology to the insertion site. In some embodiments, each homology arm is 100-1000 base pairs long. In some embodiments, the homology arm is 500 base pairs long. In some embodiments, the homology arms have sequence homology to exon 3 of the TRAC gene. In some embodiments, the homology arms have sequence capable of hybridizing to a sequence in exon 3 of the TRAC gene.

[0067] The present invention involves amplification by polymerase chain reaction (PCR, see U.S. 4,683,195). The methods and compositions for performing a polymerase chain reaction are described in PCR Strategies (Innis et al., 1995, Academic Press, San Diego, Calif.) at Chapter 14; or PCR Protocols: A Guide to Methods and Applications (Innis etal., Academic Press, N Y, 1990). The amplification utilizes an upstream primer and a downstream primer. In some embodiments, both primers are target-specific primers, i.e., primers comprising a sequence complementary to the target sequence as described herein below. The borders of a given amplicon are typically defined by the position of the complementary portion of the forward and reverse primers used for amplification. In some embodiments, one or more rounds of amplification utilize primer pairs wherein at least one primer is a universal primer. In some embodiments, universal primer binding sites are present in the 5 ’-portion (“tail”) of the target-specific primers used for prior rounds of amplification. Both universal primers and target-specific primers can have additional sequence elements in the 5 ’-tail. In some embodiments, the additional elements are utilized in optional downstream analysis steps such as capture or sequencing of the amplification products.

[0068] The methods and compositions of the invention involve a nucleic acid polymerase. Especially suitable for the polymerase chain reaction are thermostable polymerases that are stable to heat or heat-resistant and retain sufficient enzymatic activity when exposed to elevated temperatures for the time necessary to effect denaturation of double-stranded nucleic acids during PCR. In some embodiments, the following thermostable polymerases can be used: from Thermus species (e.g., T. flavus, T. ruber, T. thermophilus, T. lacteus, T. rubens, T. aquations), Bacillus stearothermophilus, Thermotoga maritima, Methanothermus fervidus, KOD polymerase, TNA1 polymerase, Thermococcus sp. 9 degrees N-7, T4, T7, phi29, Pyrococcus furiosus, P. abyssi, T. gorgonarius, T. litoralis, T. zilligii, T. sp. GT, P. sp. GB-D, KOD, Pfu, T. gorgonarius, T. zilligii, T. litoralis and Thermococcus sp. 9N-7 polymerases. Thermococcus litoralis (Vent, GenBank: AAA72101), Pyrococcus furiosus (Pfu, GenBank: D12983, BAA02362), Pyrococcus woesii,Pyrococcus GB-D (Deep Vent, GenBank: AAA67131), Thermococcus kodakaraensis KODI (KOD, GenBank: BD175553, BAA06142; Thermococcus sp. strain KOD (Pfx, GenBank: AAE68738)), Thermococcus gorgonarius (Tgo, Pdb: 4699806), Sul folobus solatari cus (GenBank: NC002754, P26811), Aeropyrum pernix (GenBank: BAA81109), Archaeglobus fulgidus (GenBank: 029753), Pyrobaculum aerophilum (GenBank: AAL63952), Pyrodictium occultum (GenBank: BAA07579, BAA07580), Thermococcus 9 degree Nm (GenBank: AAA88769, Q56366), Thermococcus fumicolans (GenBank: CAA93738, P74918), Thermococcus hydrotherm alis (GenBank: CAC 18555), Thermococcus sp. GE8 (GenBank: CAC 12850), Thermococcus sp. JDF-3 (GenBank: AX135456; WO0132887), Thermococcus sp. TY (GenBank: CAA73475), Pyrococcus abyssi (GenBank: P77916), Pyrococcus glycovorans (GenBank: CAC 12849), Pyrococcus horikoshii (GenBank: NP 143776), Pyrococcus sp. GE23 (GenBank: CAA90887), Pyrococcus sp. ST700 (GenBank: CAC 12847), Thermococcus pacificus (GenBank: AX411312.1), Thermococcus zilligii (GenBank: DQ3366890), Thermococcus aggregans, Thermococcus barossii, Thermococcus celer (GenBank: DD259850.1), Thermococcus profundus (GenBank: E14137), Thermococcus sicu (GenBank: DD259857.1), Thermococcus thioreducens, Thermococcus onnurineus NA1, Sulfolobus acidocaldarium, Sul folobus tokodaii, Pyrobaculum calidifontis, Pyrobaculum islandicum (GenBank: AAF27815), Methanococcus jannaschii (GenBank: Q58295), Desulforococcus species TOK, Desulfurococcus, Pyrolobus, Pyrodictium, Staphylothermus, Vulcanisaetta, Methanococcus (GenBank: P52025) and other archaeal B polymerases, such as GenBank AAC62712, P956901, BAAA07579)).

[0069] In some embodiments, the PCR used herein is qualitative, i.e., determines whether or not a target nucleic acid is present in a sample without quantifying the amount (or copy number) of starting target nucleic acid molecules in the sample. In some embodiments, the PCR used herein is quantitative, i.e., determines the presence and the amount (or copy number) of starting target nucleic acid molecules in the sample. In some embodiments, the PCR used herein is real-time PCR (rtPCR, see U.S. 5,994,056, and U.S. 5,210,015). This method utilizes fluorescent detection and reports the number of amplification cycles required to achieve a threshold amount of amplicon in a PCR mixture. In some embodiments, the fluorescent emission detected in the course of rtPCR results from incorporation of an intercalating dye into newly synthesized double-stranded DNA. In some embodiments, the fluorescent emission detected in the course of rtPCR results from cleavage of a fluorescently labeled probe by the 5 ’-3’ nuclease activity of the elongatingpolymerase, and the cleavage separates the donor fluorophore from an acceptor (quencher) fhiorophore allowing measurable fluorescence to occur.

[0070] In some embodiments, the PCR used herein is digital PCR. Digital PCR (dPCR) is a method comprising partitioning the sample into partitions, each partition becoming a micro reaction chamber where PCR takes place. In dPCR each partition contains one or zero target nucleic acid molecules. Each partition comprises amplification reagents including DNA polymerase, nucleoside triphosphates, co-factors (e.g., Mg2+or Mn2+), extendable primers, and optionally, labeled detection probes. The amplification reagents are added to the partitions (e.g., by combining sample droplets with reagent droplets) or mixed with the sample prior to partitioning of the formed reaction mixture into partitions. Digital PCR can be performed as end-point PCR so that partitions containing no amplicon represent partitions where no target nucleic acid was present, and partitions containing any detectable amount of amplicon represent partitions where the target nucleic acid was present. Counting the number or partitions in each category produces an estimate of the number or target nucleic acids in the original sample.

[0071] In some embodiments, the partitions are water-in-oil droplets. Such digital PCR is referred to as digital droplet PCR or ddPCR. The droplets can be formed by flowing an aqueous stream of sample into a junction into which partitioning lipid (e.g., fluorinated hydrocarbon oil) is also flown so that oil-encapsulated droplets are formed. In some embodiments, the droplets are 60 to 200 pm in diameter. In some embodiments, the encapsulating oil is thermostable. In some embodiments, the partitions are microtubes, microwells or nanowells. In some embodiments, each partition includes a solid support, e.g., a bead (see U.S. 9,388,465). In some embodiments, the target nucleic acid inside the partition is attached to the solid support. In some embodiments, one or more amplification primers are attached the solid support while the sample nucleic acid is present in solution within the partition. In some embodiments, the attachment is via incorporating chemical groups into nucleic acids (e.g., biotin, a single nucleic acid strand) and conjugating complementary chemical groups to the bead (e.g., streptavidin, a complementary nucleic acid strand).

[0072] In some embodiments, the droplets are arranged in a monolayer and the monolayer is subjected to temperature cycling to enable the PCR process inside the droplets. In some embodiments, the detection also takes place in the monolayer by detecting the fluorescence emitted from the monolayer.

[0073] The invention involves a PCR assay for detecting a gene insertion into a specific site in the cellular genome. A PCR assay involves sequence-specific primer oligonucleotides or “primers.” In some embodiments, one or more sets of amplification primers with an optional detection probe target a terminal region of the insert that includes the terminus of the inserted sequence and the flanking genomic region, t?. ., the 5’-flanking genomic region or the 3’-flanking genomic region. In some embodiments, the insertion is designed to occur in the TRAC locus and the 5’- and 3’ - flanking genomic regions are sequences of the TRAC locus. Such a design ensures that both the presence and the correct location of the inserted sequence in the genome are detected by the PCR assay. In a primer pair designed according to this principle the first primer hybridizes to a genomic sequence (z.e., a sequence present in both engineered cell and a wild-type cell) and the second primer hybridizes to an insert sequence (z.e., a sequence present only in the engineered cell). In some embodiments, only one primer pair, e. ., a primer pair targeting the 5’-flanking genomic region and the 5 ’-end of the insert sequence (“left-side PCR”), or a primer pair targeting the 3 ’-flanking genomic region and the 3 ’-end of the insert sequence (“right-side PCR”) is used to detect the presence of the inserted sequence at the desired location. In some embodiments, two primer pairs are used in succession or simultaneously, e.g., a primer pair targeting the 5 ’-flanking genomic region and the 5 ’-end of the insert, and a primer pair targeting the 3 ’-flanking region and the 3 ’-end of the insert.

[0074] In embodiments where probe-based detection is used, the PCR further includes a probe oligonucleotide or “probe.” The design and hybridization site of a probe requires that the probe specifically hybridize to the amplicon produced by the primer pair. The probe may be designed to hybridize to either a genomic sequence (z.e., a sequence present in both an engineered cell and a wild-type cell) or an insert sequence (z.e., a sequence present only in the engineered cell) or hybridize to both a portion of the genomic sequence and a portion of the insert sequence.

[0075] Examples of the PCR assay designs are shown in Figure 1. An exemplary design of a PCR assay that targets the 3 ’-portion of the insert sequence (“right-side PCR”) includes a pair of amplification primers comprising a first primer hybridizing to a genomic sequence adjacent to the insertion site at the 3 ’-side (“3 ’-genomic sequence,” e.g., TRAC sequence), and a second primer hybridizing to the polyadenylation signal sequence in the inserted CAR expression construct. In some embodiments, the primer pair consists essentially of SEQ ID NO: 1 and SEQ ID NO: 2, and the probe consists essentially of SEQ ID NO:3. This primer pair produces a 666 base pair amplicon.In some embodiments, the PCR assay further comprises a detection probe hybridizing to an insert sequence or a genomic sequence. In some embodiments, the probe hybridizes to a genomic sequence in the 3’ -TRAC sequence. An exemplary design of a PCR assay that targets the 5 ’-portion of the insert sequence (“left-side PCR”) includes a pair of amplification primers comprising a first primer hybridizing to the promoter sequence in the CAR expression construct, and a second primer hybridizing to a genomic sequence adjacent to the insertion site at the 5 ’-side (“5 ’-genomic sequence,” e.g., TRAC sequence). In some embodiments, the primer pair consists essentially of SEQ ID NO: 4 and SEQ ID NO: 5, and the probe consists essentially of SEQ ID NO: 6. This primer pair produces a 626 base pair amplicon. In some embodiments, the PCR assay further comprises a detection probe hybridizing to an insert sequence or a genomic sequence. In some embodiments, the probe hybridizes to a genomic sequence in the 5’ -TRAC sequence.

[0076] In some embodiments, the PCR assay includes two pairs of amplification primers: a first pair comprising a first primer hybridizing to a 3 ’-genomic sequence, and a second primer hybridizing to the polyadenylation signal sequence in the CAR expression construct; and a second pair comprising a first primer hybridizing to the promoter sequence in the CAR expression construct, and a second primer hybridizing to a 5 ’-genomic sequence. In some embodiments, the PCR assay further comprises two detection probes: the first probe hybridizing to a 5’-genomic sequence, and the second probe hybridizing to a 3 ’-genomic sequence.

[0077] In some embodiments, the PCR assay further includes a pair of amplification primers hybridizing to a control nucleic acid sequence. In some embodiments, the control nucleic acid sequence is a genomic sequence present in both wild type cells and engineered cells. In some embodiments, the control sequence is a single-copy sequence, i.e., is present once in the haploid genome and twice in the diploid genome of the cell. In some embodiments, the control sequence is located on a different chromosome from the insertion cite for the CAR expression construct. In some embodiments, the PCR reaction further comprises a probe hybridizing to the control sequence.

[0078] In some embodiments, the PCR assay further includes one or more amplification primers hybridizing to vector-specific sequences. Such sequences are present in the vector used to deliver the CAR expression construct but not present in the genome of engineered cells after the integration of the CAR expression construct. An example of the assay detecting residual vector constructs in shown in Figure 2. Inside the viral vector, the homology arm (“TRAC”) sequencesare flanked by viral inverted terminal repeats (ITRs). As shown in Figure 2, the “outer” primer may be designed to hybridize to an ITR sequence. In some embodiments, the first primer hybridizes to a sequence in the CAR expression construct, while the second primer hybridizes to a viral ITR sequence. Optionally, this ITR primer may be paired with the same internal primer used and the same probe used in the insert-detection assay. In some embodiments, the vectorspecific sequence if the inverted terminal repeat (ITR) of the AAV. In some embodiments, the primer consists essentially of SEQ ID NO: 7.

[0079] In some embodiments, two or more PCR amplifications occur simultaneously in a single reaction volume, e.g., two or more of the 5’-targeting assay, the 3’-targeting assay, the control gene assay, and the vector-targeting assay.

[0080] In some embodiments, the PCR is performed as quantitative PCR. The quantitative PCR assay compares a signal generated by the CAR expression construct amplification to a signal generated by the control nucleic acid amplification to determine copy number of the CAR expression construct in the cellular genome.

[0081] In some embodiments, the quantitative PCR assay compares a signal generated by the CAR expression construct amplification to a signal generated by the virus-specific amplification to determine number of virus particles present in the cellular preparation.

[0082] An amplification control can be used to control for satisfactory PCR reagents and conditions. In some embodiments, an amplification control is a plasmid comprising at least one copy of the CAR expression construct. In some embodiments, the control plasmid further comprises the flanking genomic sequences on each side of the CAR expression construct. In some embodiments, the control plasmid also includes the control nucleic acid at a 1 : 1 copy ratio with the CAR expression construct.

[0083] In some embodiment, a control cellular sample is used. In some embodiments, control cells are wild-type cells where no insert is present and no virus was introduced. In some embodiments, control cells are cells engineered to disrupt the TRAC gene but lacking any CAR insertions (“TRAC KO cells”).

[0084] In some embodiments, the invention is a reaction mixture for performing a PCR assay for detecting a gene insertion into a specific site in the cellular genome. The reaction mixture comprises at least one primer pair where the first primer hybridizes to a genomic sequence (i.e., a sequence present in both engineered cell and a wild-type cell) and the second primer hybridizes toan insert sequence (i.e., a sequence present only in the engineered cell). In some embodiments, the reaction mixture comprises only one primer pair, e.g., a primer pair targeting the 5’ -flanking region and a 5 ’-end adjacent insert sequence, or a primer pair targeting the 3 ’-flanking region and a 3’- end adjacent insert sequence. In some embodiments, the reaction mixture comprises two primer pairs, e.g., a primer pair targeting a 5’-flanking region and a 5’-end adjacent insert sequence, and a primer pair targeting a 3 ’-flanking region and a 3 ’-end adjacent insert sequence.

[0085] In some embodiments, the reaction mixture comprises a first primer hybridizing to a 3’-genomic sequence, the primer consisting essentially of SEQ ID NO: 1, and a second primer hybridizing to the polyadenylation signal sequence in the inserted CAR expression construct, the primer consisting essentially of SEQ ID NO: 2.

[0086] In some embodiments, the reaction mixture comprises a first primer hybridizing to the promoter sequence in the CAR expression construct, the primer consisting essentially of SEQ ID NO: 4, and a second primer hybridizing to a 5’-genomic sequence, the primer consisting essentially of SEQ ID NO: 5.

[0087] In some embodiments, the reaction mixture comprises a labeled detection probe. The probe may be designed to hybridize to either a genomic sequence (i.e., a sequence present in both an engineered cell and a wild-type cell) or an insert sequence i.e., a sequence present only in the engineered cell) or hybridize to both a portion of the genomic sequence and a portion of the insert sequence. In some embodiments, the reaction mixture further comprises a detection probe hybridizing to a 3 ’-genomic sequence, the probe consisting essentially of SEQ ID NO: 3. In some embodiments, the reaction mixture further comprises a detection probe hybridizing to a 5’- genomic sequence, the probe consisting essentially of SEQ ID NO: 6.

[0088] In some embodiments, the reaction mixture further includes a pair of amplification primers hybridizing to a control nucleic acid sequence and a probe capable of detecting the control nucleic acid sequence.

[0089] In some embodiments, the reaction mixture further includes amplification primers hybridizing to vector-specific sequences. In some embodiments, the vector-specific primer hybridizes to the inverted terminal repeat (ITR) of AAV. In some embodiments, the vector-specific primer consists essentially of SEQ ID NO: 7.

[0090] In some embodiments, a control reaction mixture includes an amplification control nucleic acid. In some embodiments, the reaction mixture comprises a plasmid comprising at leastone copy of the CAR expression construct, and optionally, the flanking genomic sequences on each side of the CAR expression construct when it is integrated into the cellular genome. In some embodiments, the control gene is RPP30.

[0091] In some embodiments, a control reaction mixture includes control sample nucleic acid, e.g., nucleic acid isolated from control cells engineered to disrupt the TRAC gene but lacking any CAR insertions (TRAC KO cells).

[0092] In some embodiments, a control reaction mixture includes a plasmid control comprising one or more nucleic acid sequences substantially identical to the nucleic acid sequences to be amplified from the cellular genome.

[0093] In some embodiments, the nucleic acid sequences described herein are sequenced. In some embodiments, isolated DNA from the engineered cell preparation is sequenced. In some embodiments, amplicons prepared according to the methods described herein are sequenced.

[0094] Any one of a number of sequencing technologies or sequencing assays can be utilized. The term "Next Generation Sequencing (NGS)" as used herein refers to sequencing methods that allow for massively parallel sequencing of clonally amplified molecules and of single nucleic acid molecules.

[0095] Non-limiting examples of sequence assays that are suitable for use with the methods disclosed herein include nanopore sequencing (U.S. Pat. Publ. Nos. 2013 / 0244340, 2013 / 0264207, 2014 / 0134616, 2015 / 0119259 and 2015 / 0337366), Sanger sequencing, capillary array sequencing, thermal cycle sequencing (Sears etal., Biotechniques, 13:626-633 (1992)), solid-phase sequencing (Zimmerman etal., Methods Mol. Cell Biol., 3:39-42 (1992)), sequencing with mass spectrometry such as matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI- TOF / MS; Fu et al., Nature Biotech., 16:381-384 (1998)), sequencing by hybridization (Drmanac et al., Nature Biotech., 16:54-58 (1998), and NGS methods, including but not limited to sequencing by synthesis (e.g., HiSeq™, MiSeq™, or Genome Analyzer, each available from Illumina), sequencing by ligation (e.g., SOLiD™, Life Technologies), ion semiconductor sequencing (e.g., Ion Torrent™, Life Technologies), and SMRT® sequencing (e.g., Pacific Biosciences).

[0096] Commercially available sequencing instruments include sequencing-by-hybridization platforms from Affymetrix Inc. (Sunnyvale, Calif.), sequencing-by-synthesis platforms from Illumina / Solexa (San Diego, Calif.) and Helicos Biosciences (Cambridge, Mass.), sequencing-by- ligation platform from Applied Biosystems (Foster City, Calif). Other sequencing instrumentsinclude, but are not limited to, the Ion Torrent technology from ThermoFisher Scientific (Waltham, Mass.), and nanopore-based instruments from Oxford Nanopore Technologies (Oxford, UK).

[0097] In some embodiments, the invention utilizes an adaptor added to one or both ends of a nucleic acid or nucleic acid strand. Adaptors of various shapes and functions are known in the art (see e.g., PCT / EP2019 / 05515 filed on February 28, 2019, US8822150 and US8455193). In some embodiments, the function of an adaptor is to introduce desired elements into a nucleic acid. The adaptor-borne elements include at least one of nucleic acid barcode, primer binding site or a ligation-enabling site.

[0098] The adaptor may be double-stranded, partially single stranded or single stranded. In some embodiments, a Y-shaped, a hairpin adaptor or a stem-loop adaptor is used wherein the double-stranded portion of the adaptor is ligated to the double stranded nucleic acid formed as described herein.

[0099] In some embodiments, the adaptor molecules are in vitro synthesized artificial sequences. In other embodiments, the adaptor molecules are in vitro synthesized naturally- occurring sequences. In yet other embodiments, the adaptor molecules are isolated naturally occurring molecules or isolated non naturally-occurring molecules.

[0100] The double-stranded or partially double-stranded adaptor oligonucleotide can have overhangs or blunt ends. In some embodiments, the double-stranded DNA may comprise blunt ends to which a blunt-end ligation can be applied to ligate a blunt-ended adaptor. In other embodiments, the blunt ended DNA undergoes A-tailing where a single A nucleotide is added to the blunt ends to match an adaptor designed to have a single T nucleotide extending from the blunt end to facilitate ligation between the DNA and the adaptor. Commercially available kits for performing adaptor ligation include AVENIO ctDNA Library Prep Kit or KAPA HyperPrep and HyperPlus kits (Roche Sequencing Solutions, Pleasanton, CA). In some embodiments, the adaptor ligated (adapted) DNA may be separated from excess adaptors and unligated DNA.

[0101] In some embodiments, the invention includes the use of a barcode. In some embodiments, the method of detecting epigenetic modifications includes sequencing. The nucleic acid processed as described herein is subjected to sequencing; preferably, massively parallel single molecule sequencing. Analyzing individual molecules by massively parallel sequencing typically requires a separate level of barcoding for sample identification and error correction. The use of molecular barcodes such as described in U.S. Patent Nos. 7,393,665, 8,168,385, 8,481,292,8,685,678, and 8,722,368. A unique molecular barcode is added to each molecule to be sequenced to mark molecule and its progeny (e.g., the original molecule and its amplicons generated by PCR). The unique molecular barcode (UID) has multiple uses including counting the number of original target molecules in the sample and error correction (Newman, A., et al., (2014) An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage, Nature Medicine doi: 10.1038 / nm.3519).

[0102] In some embodiments, unique molecular barcodes (UIDs) are used for sequencing error correction. The entire progeny of a single target molecule is marked with the same barcode and forms a barcoded family. A variation in the sequence not shared by all members of the barcoded family is discarded as an artefact. Barcodes can also be used for positional deduplication and target quantification, as the entire family represents a single molecule in the original sample (Newman, A., et al., (2016) Integrated digital error suppression for improved detection of circulating tumor DNA, Nature Biotechnology 34:547).

[0103] In some embodiments of the invention, the adaptor ligated to one or both ends of the barcoded target nucleic acid comprises one or more barcodes used in sequencing. A barcode can be a UID or a multiplex sample ID (MID or SID) used to identify the source of the sample where samples are mixed (multiplexed). The barcode may also be a combination of a UID and an MID. In some embodiments, a single barcode is used as both UID and MID. In some embodiments, each barcode comprises a predefined sequence. In other embodiments, the barcode comprises a random sequence. In some embodiments of the invention, the barcodes are between about 4-20 bases long so that between 96 and 384 different adaptors, each with a different pair of identical barcodes are added to a human genomic sample. In some embodiments, the number of UIDs in the reaction can be in excess of the number of molecules to be labelled. A person of ordinary skill would recognize that the number of barcodes depends on the complexity of the sample (i.e., expected number of unique target molecules) and would be able to create a suitable number of barcodes for each experiment.

[0104] In some embodiments, the sequencing step involves sequence aligning. In some embodiments, aligning is used to determine a consensus sequence from a plurality of sequences, e.g., a plurality having the same unique molecular ID (UID). The molecular ID is a barcode that can be added to each molecule prior to sequencing or if amplification step is included, prior to the amplification step. In some embodiments, a UID is present in the 5 ’-portion of the RT primer.Similarly, a UID can be present in the 5 ’-end of the last barcode subunit to be added to the compound barcode. In other embodiments, a UID is present in an adaptor and is added to one or both ends of the target nucleic acid by ligation.

[0105] In some embodiments, a consensus sequence is determined from a plurality of sequences all having an identical UID. The sequenced having an identical UID are presumed to derive from the same original molecule through amplification. In other embodiments, UID is used to eliminate artifacts, i.e., variations existing in the progeny of a single molecule (characterized by a particular UID). Such artifacts resulting from PCR errors or sequencing errors can be eliminated using UIDs.

[0106] In some embodiments, the number of each sequence in the sample can be quantified by quantifying relative numbers of sequences with each UID among the population having the same multiplex sample ID (MID). Each UID represents a single molecule in the original sample and counting different UIDs associated with each sequence variant can determine the fraction of each sequence variant in the original sample, where all molecules share the same MID. A person skilled in the art will be able to determine the number of sequence reads necessary to determine a consensus sequence. In some embodiments, the relevant number is reads per UID (“sequence depth”) necessary for an accurate quantitative result. In some embodiments, the desired depth is 5-50 reads per UID.

[0107] In some embodiments, the invention is a kit for performing a PCR assay for detecting insertion of a gene insertion into a specific site in the cellular genome. More specifically, the kit comprises reagents for detecting insertion of the CAR construct into the TRAC locus of a mammalian cell. The kit comprises an aliquot of each of a first primer hybridizing to a genomic sequence (i.e., a sequence present in both engineered cell and a wild-type cell) and a second primer hybridizing to an insert sequence i.e., a sequence present only in the engineered cell). In some embodiments, the kit further comprises a labeled detection probe. The probe may be designed to hybridize to either a genomic sequence (i.e., a sequence present in both an engineered cell and a wild-type cell) or an insert sequence (i.e., a sequence present only in the engineered cell) or hybridize to both a portion of the genomic sequence and a portion of the insert sequence.

[0108] In some embodiments, the kit comprises a first primer hybridizing to a 3 ’-genomic sequence, the primer consisting essentially of SEQ ID NO: 1, and a second primer hybridizing tothe polyadenylation signal sequence in the inserted CAR expression construct, the primer consisting essentially of SEQ ID NO: 2.

[0109] In some embodiments, the kit comprises a first primer hybridizing to the promoter sequence in the CAR expression construct, the primer consisting essentially of SEQ ID NO: 4, and a second primer hybridizing to a 5’-genomic sequence, the primer consisting essentially of SEQ ID NO: 5.

[0110] In some embodiments, the kit comprises a labeled detection probe. In some embodiments, the kit comprises a detection probe hybridizing to a 3 ’-genomic sequence, the probe consisting essentially of SEQ ID NO: 3. In some embodiments, the kit comprises a detection probe hybridizing to a 5’-genomic sequence, the probe consisting essentially of SEQ ID NO: 6.[OHl] In some embodiments, the kit further includes a pair of amplification primers hybridizing to a control nucleic acid sequence and a probe capable of detecting the control nucleic acid sequence.

[0112] In some embodiments, the kit further includes amplification primers hybridizing to vector-specific sequences. In some embodiments, the vector-specific primer consists essentially of SEQ ID NO: 7.

[0113] In some embodiments, the kit further includes an aliquot of an amplification control nucleic acid, e.g., a plasmid comprising at least one copy of the CAR expression construct, and optionally, the flanking genomic sequences on each side of the CAR expression construct when it is integrated into the cellular genome.

[0114] In some embodiments, the kit further comprises amplification buffers and reagents for performing a polymerase chain reaction, such as described e.g., in PCR Strategies (Innis et al., 1995, Academic Press, San Diego, Calif.) at Chapter 14; o PCR Protocols: A Guide to Methods and Applications (Innis et al., Academic Press, N Y, 1990).

[0115] In some embodiments, the kit further comprises reagents for sequencing the nucleic acids amplified as described herein. The sequencing reagents include one or more of barcodes, adaptors, barcoded adaptors, sequencing primers, universal primers, and nucleic acid purification regents for intermediate nucleic acid purification steps.EXAMPLES

[0116] Example 1. Detecting insertion of an anti-CD19 CAR construct by ddPCR targeting the 3 ’-portion of the insertion

[0117] In this example the ddPCR assay targeting the 3 ’-portion of the insertion was tested using a control plasmid. The control plasmid contains the CAR-expressing insert sequence flanked by additional TRAC genomic sequence and further contains a control amplicon sequence at a 1 : 1 ratio. The control plasmid DNA was extracted using NucleoSpin Plasmid, Mini kit for plasmid DNA (Macherey-Nagel). The plasmid was diluted to various concentrations within the desired assay range using 0.1% Pluronic F68 solution. The amplification primers were SEQ ID NO: 2 (TRAC) and SEQ ID NO: 1 (BGH polyadenylation site in the insert). The probe was designed for the TRAC sequence (SEQ ID NO: 3). The PCR was assembled according to Table 1 and droplets were created using the Automated Droplet Generator (Bio-Rad, Hercules, Cal ). The PCR was performed in a Cl 000 Touch Thermal Cycler (Bio-Rad) using the thermal cycling conditions in Table 2. Results are shown in Table 3. Resulting ratios of FAM copies to HEX copies approaching 1 indicate optimal assay specificity of the multiplex assay.

[0118] Table 1. 3 ’-assay PCR components

[0119] Table 2. Thermocycling profde

[0120] The results are shown in Table 3.

[0121] Table 3. 3 ’-assay specificityFAM - insertion-specific probe,HEX - control genomic probe.Droplets - accepted droplets.Pos-PCR-positive dropletsNeg - PCR-negative dropletRatio: ratio of insert / control positive droplets in the sample

[0122] Example 2. Detecting the complete insertion in genomic DNA by targeting the 3 ’- portion and the 5 ’-portion of the insert by ddPCR

[0123] In this example the ddPCR assay targeting both the 3 ’-portion and the 5’-portion of the insert was applied to genomic DNA isolated from engineered cells. The cell samples were CB-010 (engineered human donor-derived T cells with the CAR (Figure 1) inserted into the TRAC locus), TRAC KO cells (control with no CAR inserted into the cleaved TRAC locus, no exposure to the AAV vector), and wild-type cells from the same donor. A separate set of CAR-T cells and control cells was manufactured by two vendors (M and W in Table 4).

[0124] Genomic DNA was extracted using the DNeasy Blood & Tissue QIAcube Kit (Qiagen, Valencia, Cal.) and normalized to 5ng / pL. The ddPCR reactions were performed as described in Example 1. The 3’-assay primers were SEQ ID NO: 2 and SEQ ID NO: 1, and the probe was SEQ ID NO: 3 (Example 1). The 5 ’-assay primers were SEQ ID NO: 4 (EFl promoter) and SEQ ID NO: 5 (TRAC sequence), and the probe was SEQ ID NO: 6 (TRAC sequence).

[0125] The results are shown in Table 4. The number of 5 ’-end-derived amplicons, and the number of 3 ’-end-derived amplicons were compared to the number of amplicons from the controlgenomic sequence to get the “Ratio CAR left” and “Ratio CAR right.” The numbers were similar for both ends of the insert indicating insertion of the entire construct into the CAR-T cells tested.

[0126] Example 3. Detecting residual AA V

[0127] In this example the ddPCR assay targeted the residual AAV6 with the CAR insert that may be present in the CAR-T cell preparation. DNA was isolated from cell cultures as described in Example 2. For the vector construct-derived amplicons, the amplification primers were SEQ ID NO: 7 (AAV ITR) and SEQ ID NO: 1), and the probe was (SEQ ID NO: 3). PCR was set up as in Example 1 (Table 1 and Table 2). The design of the 3’-assay and the 5’-assay was according to Example 2.

[0128] The results are shown in Table 4.

[0129] Table 4. Detecting both ends of the insert and residual AA V.

[0130] While the invention has been described in detail with reference to specific examples, it will be apparent to one skilled in the art that various modifications can be made within the scopeof this invention. Thus, the scope of the invention should not be limited by the examples described herein, but by the claims presented below.

[0131] INFORMAL SEQUENCE LISTING

Claims

We Claim:

1. A method of detecting the presence of a chimeric antigen receptor (CAR)-expressing nucleic acid construct at an integration site in a genome of an engineered cell, the method comprising: contacting a sample comprising nucleic acids from one or more engineered cells with amplification primers, wherein the first amplification primer is capable of hybridizing to a sequence present in the genome of wild type cells adjacent to the integration site, and the second amplification primer is capable of hybridizing to a sequence in the CAR-expressing nucleic acid construct, and a polymerase, and performing an amplification by the polymerase chain reaction (PCR) to produce an amplicon, wherein the presence or absence of the amplicon is indicative of the presence or absence of the integrated CAR-expressing nucleic acid construct at the integration site.

2. The method of claim 1, wherein the presence or absence of the amplicon is detected by detecting fluorescence of a labeled probe.

3. The method of claim 1, wherein the CAR-expressing nucleic acid construct comprises in the following order, a promoter, a CAR-encoding sequence, and a polyadenylation signal.

4. The method of claim 1, wherein the integration site is in the T cell receptor alpha constant (TRAC) gene.

5. The method of claim 1, wherein the CAR is an anti-CD19 CAR.

6. The method of claim 2, wherein the first amplification primer consists essentially of SEQ ID NO: 2, the second amplification primer consists essentially of SEQ ID NO: 1, and the probe consists essentially of SEQ ID NO: 3.

7. The method of claim 2, wherein the first amplification primer consists essentially of SEQ ID NO: 5, the second amplification primer consists essentially of SEQ ID NO: 4, and the probe consists essentially of SEQ ID NO: 6.

8. The method of claim 2, further comprising amplifying a control nucleic acid sequence present in the genome of wild type cells and in the genome of engineered cells and detecting the presence of the control nucleic acid with a control probe.

9. The method of claim 8, further comprising comparing the fluorescence of the probe detecting the CAR-expressing nucleic acid and the fluorescence of the probe detectingthe control nucleic acid, thereby determining copy number of the CAR-expressing nucleic acid in the engineered cell.

10. The method of claim 1, wherein the CAR-expressing nucleic acid has been introduced into the engineered cell with the use of an AAV vector, the method further comprising amplifying a nucleic acid sequence present in the AAV vector, and detecting the presence of the AAV vector.

11. The method of claim 10, wherein the nucleic acid sequence present in the AAV vector is amplified with a first primer capable of hybridizing to the inverted terminal repeat (ITR) of the AAV, and a second primer, and the presence of the AAV is detected with a fluorescently labeled probe.

12. The method of claim 11, wherein the first primer consists essentially of SEQ ID NO: 7.

13. The method of claim 11, wherein the second primer is selected from the sequence consisting essentially of SEQ ID NO: 1 and the sequence consisting essentially of SEQ ID NO: 4.

14. The method of claim 11, further comprising comparing the fluorescence of the probe detecting the nucleic acid sequence present in the AAV vector and the fluorescence of the probe detecting the control nucleic acid, thereby determining copy number of the AAV in the sample.

15. The method of claim 1, wherein the PCR is digital PCR.

16. A reaction mixture for detecting the presence of chimeric antigen receptor (CAR)- expressing nucleic acid construct at an integration site in a genome of an engineered cell, the reaction mixture comprising: nucleic acids from one or more engineered cells, a first amplification primer capable of hybridizing to a sequence present in the genome of wild type cells adjacent to the integration site, and a second amplification primer capable of hybridizing to a sequence in the CAR-expressing nucleic acid construct.

17. The reaction mixture of claim 16, wherein the first amplification primer consists essentially of SEQ ID NO: 2 and the second amplification primer consists essentially of SEQ ID NO: 1.

18. The reaction mixture of claim 16, wherein the first amplification primer consists essentially of SEQ ID NO: 5 and the second amplification primer consists essentially of SEQ ID NO: 4.

19. The reaction mixture of claim 16, further comprising one or more labeled probe selected from a sequence consisting essentially of SEQ ID NO: 3 and a sequence consisting essentially of SEQ ID NO: 6.

20. The reaction mixture of claim 16, further comprising a first and second control amplification primers capable of amplifying a control nucleic acid sequence present in the genome of wild type cells and in the genome of engineered cells, and a control probe capable detecting amplification of the control nucleic acid.

21. The reaction mixture of claim 16, further comprising a primer capable of amplifying an AAV sequence, the primer consisting essentially of SEQ ID NO: 7.

22. A kit for detecting the presence of chimeric antigen receptor (CAR)-expressing nucleic acid construct at an integration site in a genome of an engineered cell, the reaction mixture comprising: a first amplification primer capable of hybridizing to a sequence present in the genome of wild type cells adjacent to the integration site, and a second amplification primer capable of hybridizing to a sequence in the CAR-expressing nucleic acid construct.

23. The kit of claim 22, wherein the first amplification primer consists essentially of SEQ ID NO: 2 and the second amplification primer consists essentially of SEQ ID NO: 1.

24. The kit of claim 22, wherein the first amplification primer consists essentially of SEQ ID NO: 5 and the second amplification primer consists essentially of SEQ ID NO: 4.

25. The kit of claim 22, further comprising one or more labeled probe selected from a sequence consisting essentially of SEQ ID NO: 3 and a sequence consisting essentially of SEQ ID NO: 6.

26. The kit of claim 22, further comprising a first and second control amplification primers capable of amplifying a control nucleic acid sequence present in the genome of wild type cells and in the genome of engineered cells, and a control probe capable detecting amplification of the control nucleic acid.

27. The kit of claim 22, further comprising a primer capable of amplifying an AAV sequence, the primer consisting essentially of SEQ ID NO:

728. The kit of claim 22, further comprising nucleoside triphosphates, a thermostable nucleic acid polymerase and buffers and cofactors necessary to sustain enzymatic activity of the polymerase.

29. The kit of claim 22, further comprising reagents for preparing sequencing libraries and sequencing nucleic acids.

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