WO2025193609A1 - Methods and compositions for cost-effective assessment of nucleic acid transcripts and isoforms in cells - Google Patents

Abstract

The invention includes improved methods and compositions for detecting transcripts and transcript isoforms in individual cells among populations of cells. Methods are useful as quality control for engineered cell preparations including CAR-T cell and CAR-NK cell preparations.

Description

METHODS AND COMPOSITIONS FOR COST-EFFECTIVE ASSESSMENT OF

NUCLEIC ACID TRANSCRIPTS AND ISOFORMS IN CELLS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. provisional application serial no. 63/563,630 filed on 11 March 2024.

STATEMENT REGARDING GOVERNMENT-SPONSORED RESEARCH

[0002] None.

SEQUENCE LISTING

[0003] The application contains a Sequence Listing which has been submitted electronical ly in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on 27 February 2025, is named “CBI056.30.xml” and is 20,043 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] Whole transcriptome single cell RNA sequencing is a powerful technique yielding a wealth of information about cells and tissues. Many commercial reagents exist to facilitate the use of this technique. However, whole transcriptome sequencing of a population of individual cells requires an extremely high number of reads, resulting in many next-generation sequencing runs to gain sufficient depth of sequencing. This is expensive and time consuming. For some applications, only a subset of transcripts needs to be queried.

[0006] For example, adoptive cell therapy involves genetic engineering of immune cells so that the cells would act as a “living medicine” and repeatedly seek and destroy tumor cells or other harmful or diseased cells in the patient’s body. These cells include exogenous genes (e.g., chimeric antigen receptor, CAR) inserted into the genome. Furthermore, the cells may include a disruption (inactivation) of certain genes. It is understood that not all cells in a population of engineered cells undergo successful gene insertion and gene disruption. There is a need for reproducible and cost-effective next-generation sequencing methods of validating engineered immune cell preparations to ensure safety and potency of the preparations prior to them being administered to the patients.

[0007] SUMMARY OF THE INVENTION

[0008] In some embodiments, the invention is a method of detecting the presence or absence of one or more gene transcripts in one or more cells, the method comprising: contacting a sample comprising a plurality of cDNA molecules derived from one or more cells with one or more reverse amplification primers selected from SEQ ID NOs: 1-14, each reverse primer paired with a forward primer, amplifying a portion of selected cDNA sequences with the forward and the reverse primer, detecting the products of amplification thereby detecting the presence or absence of one or more gene transcripts. In some embodiments, the one or more cells are engineered immune cells. In some embodiments, the detecting comprises sequencing the amplification products. In some embodiments, each cDNA molecule among the cDNA molecules comprises a unique cellular barcode identifying the cell from which the cDNA molecule was derived, and a unique molecular barcode identifying the cDNA molecule. In some embodiments, the primer selected from SEQ ID NO.: 1-14 is conjugated at the 5’-end to a sequencing adaptor. In some embodiments, the sequencing adaptor is SEQ ID NO.: 15. In some embodiments, the primer is conjugated to a capture moiety, e.g., biotin.

[0009] In some embodiments, the reverse primer is SEQ ID NO: 1 and the transcript is from one or more exogenous gene introduced into the cell and having the BGH polyadenylation site. In some embodiments, the exogenous gene is a chimeric antigen receptor (CAR). In some embodiments, the exogenous gene is a fusion between beta-2 microglobulin and an HLA Class I protein.

[0010] In some embodiments, the reverse primer is SEQ ID NO: 2 and the lack of detectable transcript indicated biallelic inactivation of the TRAC gene.

[0011] In some embodiments, the reverse primer is SEQ ID NO: 2 and the reduced amount of detectable transcript indicated monoallelic inactivation of the TRAC gene. [0012] In some embodiments, the reverse primer is SEQ ID NO: 5 and the lack of detectable transcript indicated biallelic inactivation of the PDCD1 gene. In some embodiments, the reverse primer is SEQ ID NO: 5 and the reduced amount of detectable transcript indicated monoallelic inactivation of the PDCD1 gene.

[0013] In some embodiments, the reverse primer is selected from SEQ ID NO: 8-9 and the lack of detectable transcript indicated biallelic inactivation of the CBLB gene. In some embodiments, the reverse primer is selected from SEQ ID NO: 8-9 and the reduced amount of detectable transcript indicated monoallelic inactivation of the CBLB gene.

[0014] In some embodiments, the reverse primer is SEQ ID NO: 3 and the lack of detectable transcript indicated biallelic inactivation of the B2M gene. In some embodiments, the reverse primer is SEQ ID NO: 3 and the reduced amount of detectable transcript indicated monoallelic inactivation of the B2M gene.

[0015] In some embodiments, the reverse primer is SEQ ID NO: 4 and the transcript is from the PTPRC gene.

[0016] In some embodiments, the reverse primer is selected SEQ ID NOs: 6-7 and the transcript is from the ROR1 gene.

[0017] In some embodiments, the reverse primer is selected from SEQ ID NOs: 11 -14 and the transcript is from the CLL1 gene.

[0018] In some embodiments, the invention is a kit for detecting the presence or absence of one or more gene transcripts in one or more cells, the kit comprising one or more reverse amplification primers selected from SEQ ID NOs: 1-14, each reverse primer paired with a forward primer. In some embodiments, the kit further comprises reagents for amplifying and sequencing nucleic acids. In some embodiments, the primer selected from SEQ ID NO.: 1-14 is conjugated at the 5’-end to a sequencing adaptor. In some embodiments, the sequencing adaptor is SEQ ID NO.: 15. In some embodiments, the kit further comprises reagents for sequencing nucleic acids.

[0019] In some embodiments, the invention is a reaction mixture for detecting the presence or absence of one or more gene transcripts in one or more cells, the reaction mixture comprising one or more reverse amplification primers selected from SEQ ID NOs: 1-14, each reverse primer paired with a forward primer. In some embodiments, the reaction mixture further comprises reagents for amplifying nucleic acids. In some embodiments, the primer selected from SEQ ID NO.: 1-14 is conjugated at the 5’-end to a sequencing adaptor. In some embodiments, the sequencing adaptor is SEQ ID NO.: 15.

[0020] BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIGURE 1 is a diagram of an anti-CLLl CAR.

[0022] FIGURE 2 is a diagram of a fusion construct comprising a beta-2 microglobulin

(B2M) and an HLA-E peptide.

[0023] FIGURE 3 is a diagram of a full length B2M-HLA-E transcript and its commonly observed deletion isoform.

[0024] FIGURE 4 is a diagram of a target-specific primer for use in single-cell transcriptome sequencing.

[0025] FIGURE 5 is a diagram of the BGH-specific primer and its use for an exemplary engineered gene transcript.

[0026] FIGURE 6 is a diagram of the P TP RC -specific primer.

[0027] FIGURE 7 is a diagram of the PDCD 1 -specific primer.

[0028] FIGURE 8 is a diagram of CBZB-specific primers.

[0029] FIGURE 9 is a diagram of the beta-2 microglobulin-specific primer.

[0030] FIGURE 10 is a diagram of the TT C-specific primer.

[0031] FIGURE 11 is a diagram of CZL/-specific primers.

[0032] FIGURE 12 is a diagram of ?<97?7-specific primers.

[0033] DETAILED DESCRIPTION OF THE INVENITON

[0034] Definitions

[0035] The following definitions aid in understanding of this disclosure.

[0036] 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.

[0037] 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. [0038] 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). [0039] 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.

[0040] 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 (NH^SC ), 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.

[0041] The term “amplification conditions” refers to conditions in a nucleic acid amplification reaction (e.g., PCR amplification) that allow for hybridization and templatedependent 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.

[0042] The term “sequencing primer” refers to a primer that facilitates the sequencing step on a sequencing instrument. A sequencing primer binding site can be a natural or an artificial sequences typically present in an adaptor or in an outer (5’-) portion of a primer from a previous step of a sample preparation procedure.

[0043] 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. Barcodes can 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 “UMI,” 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. Another type of barcode is a cellular barcode that uniquely identifies a cell in a cell population.

[0044] The invention involves detecting mRNA expression in each cell in a cell population. In some embodiments, the mRNA expression results from a specific genetic modification in the genome of the cell, e.g., by genome editing or genome engineering of the cell. In such instances detecting the mRNA expression serves to validate success of the genetic modification. Detecting mRNA expression may involve detecting expression of both endogenous and exogenous genes. For example, detecting mRNA expression serves to validate quality and potency of a population of engineered cells such as CAR-T cells and CAR-NK cells. Both gene knock-ins (KI) and gene knock-outs (KO) can be assessed. Furthermore, detecting mRNA expression including detecting any mRNA isoforms in individual cells can shed more light on the quality of genome engineering in the cells of a cell population. At the same time, detecting mRNA expression of endogenous genes in individual cells can be used to assess the function of cells in a cell population or in a mixture of cell populations. For example, in a mixture of immune cells and target cells (e.g., tumor cells) mRNA expression of certain activation and exhaustion genes can be used assess function and potency of the immune cells. At the same time, mRNA expression of the target antigen in the target cells can be used assess the susceptibility and resistance of tumor cells to treatment with immune cells.

[0045] Whole transcriptome single cell RNA sequencing can assess gene expression in individual cells of a cell population. Commercial kits exist for this purpose (Oxford Nanopore SQK-PCS111, Oxford Nanopore Technologies, Oxford, UK). Unfortunately, the cost of getting whole transcriptome sequencing of a population of individual cells is very high if sufficient depth of sequencing is to be achieved. Some cost-saving alternatives have been proposed. Singh et al. employ hybrid capture to enrich for certain transcripts (RAGE: repertoire and gene expression by sequencing) Singh et al., (2019) High-throughput targeted long-read single cell sequencing reveals the clonal and transcriptional landscape of lymphocytes, Nature Comm. 10, 3120. Dondi et al. accomplish enrichment by using short-read sequencing data to design primers for long-read sequencing of selected transcripts. Dondi et al., (2022) Detection of isoforms and genomic alterations by high-throughput full-length single-cell RNA sequencing for personalized oncology, BioRxiv 520051. Byrne et al. are able to sequence selected transcripts and their isoforms by starting with hybrid capture of full-length cDNA with a pan-cancer probe panel. Byrne etal. (2023) Single-cell long-read targeted sequencing reveals transcriptional variation in ovarian cancer, BioRxiv 549422. Wang et al. employ biotinylated capture probes to capture transcripts of interest for long-read sequencing. Wang etal., (2023) TEQUILA-Seq: a versatile and low -cost method for targeted long-read RNA sequencing. Nature Comm. 14:4760. Lastly, Clark et al. perform presequencing enrichment by long range-PCR. Clark et al. (2020) Long-read sequencing reveals the complex splicing profile of the psychiatric risk gene CACNA1C in human brain, Molecular Psychiatry 25:37.

[0046] The innate diversity of DNA repair pathways in mammalian cells results in less than 100% efficiency of any genome editing. For example, 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 (MMEJ) 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.

[0047] Variation in mRNA transcripts is also seen in endogenous genes. It has been shown that 95% of human multi-exon genes are alternatively spliced (see Pan et al., (2008) Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing, Nat. Genet. 40:1413.

[0048] Because of innate unpredictability of DNA repair engineered immune cell preparations (such as CAR-T cells or CAR-NK cell preparations) require quality control procedures 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. These assays are time-consuming and require sacrifice of a large number of engineered cells.

[0049] Determining the quantity and sequence of transcripts in each cell serves as a validation tool for a population of engineered immune cells by providing a variety of data on individual cells including expression of the exogenous genes introduced in the cells. For example, expression of the chimeric antigen receptor (CAR) (FIGURE 1), or expression of the fusion between beta-2 microglobulin and HLA-E (FIGURE 2) designed to protect immune cells from attack by the patient’s immune system, expression of the endogenous T cell receptor (TCR). Detection of transcript isoforms is also informative (FIGURE 3). Furthermore, verify lack of transcription from disrupted (“knocked-out”) genes e.g., immune checkpoint genes, verifies successful bi-allelic inactivation of such genes. In addition, assessing mRNA transcripts of endogenous genes in the engineered immune cells and their target tumor cells sheds light on the potency and activity of a population of engineered immune cells.

[0050] This invention involves methods and compositions including target-specific primers to amplify and sequence only selected cDNA molecules from the whole transcriptome. This assay design is especially advantageous in single cell mRNA analysis as it significantly reduced the cost of mRNA sequencing. Furthermore, selective placement of primers can efficiently provide information on several mRNA isoforms. The primers are listed in Table 1. In some embodiments, the 5’-end of the primer from Table 1 is conjugated to a sequence-platform specific adaptor, e.g., SEQ ID NO: 15.

[0051] Table 1. Target-specific primers

[0052] The target-cDNA primer (including a gene-specific primer and a BGH-polyA- specific primer) can be used to amplify any selected transcript, including a gene transcript, an exogenous gene transcript as well as transcript isoforms.

[0053] The invention comprises a method of amplifying (and optionally sequencing) selected transcripts from a cDNA library derived from a population of cells. The process is illustrated in FIGURE 4. The cDNA transcript shown in FIGURE 4 comprises (in order from the 5’-end towards the 3’-end) a forward primer binding site (Rl), a cell-specific barcode, a unique molecular indentifier (UMI), a template switch oligonucleotide (TSO) portion from the reverse transcription step, the target sequence of interest (e. ., one or more gene exons), polyA/polyT sequence, and optional additional sequences from the cDNA synthesis step that may be present in commercial cDNA synthesis and sequencing kits. The target cDNA primer shown in FIGURE 4 is capable of hybridizing to the target sequence and serving as a reverse primer in amplification when paired with the forward primer binding to the forward primer binding site (Rl). As shown in FIGURE 4, the forward and the reverse primers have 5 ’-portions comprising binding sites for sequencing primers.

[0054] In some embodiments, the reverse primer is conjugated to a capture moiety. The capture moiety is a moiety capable of specifically interacting with another capture molecule. Capture moieties -capture molecule pairs include avidin (streptavidin) - biotin, antigen - antibody, magnetic (paramagnetic) particle - magnet, or nucleic acid sequence (oligonucleotide) - complementary nucleic acid sequence (oligonucleotide). An exemplary capture moiety is biotin (“Btn” in FIGURE 4).

[0055] The capture molecule can be bound to a solid support so that any nucleic acid on which the capture moiety is present is captured on solid support and separated from the rest of the sample or reaction mixture.

[0056] In some embodiments, the capture molecule comprises a capture moiety for a secondary capture molecule. For example, a capture moiety in the adaptor may be a nucleic acid sequence captured by a complementary capture oligonucleotide. The capture oligonucleotide is biotinylated (secondary capture moiety). The secondary capture molecule is avidin (streptavidin bead) on which the amplicon-capture oligonucleotide hybrid is captured.

[0057] In some embodiments, the primer targets the BGH polyadenylation signal sequence commonly used in exogenous genes (e.g., CAR expression cassettes) introduced into engineered immune cells. Exemplary use of the BGH-polyA-specific primer is illustrated in FIGURE 5.

[0058] In some embodiments, the primers are specific of gene exons. In some embodiments, the primers span exon-exon junctions to ensure that no residual genomic DNA and no pseudogene is amplified. In some embodiments, the primers are for endogenous genes (see FIGURES 6-12)

[0059] In some embodiments, the primer targets a gene that is disrupted by genome editing step in engineered immune cells. In some embodiments, the disrupted gene is an immune checkpoint gene. In some embodiments, the primer targets an immune checkpoint gene selected from PDCD1 and CBLB. The design of the 77X 777-specific primer is illustrated in FIGURE 7. The design of the CBZB-specific primer is illustrated in FIGURE 8.

[0060] In some embodiments, the primer targets the beta 2 microglobulin (B2M) gene. The design of the 7?2A-7-specific primer is illustrated in FIGURE 9. This design captures transcripts originating from the wild-type B2M gene.

[0061] In some embodiments, the primer targets the T cell receptor alpha constant (TRAC) gene. Such primers can be used to confirm lack of gene expression and therefore a successful gene knock-out. The design of the 7 specific primer is illustrated in FIGURE 10.

[0062] In some embodiments, the primer targets an endogenous gene, e.g, PTPRC. The design of the PZP7?C-specific primer ism illustrated in FIGURE 6. In some embodiments, the primer targets the gene coding for the tumor antigen targeted by the engineered immune cell, e.g., the CLL1 gene or the ROR1 gene. The design of (’/././-specific primers is illustrated in FIGURE 11. The design of 7?O7?7-specific primers is illustrated in FIGURE 12. Such primers can be used to assess interactions between the target tumor cells and the engineered immune cells.

[0063] The primers disclosed herein can be used in conjunction with any opposite-facing second primer in nucleic acid amplification reactions. For example, the second primer in the amplification reaction may be one provided by a manufacturer of a next-generation sequencing sample preparation kit. Furthermore, the primers disclosed herein may be conjugated to a 5’- sequence necessary for annealing of further amplification or sequencing primers specific to a particular next-generation sequencing platform.

[0064] In some embodiments, two or more primers disclosed herein can be used in a multiplex reaction to simultaneously assess expression of two or more genes in the cells of a cell population.

[0065] The methods and compositions disclosed herein allow sequencing of transcripts of interest for validating engineered immune cells at a significantly reduced cost and with higher throughput.

[0066] The invention includes methods of analyzing mRNA expression of human or mammalian cells including immune cells that that have been engineered for immunotherapy. The immune cells 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, CAR macrophages and the like. In some embodiments, the cells are tumor cells assessed for expression of tumor antigens targeted by engineered immune cells.

[0067] 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.

[0068] 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.

[0069] 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 blood cells such as peripheral blood mononuclear cells (PMBC) isolated from peripheral blood or 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 imnninomodulation, Th er. Apher. 1 :203-206.

[0070] 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. [0071] 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.

[0072] In some embodiments, the cells retrieved from the patient include cells of a hematological tumor.

[0073] In some embodiments the cell (e.g., CAR-T cells or CAR-NK cells) may be isolated from a patient’s solid tissue, or a solid tumor. In some embodiments, the cells analyzed are tumor cells. 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.

[0074] In some embodiments, the method of the invention is applied to control cells. In some embodiments, a control cell is not engineered (wild type, wt cell), or is a cell having undergone fewer engineering steps than the test cell. In some embodiments, the control cell is a healthy cell from the patient. In come embodiments, the control cell is a cell isolated from a healthy volunteer. In come embodiments, the control cell is a cell isolated from the same healthy donor as the test cell. In come embodiments, the control cell is a cell of an established (immortalized) cell line or a primary (not immortalized) cell culture.

[0075] In some embodiments, the method of the invention is modified to be 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.

[0076] In some embodiments, the cells are cryopreserved and are thawed prior to performing the methods described herein.

[0077] 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.

[0078] A nucleic acid construct enabling expression of an exemplary CAR is shown in FIGURE 1. In FIGURE 1 abbreviations are as follows: “MND” is the MND promoter, “SS” is signal sequence (also known as leader sequence), “CD28TM”” is a transmembrane domain of the CD28 protein, “CD28” is a co-stimulation domain derived from the CD28 protein, “CD3-zeta” is the CD3-zeta activation domain, “BGH poly A” is a polyadenylation signal, “HA” are homology arms capable of hybridizing the desired integration site in the genome.

[0079] The CAR shown in FIGURE 1 comprises an extracellular domain comprising an antigen binding region targeting CLL1. In some embodiments, the antigen-binding region comprises a single-chain variable region (scFv). In some embodiments, the CAR comprises an anti-CLLl scFv described in the International Application Publication No. W02021050857 Anti-CD371 antibodies and uses thereof. In some embodiments, the CAR is the CAR described in the International Application Publication No. WO2021050862 Antigen recognizing receptors targeting CD371 and uses thereof. In some embodiments, the CAR is the CAR described in the International Application Ser. No. PCT/US2023/079508 filed on November 13, 2023, Anti-CLLl chimeric antigen receptors, engineered cells and related methods. One of skill in the art would recognize that expression of any nucleic acid construct incorporating the BGH polyadenylation signal can be detected using the method disclosed herein, e.g., utilizing the primer of SEQ ID NO: 1.

[0080] In some embodiments, the CAR further comprises a hinge domain and the hinge domain is derived from CD28, e.g., comprises of consists essentially of the CD28 hinge sequence. [0081] 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 is derived from CD28 signal sequence, e.g., comprises of consists essentially of the CD28 signal sequence.

[0082] 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 CD8a transmembrane domain, e.g., comprises of consists essentially of the CD28 transmembrane domain, sequence. [0083] 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 comprises a CD28 cytoplasmic domain and a CD3^ chain.

[0084] 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 the MND promoter, and the polyadenylation signal is the BGH polyadenylation signal.

[0085] 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.

[0086] 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 a sequence of the TRAC gene. In some embodiments, the homology arms have sequence capable of hybridizing to a sequence in the TRAC gene. In some embodiments, the homology arms enable homologous recombination between the CAR expression construct and a sequence in the TRAC gene.

[0087] The invention includes methods of analyzing gene expression of cells that have undergone genome editing or genome engineering. In some embodiments, the genome editing, or genome engineering involves inserting an exogenous gene (e.g., a protein expression construct) into a precise location within the cellular genome. In some embodiments, the exogenous protein expression construct encodes and drives expression of a chimeric antigen receptor (CAR) described herein. To accomplish the genome editing, or genome engineering the CAR expression construct is introduced into a cell. 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. [0088] In some embodiments, expression constructs, e.g., a CAR expression construct or a gene fusion expression construct, are 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 expression construct having all the elements necessary for transcription and translation of the CAR including one or more of promoter, enhancer, polyadenylation site, and transcription terminator. In some embodiments, the CAR expression construct is 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 pairs or about 500 base pairs of sequence cabaple of hybridizing to the sequence in the cellular genome where insertion of the CAR gene is desired. In some embodiments, the homology arms have sufficient length and degree of sequence similarity to promote homologous recombination between the AAV including the CAR expression construct and the site in the cellular genome.

[0089] In some embodiments, the 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. 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 el al., Evolution and classification of the CRISP R-Cas systems. Nature Reviews Microbiology. 2011 June; 9(6): 467-477.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] 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-I, V-K and V-U. See, e.g., Makarova et al. (Nat. Rev. Microbiol., 2020, 18:67-83) and Pausch et al. (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.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] 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. [0101] 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.

[0102] 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.

[0103] 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 Cast 2a, or Type I (CASCADE) nuclease) 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 or U.S. Patent No. 10,227,576, Engineered CASCADE components and CASCADE complexes.

[0104] 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. [0105] 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 I 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 double-strand breaks with TAL effector nucleases, Genetics. 186 (2): 757-61.

[0106] In some embodiments, 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., Molecidar Cloning: A Laboratory Manual, 1989, 2nd Ed., and further editions, 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., kits sold by 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. [0107] In some embodiments, the nucleic acids need to be separated from reagents involved in one or more steps of the method disclosed herein. In some embodiments, such separations utilize particles that selectively and reversibly bind and retain nucleic acids through buffer exchanges and washes. Examples of such particles include magnetic glass particles (MGP) sold under the MagNA Pure brand (Roche Life Science, Branchburg, N.J.), polymer-coated magnetic particles sold under the Dynabeads® sold under the AMPure brand (ThermoFisher Scientific, Waltham, Mass ), and polystyrene beads coated with magnetite (SPRI beads) (Beckman Coulter, Brea, Cal.)

[0108] In some embodiments, invention involves amplification of nucleic acids or isolated nucleic acids by polymerase chain reaction (PCR, see U.S. Patent No. 4,683,195). The methods and compositions for performing a polymerase chain reaction are described in PCR Strategies (Innis e/ c//., 1995, Academic Press, San Diego, Calif.) at Chapter 14; or PCR Protocols: A Guide to Methods and Applications (Innis et al., 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.

[0109] 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. ., T. flavus, T. ruber, T. thermophilus, T. lacteus, T. rubens, T. aquaticus), 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), Sulfolobus solataricus (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 alls (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 siculi (GenBank: DD259857.1), Thermococcus thioreducens, Thermococcus onnurineus NA1, Sulfolobus acidocaldarium, Sulfolobus 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)).

[0110] 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.

[0111] In some embodiments, the droplets contain identical PCR reagents. In some embodiments, each droplet contains a unique barcoded primer that is unique to the droplet.

[0112] 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).

[0113] 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 and involves detecting fluorescence emitted from the monolayer.

[0114] In some embodiments, nucleic acids or RNA or mRNA is isolated from cells by any of the methods known in the art or described herein. The isolated nucleic acids are subjected to a whole-transcriptome amplification using a poly-dT primer and a template-switch oligonucleotide (TSO) to enable synthesis of the first and the second strands of a whole- transcriptome cDNA library. In some embodiments, the nucleic acids are analyzed in individual cells compartmentalized or separated from each other. In some embodiments, the cells are placed into nanodroplets. In some embodiments, the nanodroplets contain the reagents necessary for lysis of cells and reverse transcription, including a poly-dT primer and a template-switch oligonucleotide (TSO) to enable synthesis of the first and the second strands of a whole- transcriptome cDNA library in each droplet. In some embodiments, the TSO includes a 5 ’-portion comprising various elements such as a cell-specific barcode to trace each transcript to its cell of origin, and a unique molecular identifier (UMI) to mark each transcript. In some embodiments, the TSO and the poly-dT primer also comprise 5’-portions that comprise a primer-binding site for an amplification primer.

[0115] In some embodiments, the cDNA library, e.g., a whole-transcriptome cDNA library is amplified prior to sequencing. The instant invention utilizes one or more target-specific primers to assess the presence of certain transcripts and transcript isoforms in one or more cells of a population of cells. The process utilizes sequence elements illustrated in FIGURE 4. First, cDNA from a mixture of cells (including cells encapsulated in droplets now broken to pool the barcoded cDNAs) is subjected to the first round of amplification. As shown in FIGURE 4, forward primer has a binding site in the 5 ’-portion of the TSO and on the 5 ’-flank of the cDNA molecule, and the reverse primer is a target specific primer of the instant invention (see Table 1). In some embodiments, only the reverse (target-specific) primer has an affinity tag, e.g., biotin. Next, only the amplicons including the target-specific primer are captured using affinity capture (e.g., streptavidin-coated beads in the case of biotin). The remainder of the cDNA library is left behind and not subjected to sequencing. Sequencing only the selected nucleic acids allows for a cost- effective way of assessing the desired transcripts. As shown in FIGURE 4, the forward and reverse primers in the first round of amplification have 5’-portions comprising binding sites for sequencing primers. A sequencing primer reflects the choice of the sequencing method and sequencing platform. For example, the sequencing primer may be specific to any platform available for nextgeneration sequencing, for example, any Oxford Nanopore platform, Pacific Biosciences platform, or Illumina platform to name a few. Next, selected products of the first amplification are subjected to the second round of amplification utilizing the forward and reverse primers specific to the sequencing platform. The products of the second round of amplification are optionally purified and subjected to the sequencing step.

[0116] In some embodiments, a control population of cells is used along with the test population of cells. In some embodiments, the control population of cells is also a donor-derived population of lymphocytes that has not been subjected to any genome modification (wild-type cells). In some embodiments, the control population of cells has fewer than all of the genome modifications than the test cell population. In some embodiments, the control cell population has gene knock out but without the corresponding gene knock in. acid, e.g., have a disruption of the TRAC gene but lack any CAR insertions into the TRAC locus (TRAC KO cells).

[0117] The invention includes methods of sequencing nucleic acids. Any one of a number of sequencing technologies or sequencing assays and instruments 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.

[0118] 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 et al., 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).

[0119] 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 instruments include, but are not limited to, the Ion Torrent technology from ThermoFisher Scientific (Waltham, Mass.), and nanopore-based instruments from Oxford Nanopore Technologies (Oxford, UK).

[0120] The non-limiting example presented herein involves the use of nanopore sequencing, specifically, sequencing using the regents, methods, and instruments available from Oxford Nanopore Technologies (Oxford, UK).

[0121] In some embodiments, the sequencing step 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. ., U.S. Patent Nos. 8,822,150, 8,455,193, 9,551,023, 11,479,815, 11,512,308, and 10,280,459). In some embodiments, the function of an adaptor is to introduce desired elements into a nucleic acid. In some embodiments, the adaptor-borne elements are structural and include a primer binding site, a ligation-enabling site and a capture site such as transmembrane pore-binding site or a capture primer-binding site. In some embodiments, the adaptor-borne elements are information elements including a sample barcode and a molecular barcode.

[0122] 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.

[0123] 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.

[0124] 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.

[0125] 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, 8,722,368 and 10,280,459. 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., etal., (2014) An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage, Nature Medicine 20:548).

[0126] 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., el al., (2016) Integrated digital error suppression for improved detection of circulating tumor DNA, Nature Biotechnology 34:547). [0127] 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 sample ID (multiplex ID, MID) 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.

[0128] In some embodiments, the sequencing step involves sequence analysis. In some embodiments, the analysis comprises determining the presence of the nucleic acid, i.e., whether the nucleic acid has been successfully inserted into the cellular genome during the genome engineering step. In some embodiments, the analysis further comprises determining the exact sequence of the nucleic acid inserted into the cellular genome thereby determining the accuracy of the genome engineering step.

[0129] 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.

[0130] 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.

[0131] 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.

[0132] In some embodiments, the invention is a kit for performing a cDNA sequencing assay. More specifically, the kit comprises reagents for detecting the presence and isoforms of certain mRNA transcripts in cells, e.g., populations of engineered immune cells. The kit comprises aliquots of each of one or more primers from Table 1 (SEQ ID NOs: 1-14) and one or more other reagent for reverse transcription, amplification and sequencing of nucleic acids. In some embodiments, the 5’-end of the primer from Table 1 is conjugated to a sequence-platform specific adaptor, e.g., SEQ ID NO: 15. In some embodiments, the kit further comprises one or more of poly-dT primer, reverse transcriptase tempi ate- switch oligonucleotide (TSO), the TSO comprising at the 5’-end, an adaptor comprising one or more barcodes and a sequencing primer binding site, a reverse primer (to be paired with the primer from Table 1, and sequencing primer. In some embodiments, the kit further comprises one or more enzymes selected from reverse transcriptase and thermostable nucleic acid polymerase. 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; or PCR Protocols: A Guide to Methods and Applications (Innis et al., Academic Press, N Y, 1990).

[0133] In some embodiments, the invention is a reaction mixture for performing a cDNA sequencing assay. More specifically, the reaction mixture comprises reagents for detecting the presence and isoforms of certain mRNA transcripts in cells, e.g., populations of engineered immune cells. The reaction mixture comprises one or more primers from Table 1 (SEQ ID NOs: 1-14) and a reverse primer. In some embodiments, the 5’-end of the primer from Table 1 is conjugated to a sequence-platform specific adaptor, e.g., SEQ ID NO: 15. The reaction mixture further comprises a cDNA molecule comprising on the 5 ’-flank, a reverse transcriptase templateswitch oligonucleotide (TSO) conjugated to an adaptor comprising one or more barcodes and a sequencing primer binding site. On the 3 ’-flank, the cDNA molecule comprises a poly-dT sequence conjugated to a primer binding site. In some embodiments, the reaction mixture further comprises one or more enzymes selected from reverse transcriptase and thermostable nucleic acid polymerase. In some embodiments, the reaction mixture further comprises amplification buffers and reagents for performing a polymerase chain reaction, such as described e.g., in PCR Strategies (Innis etal., 1995, Academic Press, San Diego, Calif.) at Chapter 14; or PCR Protocols: A Guide to Methods and Applications (Innis et al., Academic Press, N Y, 1990).

[0134] EXAMPLES

[0135] Example 1. Sequencing selected genes from a whole-transcriptome single cell cDNA library

[0136] A. Cells and cell preparation

[0137] The anti-CLLl CAR-T cells were engineered as described in PCT/US2023/079508. Frozen cells were thawed, washed, and resuspended in PBS containing BSA. The numbers of cells recommended in manual for the lOx Genomics Chromium Next GEM Single Cell 5’ v2 kit were used as described below.

[0138] B. Single cell cDNA library

[0139] The samples were prepared using the lOx Genomics Chromium Next GEM Single Cell 5’ v2 kit (10X Genomics, Pleasanton, Cal.). Briefly, nanodroplets containing gel beads in emulsion (GEMs) were mixed with cells diluted to a concentration that ensures that most droplets do not contain more than one cell. Each GEM comprised a poly-dT primer and a template-switch oligonucleotide (TSO) to enable synthesis of the cDNA library in each droplet. The reverse transcription primer includes a cell-specific “10X” barcode, a unique molecular barcode (UMI), and an amplification primer binding site (Rl).

[0140] C. Amplification of the library and preparation for sequencing [0141] Next, the nanodroplets were broken and the barcoded cDNA was pooled, and a portion of the pool was amplified in the first round of PCR with biotinylated primers. The forward primer was the primer from the Oxford Nanopore kit SQK-PCS111 (Oxford Nanopore Technologies (ONT), Oxford, UK) capable of binding to the R1 primer binding site, and the reverse primer was selected from Table 1 The reverse primer included a target-specific portion and an ONT primer binding site for the second round of PCR. The Long Amp Taq Hot Start 2x Master mix (New England Biolabs, Ipswich, Mass.) was used for the PCR. The amplified nucleic acid is then purified using AMPure XP beads (Beckman Coulter, Brea, Cal.), a rotator mixer and 70% ethanol. The sample is eluted into molecular biology grade water. Streptavidin M280 Dynabeads® (ThermoFisher Scientific, Waltham, Mass.) are washed and mixed with the sample on a rotator mixer. The sample is then washed again using lx wash/bind buffer containing Tris- HC1 pH7.5, NaCl, EDTA pH8 and molecular biology grade water. The sample is then washed with Tris-HCl pH7.5.

[0142] The pulled down sample was placed in a second round of PCR with a forward and reverse primers provided in ONT kit SQK-PCS 111. This amplification reaction also used the Long Amp Taq Hot Start 2x Master mix. The amplified reaction was also purified using AMPure XP beads as described above and eluted into Elution Buffer provided in the ONT kit.

[0143] D. Sequencing the cDNA library

[0144] The amplified library was quantified and qualified using Qubit dsDNA High Sensitivity kit (ThermoFisher Scientific) and the Agilent Bioanalayzer 12000 kit (Agilent, Santa Clara, Cal.). An adapter provided in the Oxford Nanopore kit was added to the library. The library was then loaded onto a flushed Oxford Nanopore MinlON FLO-MIN106D flow cell in conjunction with the sequencing buffer and loading beads, both provided in Oxford Nanopore kit. The library was sequenced using an Oxford Nanopore Technologies GridlON instrument. The basecalling was performed live on the instrument. The Oxford Nanopore’s Epi2Me wf-single-cell pipeline (with appropriate modifications to input parameters) was used to analyze data. Results are shown in Table 2.

[0145] Table 2. On-target sequence reads.

[00130] Example 2. Detecting full-length and isoform transcripts of the B2M-HLA-E fusion in engineered immune cells.

[00131] In this experiment, the method and primers described in Example 1 were applied to analysis and validation of manufactured lots of donor-derived engineered immune cells.

[00132] A. Cells and cell preparation

[00133] The anti-CLLl CAR-T cells were engineered as described in PCT/US2023/079508. Frozen cells were thawed, washed, and resuspended in PBS containing BSA. The numbers of cells recommended in manual for the lOx Genomics Chromium Next GEM Single Cell 5’ v2 kit were used as described below.

[00134] B. Single-cell cDNA library

[00135] The procedure used to generate and sequence amplified cDNA molecules from single-cell RNA transcripts utilized the single cell analysis technology (10X Genomics) combined with an antibody cocktail targeting a number of leukocyte cell surface antigens including principal lineage antigens and isotype control antibodies (TotalSeq™-C antibodies (BioLegend, San Diego, Cal.). These antibodies generate nucleotide transcript counts (antibody-derived tags (ADTs)) that are given the same cell barcode as the rest of the transcripts in that cell in the 10X Genomics workflow.

[00136] To perform the assay, cell suspensions were loaded onto the Chromium X instrument (10X Genomics, Pleasanton, Cal.) and processed as described in Example 1 .

[00137] C. Amplification of the library and preparation for sequencing

[00138] Following reverse transcription, the resulting cDNA library was amplified with the forward primer from the ONT kit SQK-PCS111 capable of binding to the R1 primer binding site, and the reverse primer selected from Table 1. The reverse primer included a target-specific portion and an ONT primer binding site for the second round of PCR. The fraction of the amplified library was used to generate a first sequencing library for long-read sequencing as described in Example 1. The long-read sequencing was performed on the GridlON platform (Oxford Nanopore Technologies, Oxford, UK) according to the manufacturer’s instructions. [00139] A separate fraction of the amplified library was used to generate a second sequencing library for short-read \sequencing and ADT library. The second round of PCR and short-read sequencing were performed on the Illumina platform (Illumina, San Diego, Cal.) according to the manufacturer’s instructions. The raw sequencing BCL files were demultiplexed, aligned to custom GRCh38 references and normalized using the corresponding sequencing instrument manufacturer’s software. Results are shown in Table 3. In Table 3, “No BHE” refers to cells where no B2M-HLA-E fusion transcripts are detectable; “Full BHE” refers to cells expressing the full-length B2M-HLA-E fusion; “Partial BHE” refers to cells expressing the B2M- HLA-E fusion with a deletion; “Both BHE” refers to cells expressing both the full-length and the deleted B2M-HLA-E fusion.

Table 3. B2M-HLA-E transgene isoforms in CAR-T cell preparations.

[0146] 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 scope of this invention. Thus, the scope of the invention should not be limited by the examples described herein, but by the claims presented below.

[0147] INFORMAL SEQUENCE LISTING

Claims

CLAIMS We Claim:

1. A method of detecting the presence or absence of one or more gene transcripts in one or more cells, the method comprising: a. contacting a sample comprising a plurality of cDNA molecules derived from one or more cells with one or more reverse amplification primers selected from SEQ ID NOs: 1-14, each reverse primer paired with a forward primer, b. amplifying a portion of selected cDNA sequences with the forward and the reverse primer, c. detecting the products of amplification produced in step b. thereby detecting the presence or absence of one or more gene transcripts.

2. The method of claim 1, wherein the one or more cells are engineered immune cells.

3. The method of claim 1, wherein the detecting comprises sequencing the amplification products.

4. The method of claim 1, wherein each cDNA molecule among the cDNA molecules comprises a unique cellular barcode identifying the cell from which the cDNA molecule was derived, and a unique molecular barcode identifying the cDNA molecule.

5. The method of claim 1, wherein the primer selected from SEQ ID NO.: 1-14 is conjugated at the 5 ’-end to a sequencing adaptor.

6. The method of claim 5. wherein the sequencing adaptor is SEQ ID NO.: 15.

7. The method of claim 1, wherein tire primer is conjugated to a capture moiety.

8. Tire method of claim 7, wherein tire capture moiety is biotin.

9. Tire method of claim 1, wherein tire reverse primer is SEQ ID NO: 1 and the transcript is from one or more exogenous gene introduced into tire cell and having the BGH poly adenylation site.

10. The method of claim 9, wherein the exogenous gene is a chimeric antigen receptor (CAR).

11. The method of claim 9, wherein the exogenous gene is a fusion between beta-2 microglobulin and an HLA Class I protein.

12. Tire method of claim 1, wherein tire reverse primer is SEQ ID NO: 2 and the lack of detectable transcript indicated biallclic inactivation of the TRAC gene.

13. The method of claim 1, wherein the reverse primer is SEQ ID NO: 2 and the reduced amount of detectable transcript indicated monoallelic inactivation of the TRAC gene.

14. The method of claim 1, wherein the reverse primer is SEQ ID NO: 5 and the lack of detectable transcript indicated biallelic inactivation of the PDCD1 gene.

1 . The method of claim 1, wherein the reverse primer is SEQ ID NO: 5 and the reduced amount of detectable transcript indicated monoallelic inactivation of the PDCD1 gene.

16. Tire method of claim 1, wherein tire reverse primer is selected from SEQ ID NO: 8-9 and the lack of detectable transcript indicated biallelic inactivation of tire CBLB gene.

17. The method of claim 1, wherein the reverse primer is selected from SEQ ID NO: 8-9 and the reduced amount of detectable transcript indicated monoallelic inactivation of the CBLB gene.

18. The method of claim 1, wherein tire reverse primer is SEQ ID NO: 3 and the lack of detectable transcript indicated biallelic inactivation of the B2M gene.

19. Tire method of claim 1, wherein tire reverse primer is SEQ ID NO: 3 and the reduced amount of detectable transcript indicated monoallelic inactivation of the B2M gene.

20. The method of claim 1, wherein the reverse primer is SEQ ID NO: 4 and the transcript is from the PTPRC gene.

21. Tire method of claim 1, wherein tire reverse primer is selected SEQ ID NOs: 6-7 and the transcript is from the ROR1 gene.

22. Tire method of claim 1, wherein tire reverse primer is selected from SEQ ID NOs: 11-14 and the transcript is from the CLL1 gene.

23. A kit for detecting tire presence or absence of one or more gene transcripts in one or more cells, the kit comprising one or more reverse amplification primers selected from SEQ ID NOs: 1-14, each reverse primer paired with a forward primer.

24. Tire kit of claim 23, further comprising reagents for amplifying and sequencing nucleic acids.

25. Tire kit of claim 23, wherein the primer selected from SEQ ID NO.: 1-14 is conjugated at the 5'- end to a sequencing adaptor.

26. The kit of claim 23. wherein the sequencing adaptor is SEQ ID NO.: 15.

27. The kit of claim 23, further comprising reagents for sequencing nucleic acids.

28. A reaction mixture for detecting the presence or absence of one or more gene transcripts in one or more cells, the reaction mixture comprising one or more reverse amplification primers selected from SEQ ID NOs: 1-14, each reverse primer paired with a forw ard primer.

29. The reaction mixture of claim 28, further comprising reagents for amplifying nucleic acids.

30. The reaction mixture of claim 28. wherein the primer selected from SEQ ID NO.: 1-14 is conjugated at the 5 ’-end to a sequencing adaptor.

31. The reaction mixture of claim 28, wherein the sequencing adaptor is SEQ ID NO.: 15.