Compositions and methods for quantifying recombining vector nucleic acid integration

Abstract

In certain aspects, the present disclosure relates to methods of quantifying integration of a recombinant vector nucleic acid into the genome of a target cell. The present disclosure also provides compositions and kits comprising particular primers and probes for performing the quantification.

Description

[0001] This application is a divisional application of the invention patent filed on March 8, 2021, with application number 202180020179.5 (PCT / EP2021 / 055816) and entitled "Composition and Method for Quantitative Integration of Nucleic Acids into Recombinant Vectors".

[0002] Related applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 987019, filed March 9, 2020, and U.S. Provisional Patent Application No. 63 / 148300, filed February 11, 2021, the entire contents of which are incorporated herein by reference. Background Technology

[0003] Recent advances in understanding the delivery and integration of genomic materials into the genome of target cells have enormous potential to transform the standard of care for a wide range of diseases. Chimeric antigen receptor (“CAR”) T-cell therapy has shown potential for treating B-cell malignancies and other cancers such as lymphocytic leukemia, B-cell lymphoma, sarcoma, neuroblastoma, and certain solid tumor cancers (see, for example, Sadelain et al., Cancer Discovery, Vol. 3: pp. 388–398, 2013, and Titov et al., Cancer, Vol. 12: pp. 125–146, 2020), and typically involves modifying T cells in vitro to generate CAR-expressing T cells. Most engineered T-cell gene manipulation methods for human applications utilize retroviruses, such as lentiviruses, for the stable expression of chimeric antigen receptors (CAR) (Jena et al., 2010; Ertl et al., 2011; Kohn et al., 2011). However, one of the challenges of current integration technologies using viral vector delivery is determining whether the vector has not only entered the cell, but also whether the transgene has been successfully integrated into the host genome (a process known as “viral integration”) and how many times (or the frequency of viral integration).

[0004] Southern blotting was initially the preferred technique for measuring the copy number integration of retroviral vector sequences, but it was slow and costly due to its manual nature. Recently, researchers have developed quantitative PCR (“qPCR”) methods to determine transduction efficiency and copy number. Some methods cannot distinguish between free, unincorporated vectors and integrated vectors, and therefore only provide an estimate of the copy number. See, for example, Charrier et al., Gene Therapy, Vol. 18: pp. 479–487, 2011. Some other methods use primers specific to particular transgenes. See also... Hum.Gene Ther.Volume 14: pp. 497-507, 2003. However, these methods either fail to produce accurate copy numbers or are costly due to the need to design different specific primers for each transgene. Summary of the Invention

[0005] This disclosure provides methods for quantifying the integration of recombinant vector nucleic acids (typically including transgenes) into the genome of target cells, methods that are not limited to or specific to any particular transgene. This disclosure also provides compositions and kits including specific primers and probes for performing this quantification.

[0006] In one aspect, this disclosure provides a method for quantifying the integration of recombinant vector nucleic acid into a cell genome, the method comprising: (a) providing a biological sample containing a host cell genome; (b) amplifying the genomic DNA of the biological sample using a primer pair containing a first oligonucleotide primer and a second oligonucleotide primer using a quantitative amplification technique, wherein at least one oligonucleotide primer of the primer pair specifically hybridizes to the integrated recombinant vector polynucleotide sequence; and (c) quantifying the genomic nucleic acid amplified in step (b).

[0007] In some implementations, quantification includes determining the copy number. In some implementations, the quantitative amplification technique is quantitative PCR. In some implementations, the quantitative amplification technique is digital PCR. In some implementations, the quantitative amplification technique is droplet digital PCR. In some implementations, the quantitative amplification technique is endpoint PCR.

[0008] In some embodiments, quantifying the integration of the recombinant vector nucleic acid into the host cell genome further includes comparing the copy number of the integrated recombinant vector sequence in the biological sample with a reference polynucleotide sequence. In some embodiments, the reference polynucleotide sequence encodes a housekeeping protein. In some embodiments, the housekeeping protein is albumin. In some embodiments, the copy number of the integrated recombinant vector sequence and the copy number of the reference polynucleotide sequence are measured in a multiplex manner. In some embodiments, the copy number of the integrated recombinant vector sequence and the copy number of the reference polynucleotide sequence are measured in a singlex manner.

[0009] In some embodiments, the method further includes evaluating the effectiveness of the assay for quantifying recombinant vector nucleic acid integration by assessing one or more assay acceptance criteria selected from the group consisting of: (a) the threshold cycling of both the provirus and the reference polynucleotide sequence is indeterminate in all replicates of the control without template DNA; (b) the correlation coefficient of the standard curves of both the provirus and the reference polynucleotide sequence generated by linear regression using standard samples is greater than or equal to 0.97; (c) the copy values ​​of the provirus and the reference polynucleotide sequence estimated from the slope of said standard curves indicate PCR efficiency between 90% and 110%; and (d) in replicates of either of the standard samples, the threshold cycling of both the provirus and the reference polynucleotide sequence is indeterminate. (e) The threshold cycle of either nucleotide sequence is indeterminate; (f) The average threshold cycle of both the provirus and reference polynucleotide sequences in the baseline standard sample is less than or equal to 22.0; (g) The standard deviation of the threshold cycle of both the provirus and reference polynucleotide sequences in each standard sample is less than or equal to 0.60; (h) The average measured copy number of the reference polynucleotide sequence in the one or more positive control samples is within 30% of the nominal expected value; (i) The measured average VCN / cell value of the one or more positive control samples is within 30% of the nominal expected VCN / cell value of each control; and (ii) The coefficient of variation of the VCN / cell value of the one or more positive control samples is less than or equal to 20%.

[0010] In some embodiments, the method further includes evaluating the effectiveness of quantitative recombinant vector nucleic acid integration in a sample by assessing one or more sample acceptance criteria selected from the group consisting of: (a) the mean copy value of the reference polynucleotide sequence in the sample is within 30% of the expected value of 30,303,030 copies; (b) if the genomic DNA (gDNA) concentration of the sample is less than 0.02 µg / µL, the expected copy value of the reference polynucleotide sequence in the sample is calculated based on the amount of DNA actually loaded into the reaction; (c) the mean pre-target viral copy value in the sample is within the validation range of the measured copy value; (d) the mean pre-target viral copy value in the sample is between 121,212,121 and 193,939 copies; (e) the coefficient of variation of the VCN / cell value of the target sample replica is less than or equal to 20%; and (f) the standard deviation of the cycling thresholds of both the pre-target virus and the target reference polynucleotide sequence in the sample is less than or equal to 0.60.

[0011] In some embodiments, the recombinant vector contains a transgene. In some embodiments, the recombinant vector is a gene therapy vector. In some embodiments, the recombinant vector is a viral vector. In some embodiments, the recombinant vector is a retroviral vector. In some embodiments, the recombinant vector is a lentiviral vector. In some embodiments, the lentivirus on which the lentiviral vector is based is human immunodeficiency virus 1 (HIV-1) or human immunodeficiency virus 2 (HIV-2).

[0012] In some embodiments, the method for quantifying the integration of recombinant vector nucleic acid into the cell genome further includes methods for identifying the transgene. In some embodiments, the methods for identifying the transgene include: (a) providing a biological sample containing the host cell genome; (b) amplifying the genomic DNA of the biological sample using a primer pair containing a first oligonucleotide primer and a second oligonucleotide primer using a quantitative amplification technique, wherein at least one oligonucleotide primer of the primer pair specifically hybridizes to the transgene; and (c) detecting and / or quantifying the genomic nucleic acid amplified in step (b). In some embodiments, the transgene is a polypeptide encoded by a nucleic acid sequence containing the sequence of SEQ ID NO: 19 or 21.

[0013] In some embodiments, the oligonucleotide primer that specifically hybridizes to the integrated recombinant vector polynucleotide sequence comprises the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 5, or SEQ ID NO: 14. In some embodiments, the second oligonucleotide primer for amplifying the recombinant vector nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 2, SEQ ID NO: 6, or SEQ ID NO: 15.

[0014] In some embodiments, oligonucleotide primers that specifically hybridize to the integrated recombinant vector polynucleotide sequence hybridize specifically to the LTR sequence of the integrated recombinant vector sequence. In some embodiments, oligonucleotide primers that specifically hybridize to the integrated lentiviral vector polynucleotide sequence hybridize specifically to the U3 region of the 5' LTR of the lentiviral vector nucleic acid sequence. In some embodiments, oligonucleotide primers that specifically hybridize to the integrated lentiviral vector polynucleotide sequence hybridize specifically to the U3 and R regions of the 5' LTR of the lentiviral vector nucleic acid sequence. In some embodiments, oligonucleotide primers that specifically hybridize to the integrated lentiviral vector polynucleotide sequence hybridize specifically to the PBS region of the 5' LTR of the lentiviral vector nucleic acid sequence. In some embodiments, oligonucleotide primers that specifically hybridize to the integrated lentiviral vector polynucleotide sequence hybridize specifically to the psi(Ψ) packaging signal. In some embodiments, the primers do not specifically hybridize to naturally occurring retroviral nucleic acid sequences.

[0015] In some embodiments, the method utilizes a detectable nucleic acid probe that specifically hybridizes with the amplified recombinant vector nucleic acid. In some embodiments, the probe that specifically hybridizes with the recombinant vector nucleic acid comprises a nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 16. In some embodiments, the probe for the integrated recombinant vector nucleic acid specifically hybridizes with the LTR sequence of the integrated recombinant vector nucleic acid. In some embodiments, the probe for the lentiviral vector nucleic acid used in step (b) specifically hybridizes with the U3 and R regions of the 5' LTR of the lentivirus. In some embodiments, the probe for the lentiviral vector nucleic acid used in step (b) specifically hybridizes with the U5 and PBS regions of the 5' LTR of the lentivirus. In some embodiments, the probe for the lentiviral vector nucleic acid used in step (b) specifically hybridizes with the PBS region of the 5' LTR of the lentivirus. In some embodiments, the probe for the lentiviral vector nucleic acid used in step (b) specifically hybridizes with the R and U5 regions of the 5' LTR of the lentiviral vector nucleic acid sequence. In some embodiments, step (b) utilizes an intercalation dye. In some implementations, the embedded dye is SYBR Green.

[0016] In some embodiments, the biological sample is a cell sample or a tissue sample. Specifically, in some embodiments, the tissue sample is blood, plasma, serum, saliva, or a tissue biopsy. In some embodiments, the sample is from a subject. In some embodiments, the subject is a human. In some embodiments, the recombinant vector nucleic acid sequence contains a transgene. In some embodiments, the transgene encodes a chimeric antigen receptor. In some embodiments, the chimeric antigen receptor contains the amino acid sequence of SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22. In some embodiments, the chimeric antigen receptor is a polypeptide encoded by a nucleic acid sequence containing the sequence of SEQ ID NO:19 or SEQ ID NO:21. In some embodiments, the chimeric antigen receptor recognizes BCMA, KLK2, or GPRC5D.

[0017] In some embodiments, the method further includes utilizing at least one pair of oligonucleotide primers that specifically amplify a reference polynucleotide sequence.

[0018] On the other hand, this disclosure provides a method for monitoring the transduction efficiency of recombinant vector nucleic acids, the method comprising: a) providing one or more biological samples containing genomic DNA transduced by recombinant vector nucleic acids, wherein a portion of the recombinant vector nucleic acid is integrated into the genomic DNA; and b) quantifying the recombinant vector nucleic acid integrated into the host cell genome according to the method of this disclosure. In some embodiments, the method further comprises comparing the copy number of the recombinant vector sequence of the biological sample with a reference.

[0019] In another aspect, this disclosure provides a method for batch release testing of cell products transduced by a recombinant vector, the method comprising: a) providing one or more biological samples from each batch of cell products containing genomic DNA transduced by a recombinant vector; b) quantifying the recombinant vector nucleic acid integrated into the host cell genome in each biological sample according to the method of this disclosure; c) comparing the copy number of the integrated recombinant vector sequence quantified for the biological sample in step (b) with a reference; and d) releasing the copy number of the integrated recombinant vector sequence therein through a predetermined standard batch.

[0020] In another aspect, this disclosure provides a method for quantifying the integration of retroviral vector nucleic acid into a cellular genome, the method comprising: (a) providing a biological sample containing a host cell genome; (b) amplifying the genomic DNA of the biological sample using a primer pair containing a first oligonucleotide primer and a second oligonucleotide primer using a quantitative amplification technique, wherein at least one oligonucleotide primer of the primer pair specifically hybridizes to the integrated retroviral vector polynucleotide sequence; and (c) quantifying the genomic nucleic acid amplified in step (b), wherein quantifying the integration of retroviral vector nucleic acid into the host cell genome comprises comparing the amplified retroviral vector nucleic acid with a reference ratio, wherein the retroviral vector nucleic acid is a lentiviral vector sequence, and wherein the oligonucleotide primer pair comprises the nucleic acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2, respectively, or the nucleic acid sequences of SEQ ID NO: 5 and SEQ ID NO: 6, respectively, or the nucleic acid sequences of SEQ ID NO: 14 and SEQ ID NO: 15, respectively. In some embodiments, a detectable probe comprising a nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 16 is used quantitatively. In another aspect, this disclosure provides an oligonucleotide comprising a nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.

[0021] In another aspect, this disclosure provides a kit for measuring the copy number of an integrated recombinant vector nucleic acid sequence, the kit comprising: primers for a primer pair that specifically hybridize with the integrated recombinant vector nucleic acid, wherein the primer pair specifically amplifies a portion of the integrated recombinant vector nucleic acid; and a detectable nucleic acid probe that specifically hybridizes with the amplified recombinant vector nucleic acid.

[0022] In another aspect, this disclosure provides a kit for measuring the copy number of an integrated recombinant vector nucleic acid sequence, the kit comprising: a forward primer that specifically hybridizes with an integrated recombinant vector nucleic acid containing a nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 5, or SEQ ID NO: 14; a reverse primer that specifically hybridizes with an integrated recombinant vector nucleic acid containing a nucleic acid sequence of SEQ ID NO: 2, SEQ ID NO: 6, or SEQ ID NO: 15; and a detectable probe containing a nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 16.

[0023] This disclosure covers all combinations of any of the foregoing aspects and embodiments, as well as combinations of any of the embodiments set forth in the detailed description and examples. Attached Figure Description

[0024] For the purpose of illustrating the invention, the accompanying drawings depict certain embodiments of the present disclosure. However, the present disclosure is not limited to the precise arrangement and means of the embodiments depicted in the drawings.

[0025] Figure 1A An exemplary lentiviral vector transfer plasmid containing a transgene encoding a chimeric antigen receptor (CAR) is shown. Figure 1B The image depicts cells after a portion of the vector has been integrated into target cells, and after the integrated transgene has been expressed on the cell surface. (Example) Figure 1A As described, the lentiviral vector transgene encodes a CAR flanked by 5'-LTR (site C) and 3'-LTR (site D). For example... Figure 1B As depicted, when a lentiviral vector is incorporated into the genome of a cell, a portion of the 5'-LTR region (site A) is replaced by the ΔU3 portion of the 3'-LTR sequence. Rectangular black boxes indicate exemplary primer and / or probe binding sites, and "X" indicates the absence of a primer binding site to produce a quantitatively megason. The integrated 5'-LTR (site a) has a ΔU3 site, while the transfer plasmid 5'-LTR (site C) does not.

[0026] Figure 2Alignment of selected primers and probes of this disclosure with exemplary lentiviral vector sequences is shown. The following sequences are depicted: example lentiviral vector sequence (SEQ ID NO: 13); 5LTR_to 1 and 2_F primers (SEQ ID NO: 1); 5LTR_to 1_probe (SEQ ID NO: 3); 5LTR_to 1 and 2_R primers (SEQ ID NO: 2); 5LTR_to 2_probe (SEQ ID NO: 4); 5LTR_to 3_F primer (SEQ ID NO: 5); 5LTR_to 3_probe (SEQ ID NO: 7); 5LTR_to 3_R primer (SEQ ID NO: 6), 5LTR_to 4_F primer (SEQ ID NO: 14); 5LTR_to 4_R primer (SEQ ID NO: 15); 5LTR_to 4_probe (SEQ ID NO: 16).

[0027] Figures 3A to 3B The results of side-by-side qPCR (3A) and flow cytometry (3B) quantification of GPRc5d CAR-T in blood are shown. Detailed Implementation

[0028] Overview This invention provides a method for detecting and / or quantifying the integration of recombinant vector nucleic acids and thus detecting and / or quantifying transgenes delivered into the genome of host cells. There is a need for specific detection and / or quantification of transgene sequences delivered by recombinant vectors in transduced cells such as CAR-T cells. To this end, the inventors have developed a method for detecting and / or quantifying the integration of recombinant vector nucleic acids (such as CAR) encoding transgenes, but not for detecting and / or quantifying residual plasmids used to deliver transgenes or unintegrated recombinant vector nucleic acid sequences. The ability to identify whether a vector has been successfully incorporated into cells with transgene-independent sequences allows for more accurate, universal, and inexpensive quantification using quantitative PCR, digital PCR, or other quantitative methods. This disclosure provides methods and kits for identifying and quantifying genetic information delivered and incorporated via recombinant vectors by utilizing changes in the 5'-LTR sequence relative to the 5'-LTR sequence in the transfer plasmid vector after integration into the host genome. This information, including copy number, is crucial for understanding and optimizing transfection or transduction conditions. The method of this invention facilitates monitoring the transduction efficiency and in vivo characterization of lentiviral vectors. Therefore, this disclosure provides an improved method for quantifying the integration of recombinant vector nucleic acids into the genome of target cells. Although the exemplary embodiments described herein involve the detection of integrated retroviral vector nucleic acids, the methods of the present invention can be applied to any recombinant nucleic acid vector sequence that can be distinguished from unintegrated nucleic acids when integrated into the host genome.

[0029] Throughout this application, various documents have been cited. The full disclosure of these documents is incorporated herein by reference.

[0030] Unless otherwise defined herein, the scientific and technical terms used in connection with this invention shall have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include plural terms, and plural terms shall include singular terms. Generally, the nomenclature and techniques used herein in conjunction with cell and tissue culture, molecular biology, cell and cancer biology, virology, immunology, microbiology, genetics, and protein and nucleic acid chemistry are those well-known and commonly used in the art. Each embodiment of the invention described herein may be used alone or in combination with one or more other embodiments of the invention.

[0031] Unless otherwise stated, the methods and techniques of this invention are generally carried out according to conventional methods well known in the art, and as described in the various general and more specific references cited and discussed in this specification. Unless otherwise stated, the methods and techniques of various embodiments are generally carried out according to methods well known in the art, such as molecular biology, cell biology, biochemistry, microarrays, and sequencing technologies, and as described in the various general and more specific references cited and discussed in this specification.See, for example, Motulsky, “Intuitive Biostatistics”, Oxford University Press, Inc., 1995; Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates, 1992, with a supplement in 2003; Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1990; Coffin et al., Retroviruses, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1997; Bast et al., Cancer Medicine, 5th ed., edited by Frei and Emil, BC Decker Inc., Hamilton, Canada, 2000; Lodish et al., Molecular Cell Biology, 4th edition, WH Freeman & Co., New York, 2000; Griffiths et al., Introduction to Genetic Analysis, 7th edition, WH Freeman & Co., New York, 1999; Gilbert et al., Developmental Biology, 6th edition, Sinauer Associates, Inc., Sunderland, MA, 2000; and Cooper, The Cell AMolecular Approach, 2nd edition, Sinauer Associates, Inc., Sunderland, MA, 2000.

[0032] Chemical terms used in this article are used in accordance with the usual usage in the field, as illustrated in the McGraw-Hill Dictionary of Chemical Terms (edited by Parker S., McGraw-Hill, San Francisco, CA, 1985).

[0033] All of the above and any other publications, patents and published patent applications mentioned in this application are incorporated herein by reference. In case of any conflict, this specification and its express definitions shall prevail.

[0034] definition As used herein, the terms “polynucleotide,” “nucleic acid,” and “nucleic acid molecule” are used interchangeably. They refer to nucleotides of any length, in polymeric form, of DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), DNA-RNA hybrids, and their analogues. Nucleic acid molecules can be nucleotides, oligonucleotides, double-stranded DNA, single-stranded DNA, multi-stranded DNA, complementary DNA, genomic DNA, non-coding DNA, messenger RNA (mRNA), microRNA (miRNA), small nucleolar RNA (snoRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), small interfering RNA (siRNA), heteronuclear RNA (hnRNA), or small hairpin RNA (shRNA). In some embodiments, these methods may be performed on nucleic acid samples such as DNA or RNA (e.g., genomic DNA).

[0035] Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be conferred before or after polymer assembly. The nucleotide sequence can be interrupted by non-nucleotide components. Polynucleotides can be further modified, such as by conjugation with labeled components. The term "recombinant" polynucleotide refers to a genomic, cDNA, semi-synthetic, or synthetically derived polynucleotide that is not found in nature or is linked to another polynucleotide in a non-natural arrangement. Polynucleotides can be operatively linked to "expression control sequences," which are nucleotide sequences that regulate gene expression.

[0036] The term "endogenous" refers to proteins, nucleic acids, cells, or another molecule originating from within the subject's body.

[0037] The term "exogenous" refers to a protein, nucleic acid, cell, or another molecule derived from an external source of the subject. Non-limiting examples of exogenous molecules include: recombinant proteins, plasmids, viruses, cells from donor subjects, tissues from donor subjects, organs from donor subjects, or synthetic chemicals.

[0038] Throughout this specification, the word “including” or variations thereof such as “comprising” or “containing” shall be understood to imply inclusion of the stated integer or group of integers, but not to exclude any other integer or group of integers.

[0039] Furthermore, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the / that” include plural references.

[0040] The term "including" is used to mean "including but not limited to". "Including" and "including but not limited to" are used interchangeably.

[0041] The terms “subject” and “individual” are used interchangeably and refer to any animal, such as a dog, cat, bird, livestock, and especially a mammal, and preferably a human.

[0042] The term "recombinant vector nucleic acid" refers to a nucleic acid sequence containing a vector for delivering nucleic acid, which includes at least one modification compared to a naturally occurring sequence, and may contain a transgene encoding a foreign protein that will be expressed in the genome of a transduced cell.

[0043] The term "retroviral vector nucleic acid" refers to a nucleic acid sequence that is at least a portion of a retroviral vector, includes at least one modification compared to a naturally occurring retroviral sequence, and may contain a transgene encoding a foreign protein to be expressed in the genome of the transduced cell, such as a portion of a lentiviral vector transfer plasmid. A "retroviral vector nucleic acid sequence" does not contain naturally occurring retroviral nucleic acids unrelated to the retroviral vector. Retroviral vector nucleic acids that have been integrated into the host cell genome are sometimes referred to as "proviral nucleic acids."

[0044] The term "retroviral vector" refers to a vector containing structural and functional genetic elements primarily derived from retroviruses.

[0045] As used herein, the term "retrovirus" refers to an RNA virus that transcribes its genomic RNA into a linear double-stranded DNA copy and subsequently covalently integrates its genomic DNA into the host genome. Exemplary retroviruses suitable for specific implementations include, but are not limited to: Moloney murine leukemia virus (MoMLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibberish leukemia virus (GaLV), feline leukemia virus (FLV), foam virus, Freund's murine leukemia virus, murine stem cell virus (MSCV), Rous sarcoma virus (RSV), and lentiviruses.

[0046] The term "lentiviral vector" is a subset of retroviral vectors, referring to a vector containing structural and functional genetic elements primarily derived from lentiviruses. In some embodiments, lentiviral vectors are generated according to known methods. See, for example, Kutner et al., BMC Biotechnol., 2009; Kutner et al., Nat. Protoc., 2009. This invention includes recombinant, retroviral, and lentiviral vector constructs expressing transgenes that can be directly transduced into cells. In certain embodiments, lentiviral vectors are used to deliver polynucleotides encoding CARs into cells.

[0047] According to certain specific embodiments considered herein, the majority or all of the viral vector backbone sequence is derived from lentiviruses, such as HIV-1. However, it should be understood that many different sources of retroviral and / or lentiviral sequences can be used or combined, and numerous substitutions and alterations to certain lentiviral sequences can be adapted without impairing the transfer vector's ability to perform the functions described herein. Furthermore, a variety of lentiviral vectors are known in the art; see Naldini et al. (1996a, 1996b, and 1998); Zufferey et al., 1997; Dull et al., 1998, U.S. Patents 6,013,516 and 5,994,136, many of which can be adapted to produce the viral vectors or transfer plasmids considered herein.

[0048] "Lentviral viruses" refers to the retrovirus group (or genus) that causes slowly developing diseases. Viruses included in this group include HIV (human immunodeficiency virus; including HIV types 1 and 2), the pathogen of human acquired immunodeficiency syndrome (AIDS); Wisner-Meddy disease, which causes encephalitis (Wisner disease) or pneumonia (Meddy disease) in sheep; caprine arthritis-encephalitis virus, which causes immunodeficiency, arthritis, and encephalopathy in goats; equine infectious anemia virus, which causes autoimmune hemolytic anemia and encephalopathy in equines; feline immunodeficiency virus (FIV), which causes immunodeficiency in cats; bovine immunodeficiency virus (BIV), which causes lymphadenopathy, lymphocytosis, and possible central nervous system infections in cattle; and simian immunodeficiency virus (SIV), which causes immunodeficiency and encephalopathy in subhuman primates. Diseases caused by these viruses are characterized by long incubation periods and long disease courses. Typically, the viruses latently infect monocytes and macrophages, and then spread to other cells. HIV, FIV, and SIV are also susceptible to infecting T lymphocytes (i.e., T cells). In various embodiments, the lentiviral vectors considered herein comprise one or more LTRs, and one or more of the following auxiliary elements: cPPT / FLAP, Psi(Ψ) packaging signal, leading element, polyadenylation sequence, and optionally include WPRE or HPRE as discussed elsewhere herein, insulator element, optional marker, and cell suicide gene.

[0049] As a result of LTR modification, lentiviral vectors preferably contain several safety enhancements. "Self-activating" (SIN) vectors refer to replication-defective vectors. The term "self-inactivating vector" refers to a vector in which the right (3') LTR enhancer-promoter region, known as the U3 region, has been modified (e.g., by deletion or substitution) to prevent viral transcription beyond the first round of viral replication. In some embodiments, LTR U3 is ΔU3. Therefore, the vector is able to infect and then integrate into the host genome only once, and may not be able to spread further. This is because the right (3') LTR U3 region serves as a template for the left (5') LTR U3 region during viral replication, and therefore, in the absence of the U3 enhancer-promoter, viral transcripts may not be able to replicate. If viral transcripts cannot replicate, they may not be processed or packaged into virions, thus ending the viral life cycle. Therefore, SIN vectors significantly reduce the risk of generating unwanted, replicating viruses because the right (3') LTR U3 region has been modified to prevent viral transcription beyond the first round of replication, thereby eliminating the ability of the virus to spread. Additional safety enhancements are provided by replacing the U3 region of the 5' LTR with a heterologous promoter to drive transcription of the viral genome during viral particle production. Examples of heterologous promoters that can be used include, for example, the promoters of viral simian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g., immediate early), Moloney murine leukemia virus (MoMLV), Rous sarcoma virus (RSV), and herpes simplex virus (HSV) (thymidine kinase) (TSV).

[0050] The term "long terminal repeat (LTR)" refers to a base-pair domain located at the end of certain recombinant DNA sequences, such as those of retroviruses. In their natural sequence environment, these are directly repeated and contain U3, R, and U5 regions. LTRs typically provide essential functions for the expression of recombinant genes (e.g., promotion, initiation, and polyadenylation of gene transcripts) and viral replication.

[0051] The term "R region" refers to the region within the recombinant LTR that begins at the capping group (i.e., transcription initiation) and immediately terminates before the start of the polyadenylate chain. The R region is also defined as flanking the U3 and U5 regions. The R region plays a crucial role during reverse transcription, allowing the transfer of nascent DNA from one end of the genome to the other. The term "PBS region" refers to the region downstream of the U5 region of the 5'-LTR, which serves as the tRNA. Lys The primer binding site (PBS) of the primer is essential for initiating reverse transcription.

[0052] The term "specific hybridization with integrated recombinant vector nucleic acid" refers to the specific hybridization of nucleic acid (e.g., primers) with recombinant vector nucleic acid that has been integrated into the host cell genome, but not with residual plasmids used to deliver transgenes or unintegrated recombinant vector nucleic acid sequences.

[0053] Test and measurement The efficacy and properties of the recombinant vector nucleic acids of this disclosure can be readily tested using any of a number of available in vitro or in vivo assays. Several such assays are described below. This disclosure contemplates that any of these assays, as well as other assays known in the art, can be used to test any of the recombinant vector nucleic acids of this disclosure.

[0054] In some embodiments, quantitative polymerase chain reaction (qPCR) is used to quantify the integration of recombinant vector nucleic acid into the target cell genome. In some embodiments, digital polymerase chain reaction (dPCR) is used to quantify the integration of recombinant vector nucleic acid into the target cell genome. In some embodiments, droplet digital polymerase chain reaction (ddPCR) is used to quantify the integration of recombinant vector nucleic acid into the target cell genome. In these embodiments, cultures of cells expressing the recombinant vector nucleic acid (e.g., PBMC cells) are collected and primers specific to the LTR sequence of the integrated recombinant vector nucleic acid are prepared for qPCR or dPCR. Such experiments detect the integrated recombinant vector nucleic acid sequence encoding CAR in the transduced CAR-T cell genome but do not detect residual non-integrated transfer plasmids.

[0055] Methods and uses disclosed herein This disclosure provides methods for detecting and / or quantifying the integration of recombinant vector nucleic acids into the cellular genome. In some embodiments of this method, a biological sample comprising the host cell genome is provided. Providing biological material includes providing fresh biological material, such as biological material acquired at a given time for this analytical purpose. Providing biological material also includes using previously acquired biological material collected at another point during patient care for this or other purpose, or using archived patient material. The biological material may be freshly acquired or previously acquired, and in the case of previously acquired material, it may have been stored prior to use (e.g., at room temperature, refrigerated, or frozen). Exemplary biological materials include, but are not limited to, whole blood, serum, plasma, urine, feces, cerebrospinal fluid, ascites, etc.

[0056] In some embodiments, the biological material may be purified or otherwise processed to isolate genomic DNA from the biological sample. This processing may include HPLC, size exclusion chromatography, gel electrophoresis, affinity chromatography, commercial DNA extraction or purification kits, or other purification techniques.

[0057] Methods for amplifying genomic DNA from biological samples include, but are not limited to, polymerase chain reaction (PCR), linear polymerase reaction, nucleic acid sequence-based amplification (NASBA), rolling circle amplification, digital PCR (dPCR), droplet digital PCR (ddPCR), endpoint PCR, etc., which are disclosed herein by reference in the following sources: Mullis et al., U.S. Patents 4,683,195, 4,965,188, 4,683,202, 4,800,159 (PCR); Gelfand et al., U.S. Patent 5,210,015 (using "TAQMAN"). ™ (Real-time PCR of probes); Wittwer et al., U.S. Patent 6,174,670; Kacian et al., U.S. Patent 5,399,491 (“NASBA”); Lizardi, U.S. Patent 5,854,033; Aono et al., Japanese Patent Publication JP4-262799 (Rolling Circle Amplification), etc.

[0058] In some implementations, the genomic DNA of the biological sample is then amplified using quantitative amplification techniques. Quantitative amplification techniques are known to those skilled in the art and include quantitative PCR (qPCR), digital PCR, and endpoint PCR. "qPCR" or "real-time quantitative PCR" (real-time quantitative polymerase chain reaction) refers to an experimental method that uses PCR to simultaneously amplify and quantify target nucleic acids. Various measurement chemicals (including, for example, SYBR) are used. ™ Quantification was performed using a green fluorescent dye or a fluorescent reporter oligonucleotide probe (TaqMan probe), and real-time quantification was performed using the amplified DNA accumulated in the reaction after each amplification cycle. For a description of the digital PCR method, see, for example, Hindson et al., 2011, Anal. Chem., Vol. 83, No. 22: 8604-8610; Pohl and Shih, 2004, Expert Rev. Mol. Diagn., Vol. 4, No. 1: 41-47; Pekin et al., 2011, Lab Chip, Vol. 11, No. 13: 2156-2166; Pinheiro et al., 2012, Anal. Chem., Vol. 84, No. 2: 1003-1011; Day et al., 2013, Methods, Vol. 59, No. 1: 101-107; the full text of these references are incorporated herein by reference.

[0059] As used herein, the term "primer" means an oligonucleotide that can serve as a starting point for synthesis under conditions that induce the synthesis of primer extension products complementary to the nucleic acid strand (template), namely, in the presence of a polymerization mixture containing nucleotides and DNA polymerase, and suitable temperature and pH conditions. In some embodiments, the primer is a deoxyribonucleotide and is single-stranded. Primers used in this invention may include naturally occurring dNMPs (i.e., dAMP, dGMP, dCMP, and dTMP), modified nucleotides, or non-natural nucleotides. Additionally, primers may also include ribonucleotides.

[0060] Primers should be long enough to initiate the synthesis of extended products in the presence of the polymer mixture. The appropriate primer length is determined by many factors, such as temperature, application, and primer source, and is typically 15 to 30 nucleotides. Short primer molecules generally require lower temperatures to form a sufficiently stable hybridization complex with the template.

[0061] In some embodiments, a detectable nucleic acid probe (e.g., an integrated retroviral nucleic acid amplicon or a control nucleic acid amplicon) that specifically hybridizes with the amplification product generates a detectable signal in the amplification reaction. In some embodiments, the detectable nucleic acid probe comprises a detectable reagent. In some embodiments, the detectable nucleic acid probe comprises a quencher. Detectable reagents and quenchers are disclosed in U.S. Patent Publication 20190284610, which is incorporated herein by reference.

[0062] In some implementations, fluorescence analysis can be performed by a commercial detector (e.g., Biorad's droplet reader), and the fluorescence signal of each droplet can be detected in the device, and the number of positive and negative droplets can be counted, and the analysis can be completed automatically.

[0063] In some embodiments, the term "detectable nucleic acid probe" refers to a TaqMan probe used for quantitative PCR. In some embodiments, fluorescent material (HEX, VIC, FAM dye) is attached to the probe. In some embodiments, 3lABkFQ can be used as a quencher on the 3' side of the probe. The TaqMan probe is an oligonucleotide labeled with a fluorescent material at the 5' end and a quencher material at the 3' end. The TaqMan probe specifically hybridizes with the template DNA during the annealing step, but does not show fluorescence even under light because of the quencher at the 3' end of the probe. In the following extension step, the 5' to 3' exonuclease activity of Taq DNA polymerase degrades the TaqMan probe hybridized with the template. The fluorescent material then separates from the probe, and the inhibition of the quencher is released. This principle allows for the quantitative visualization of fluorescence induced by the PCR reaction.

[0064] In some embodiments, fluorescence quenching assays can be used, wherein the probe according to the invention comprises a fluorophore and a quencher, which are positioned such that fluorescence is quenched in the absence of target nucleic acid and at a temperature below the probe's Tm. According to the invention, a variety of fluorophores can be used in the probe and primers. Available fluorophores include coumarin, fluorescein (FAM), tetrachlorofluorescein, hexachlorofluorescein, fluorescein yellow, rhodamine, BODIPY, tetramethylrhodamine, Cy3, Cy5, Cy7, eosin, Texas red, and ROX. For example, combined fluorophores such as the fluorescein-rhodamine dimer described by Lee et al. (1997, Nucleic Acids Research, Vol. 25: p. 2816) are also suitable. The fluorophore can be selected to absorb and emit within or outside the visible spectrum, such as in the ultraviolet or infrared range. Suitable quenchers described in the art include 3lABkFQ, DABCYL, and variants thereof, such as DABSYL, DABMI, and methyl red. Phosphores can also be used as quenchers because they tend to quench fluorescence when in the vicinity of certain other fluorophores. In some embodiments, a preferred quencher is 3lABkFQ. In some embodiments, a preferred fluorophore is fluorescein (FAM). In some embodiments, a preferred internal quencher is ZEN. In some embodiments, a preferred fluorophore is VIC.

[0065] In some embodiments, quantitative PCR is used to simultaneously amplify the genomic DNA of a biological sample using at least one pair of oligonucleotide primers that specifically amplify a reference polynucleotide sequence and using a primer pair comprising a first oligonucleotide primer and a second oligonucleotide primer, wherein at least one oligonucleotide primer of the primer pair specifically hybridizes to the integrated retroviral vector polynucleotide sequence. Specifically, as... Figure 1B As shown, reference polynucleotide sequences can be used to determine the number of cells present in a sample using genomic information, and thus allow for copy number estimation. This improves the accuracy of previous methods, which typically use transformation factors derived from the quality of genetic material in the sample to determine copy number. Also, Figure 1B As shown, a retroviral vector sequence including a multinucleotide encoding a CAR can be incorporated into the host cell genome. This incorporated retroviral vector sequence specifically includes the 5'-LTR δ U3 region, which is absent in the unincorporated vector, such as... Figure 1A As shown. This method involves including only... Figure 1B The primers are complementary to the 5'-LTR δ U3 region, thus exhibiting selectivity only for integrated retroviral vector sequences and not for unintegrated retroviral vectors.

[0066] In some embodiments, the retroviral vector nucleic acid is based on a member of the lentivirus genus. Specifically, lentiviruses have demonstrated strong transfection efficiency and are a validated tool for incorporating genetic information into host cells. In some embodiments, the lentivirus is human immunodeficiency virus 1 (HIV-1) or human immunodeficiency virus 2 (HIV-2). In some embodiments, primers for amplifying the integrated retroviral vector nucleic acid specifically hybridize to the LTR sequence of the integrated retroviral vector sequence. In some embodiments, primers for amplifying the retroviral vector nucleic acid specifically hybridize to the U3 region of the 5' LTR of the lentivirus. In some embodiments, primers for amplifying the retroviral vector nucleic acid specifically hybridize to the U3 and R regions of the 5' LTR of the lentivirus. In some embodiments, primers for amplifying the retroviral vector nucleic acid specifically hybridize to the PBS region of the 5' LTR of the lentivirus. In some embodiments, primers for amplifying the retroviral vector nucleic acid specifically hybridize to the PBS and R regions of the 5' LTR of the lentivirus. In some embodiments, the primers used to amplify the retroviral vector nucleic acid specifically hybridize with the psi(Ψ) packaging signal. In some embodiments, the sequence specific to the incorporated retroviral vector nucleic acid comprises the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:5, or SEQ ID NO:14. In some embodiments, the sequence specific to the incorporated retroviral vector nucleic acid comprises the nucleic acid sequence of SEQ ID NO:2, SEQ ID NO:6, or SEQ ID NO:15. In some embodiments, a detectable nucleic acid probe is used that specifically hybridizes with the amplified retroviral vector nucleic acid. Specifically, in some embodiments, the probe that specifically hybridizes with the retroviral vector nucleic acid comprises the nucleic acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, or SEQ ID NO:16. In some embodiments, the probe for specific hybridization with the nucleic acid of the retroviral vector comprising the nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 16 further comprises a fluorophore or quencher located at the 5' end, 3' end, and / or between the ninth and tenth nucleotides measured from the 5' end of the sequence. In some embodiments, the probe for specific hybridization with the nucleic acid of the retroviral vector consists of the nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 16, including a fluorophore or quencher located at the 5' end, 3' end, and / or internally. In a specific embodiment, the internal quencher is located between the ninth and tenth nucleotides measured from the 5' end of the sequence.In some embodiments, the probe has a 5' fluorophore, an internal quencher, and a 3' second quencher. In some embodiments, the preferred quencher is 3lABkFQ. In some embodiments, the preferred fluorophore is fluorescein (FAM). In some embodiments, the preferred internal quencher is ZEN. In some embodiments, the preferred fluorophore is VIC. In some embodiments, the probe that specifically hybridizes to the retroviral vector nucleic acid is / FAM / CTTTCAAGT / ZEN / CCCTGTTCGGGCGCC / 3lABkFQ / (SEQ ID NO: 12) or / 56-FAM / TGCCTTGAG / ZEN / TGCTTCAAGTAGTGTGT / 3IABkFQ / (SEQ ID NO: 17). In some embodiments, the primers amplifying a reference sequence present in all host cells contain a nucleic acid sequence that specifically hybridizes to a portion of the sequence encoding a housekeeping protein. Specifically, in some embodiments, the housekeeping protein is albumin. In some embodiments, the primer sequences that specifically hybridize to a portion of the albumin gene are SEQ ID NO: 8 and / or 9. In some embodiments, the sequence of the probe that specifically hybridizes with albumin nucleic acid is SEQ ID NO: 10. In some embodiments, the probe that specifically hybridizes with albumin nucleic acid is / 5HEX / AGGGAGA / ZEN / GATTTGTGTGGGCATGAC / 3IABkFQ / (SEQ ID NO: 11).

[0067] In this method, in some implementations, the amplified genomic nucleic acids are then detected and / or quantified, wherein the ratio of amplified retroviral vector nucleic acids to reference nucleic acids is correlated with the copy number of the retroviral vector nucleic acids integrated into the cellular genome. Specifically, as discussed above, because a primer pair is specific to polynucleotide sequences that appear only when a retroviral vector is incorporated into the host cell genome, the number of sequences counted by qPCR is proportional to the number of retroviral vectors integrated into the sample. Dividing by the number of detected reference polynucleotide sequences allows for the direct calculation of the copy number of a given biological sample.

[0068] In some embodiments, this disclosure provides a method for determining the copy number of integrated retroviral vector nucleic acid in the genome of a subject. In some embodiments of this method, a biological sample containing the subject's genomic DNA is provided. In some embodiments of this method, a portion of the integrated retroviral vector nucleic acid and a reference polynucleotide sequence in the subject's genome are amplified in the biological sample by quantitative PCR. In some embodiments of this method, (i) the amount of amplified polynucleotide containing the retroviral vector nucleic acid sequence and (ii) the amount of amplified polynucleotide containing the reference polynucleotide sequence are determined, and a ratio of (i) to (ii) is determined, wherein the ratio corresponds to the copy number of integrated retroviral vector nucleic acid.

[0069] In some embodiments, amplification includes combining a sample with a composition containing primer pairs specific to the integrated retroviral vector nucleic acid sequence and performing quantitative PCR. In some embodiments, amplification includes combining a sample with a composition containing primer pairs specific to the integrated retroviral vector nucleic acid sequence and performing quantitative amplification. In some embodiments, amplification further includes using primer pairs specific to a reference polynucleotide sequence and performing quantitative amplification, wherein the integrated retroviral vector nucleic acid sequence and the reference polynucleotide are amplified in substantially equal proportions. In some embodiments, one primer of the primer pair for the integrated retroviral vector nucleic acid sequence specifically hybridizes to the 5'LTR. In some embodiments, one primer of the primer pair for amplifying the retroviral vector nucleic acid specifically hybridizes to the U3 region of the 5'LTR. In some embodiments, one primer of the primer pair for the integrated retroviral vector nucleic acid sequence specifically hybridizes to both the U3 and R regions of the 5'LTR. In some embodiments, one primer of the primer pair for amplifying the retroviral vector nucleic acid specifically hybridizes to the PBS region of the 5'LTR. In some implementations, the integrated retroviral vector nucleic acid sequence contains a transgene. In some implementations, the transgene encodes a chimeric antigen receptor (CAR) that specifically binds to a target antigen.

[0070] This disclosure provides yet another method for monitoring the transduction efficiency of retroviral vectors. In some embodiments, one or more biological samples containing genomic DNA are provided, transduced from retroviral vector nucleic acids, wherein a portion of the retroviral vector nucleic acid is integrated into the genomic DNA. In some embodiments, the copy number of the retroviral vector sequence integrated into the genomic DNA of each biological sample is determined according to the method described herein. In some embodiments, the copy number of the retroviral vector sequence of the biological sample is compared with a reference.

[0071] This disclosure also provides a method for batch release testing of cell products transduced by retroviral vectors. In some embodiments, one or more biological samples containing genomic DNA from each batch of cell products transduced by retroviral vectors are provided. In some embodiments, the copy number of the integrated retroviral vector sequence in each biological sample is determined according to the method of this disclosure. In some embodiments, the copy number of the retroviral vector sequence in the biological sample is compared with a reference. In some embodiments, the copy number of the integrated retroviral vector sequence therein is determined through a predetermined standard of release. In some embodiments, the predetermined standard may be a copy number range or a percentage change from a control.

[0072] This disclosure also provides assay acceptance criteria and sample acceptance criteria for determining the validity of assays or samples, respectively, for quantifying recombinant vector nucleic acid integration. In some embodiments, integration of the recombinant vector nucleic acid sequence is determined by calculating a vector copy number (VCN / cell) value per cell, defined as twice the ratio of the measured amount of provirus to a reference polynucleotide sequence. In some embodiments, the amount of provirus or reference polynucleotide sequence is determined via quantitative PCR (qPCR). In some embodiments, the amount of provirus or reference polynucleotide sequence is determined via quantitative PCR (qPCR) from a threshold cycle (Ct) measured by a qPCR instrument. The threshold cycle (Ct) is defined as the number of PCR cycles required to reach the provirus or reference polynucleotide sequence per amplification cycle and is inversely proportional to the content of the corresponding polynucleotide sequence.

[0073] In some embodiments, the determination of one or more of the acceptance criteria or sample acceptance criteria utilizes three or more copies of three or more standard samples. In some embodiments, three or more standard samples, each at or about 3.20 VCN / cell, are prepared from a predetermined base standard sample by up to four 5-fold serial dilutions using a buffer as the diluent. In some embodiments, the base standard sample contains 121,212,121 copies of provirus and 75,757,576 copies of reference polynucleotide sequence. In some embodiments, for three or more copies of one or more positive control samples, the determination of one or more of the acceptance criteria or sample acceptance criteria requires comparing the measured amounts of provirus and reference polynucleotide sequence with the nominal amounts of provirus and reference polynucleotide sequence calculated during the preparation of said one or more positive control samples from separate samples of known concentrations of provirus and reference polynucleotide sequence.

[0074] In some implementations, the method further includes assessing the effectiveness of the assay for quantifying the integration of recombinant vector nucleic acid samples by evaluating one or more assay acceptance criteria. In some implementations, an assay is valid if one or more or all of the following assay acceptance criteria are met: (a) the threshold cycling of both the provirus and the reference polynucleotide sequence is indeterminate in all replicates of the control without template DNA; (b) the correlation coefficient of the standard curves of the provirus and the reference polynucleotide sequence generated by linear regression using standard samples is greater than or equal to 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, or 0.97; (c) the VCN / cell value estimated from the slope of the standard curve indicates a PCR efficiency between 80% and 120%, between 82% and 118%, between 84% and 116%, between 86% and 114%, between 88% and 116%, or between 90% and 110%; (d) in replicates of any of the standard samples, the threshold cycling of neither the provirus nor the reference polynucleotide sequence is indeterminate. The criteria are as follows: (e) the mean threshold cycle of both the provirus and the reference polynucleotide sequence in the baseline standard sample is less than or equal to 30.0, 28.0, 26.0, 24.0, or 22.0; (f) the standard deviation of the threshold cycle of both the provirus and the reference polynucleotide sequence in each standard sample is less than or equal to 0.95, 0.90, 0.85, 0.80, 0.75, 0.70, 0.65, or 0.60; (g) the mean measured copy number of the reference polynucleotide sequence in the one or more positive control samples is within 50%, 45%, 40%, 35%, or 30% of the nominal expected value; (h) the measured mean VCN / cell value of the one or more positive control samples is within 50%, 45%, 40%, 35%, or 30% of the nominal expected VCN / cell value of each control; and (i) the coefficient of variation of the VCN / cell value of the one or more positive control samples is less than or equal to 50%, 45%, 40%, 35%, 30%, 25%, or 20%.

[0075] In some embodiments, the method further includes evaluating the effectiveness of quantifying the recombinant vector nucleic acid integration of a sample by assessing one or more sample acceptance criteria. In some embodiments, a sample is considered valid if it meets one or more or all of the following sample acceptance criteria selected from the group consisting of: (a) the average copy number of the reference polynucleotide sequence in the sample is within 50%, 45%, 40%, 35%, or 30% of the expected value of 30, 303,030 copies; (b) if the genomic DNA (gDNA) concentration of the sample is less than 0.02 µg / µL, the expected copy number of the reference polynucleotide sequence in the sample is calculated based on the amount of DNA actually loaded into the reaction; (c) the average pre-target viral copy number in the sample is within... (d) The mean pre-target viral copy number in the sample is between 121, 212.121 and 193.939 copies; (e) The coefficient of variation of the VCN / cell ratio of the target sample copy is less than or equal to 50%, 45%, 40%, 35%, 30%, 25% and 20%; and (f) The standard deviation of the cycle thresholds of both the pre-target virus and the target reference polynucleotide sequence in the sample is less than or equal to 0.95, 0.90, 0.85, 0.80, 0.75, 0.70, 0.65 or 0.60.

[0076] In some embodiments, the method for quantifying the integration of recombinant vector nucleic acid into the cell genome further includes methods for identifying the transgene. In some embodiments, the method for identifying the transgene includes: (a) providing a biological sample containing the host cell genome; (b) amplifying the genomic DNA of the biological sample using a primer pair containing a first oligonucleotide primer and a second oligonucleotide primer using quantitative amplification techniques, wherein at least one oligonucleotide primer of the primer pair specifically hybridizes to the transgene; and (c) detecting and / or quantifying the genomic nucleic acid amplified in step (b). In some embodiments, the transgene encodes a chimeric antigen receptor. In some embodiments, the chimeric antigen receptor comprises the amino acid sequence of SEQ ID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 22. In some embodiments, the chimeric antigen receptor is a polypeptide encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 19 or SEQ ID NO: 21. In some embodiments, the chimeric antigen receptor recognizes BCMA, KLK2, or GPRC5D.

[0077] Reagent test kit In some embodiments, this disclosure also provides a kit for measuring the copy number of integrated recombinant vector sequences. In some embodiments, the kit may include one or more primers of the present invention. In some embodiments, the kit may include one or more probes of the present invention.

[0078] This disclosure also provides a kit for measuring the copy number of an integrated recombinant vector sequence. In some embodiments, the kit includes one or both primers of a primer pair that specifically hybridize with the integrated recombinant vector nucleic acid, wherein the primer pair specifically amplifies a portion of the integrated recombinant vector nucleic acid. In some embodiments, the kit includes a detectable nucleic acid probe that specifically hybridizes with the amplified recombinant vector nucleic acid.

[0079] This disclosure also provides another kit for measuring the copy number of an integrated recombinant vector sequence. In some embodiments, the kit includes a forward primer that specifically hybridizes to the integrated recombinant vector nucleic acid. In some embodiments, the kit includes a forward primer that specifically hybridizes to the integrated recombinant vector nucleic acid, wherein the forward primer comprises the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 5, or SEQ ID NO: 14. In some embodiments, the kit includes a reverse primer that specifically hybridizes to the integrated recombinant vector nucleic acid. In some embodiments, the kit includes a reverse primer that specifically hybridizes to the integrated recombinant vector nucleic acid, wherein the reverse primer comprises the nucleic acid sequence of SEQ ID NO: 2, SEQ ID NO: 6, or SEQ ID NO: 15. In some embodiments, the kit includes a detectable probe. In some embodiments, the kit includes a detectable probe that comprises the nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 16. Example

[0080] This disclosure will now be described in general terms, and will be more readily understood by referring to the following embodiments, which are merely illustrative of certain aspects and implementations of this disclosure and are not intended to limit it. For example, the specific constructs and experimental designs disclosed herein represent exemplary tools and methods for verifying correct functionality. Therefore, it will be apparent that any of the disclosed specific constructs and experimental designs may be substituted within the scope of this disclosure.

[0081] Example 1 Example materials and methods Using Lipofectamine 2000 (Life Technologies) and plasmids, 4 x 10⁻⁶ cells per well of a 6-well plate. 5 Transient transfection experiments were performed at a cell density of [specific value]. After 24 hours, the culture medium was removed and replaced with fresh growth medium.

[0082] qPCR was performed to determine the copy number of transfected cells. First, in the presence of tRNA (yeast tRNA; Invitrogen catalog number 657491), standards and plasmids linearized from the reference gene and 5'-LTR sequence were prepared, respectively. Second, a reference gene premix was prepared, consisting of the forward primer (SEQ ID NO: 8), the reverse primer (SEQ ID NO: 9), and the probe / 5HEX / AGGGAGA / ZEN / GATTTGTGTGGGCATGAC / 3IABkFQ / (SEQ ID NO: 11) from 1X Fast Advanced MasterMix (ThermoScientific, catalog number 4444558). Third, we prepared a 5'-LTR premix, which consisted of the forward primer (SEQ ID NO: 5), the reverse primer (SEQ ID NO: 6), and the probe / FAM / CTTTCAAGT / ZEN / CCCTGTTCGGGCGCC / 3lABkFQ / (SEQ ID NO: 12) from the 1X Fast Advanced Master Mix (ThermoScientific, catalog number 4444558).

[0083] Prepare qPCR assays by adding MasterMix, standards, QC, and samples to a multi-well plate. Run the qPCR assay on any real-time PCR instrument. qPCR assays include, but are not limited to, preparation by adding MasterMix, standards, QC, and samples to a 96-well plate. qPCR assays can be run on, but are not limited to, Quantstudio instruments, under the following conditions: 50°C for 2 min, 95°C for 10 min, 95°C for 15 sec melting, 60°C for 1 min annealing, and extension.

[0084] Example 2 : HIV-1 delivered T Encoding in cells CAR Quantitative analysis of polynucleotide incorporation Universal qPCR assays detect only the integrated retroviral vector sequence encoding any transgene in the genome of the transduced cell product, but not the transfer plasmid used to generate the lentiviral vector. Based on the unique characteristics of lentiviral vector integration into the host genome, specific qPCR assays have been developed that can quantify the copy number of lentivirally transduced cells but not quantify unintegrated vectors (see [link to assay]). Figure 1A and Figure 1B ).

[0085] The nucleic acids of retroviral vectors integrated into the cell genome can be easily quantified using the method of the present invention.

[0086] According to the method of Example 1, the integrated lentiviral vector sequence was amplified using primer and probe sets 1 to 5 (according to Table 1). Primer sets 1 to 4 amplified the lentiviral sequence in cell samples in which the vector was integrated, and not in control cell samples in which lentiviral transfer plasmids were present but not transduced (see [reference]). Figure 2 In control group 5, the human albumin (hALB) sequence was amplified in all samples containing cells.

[0087] Example 3 GPRc5d integrated in blood CAR-T Quantitative The method described in the previous embodiments was used to quantify the number of CAR transgene copies integrated in mouse studies using GPRc5d CAR-T. This study was conducted to select the final clone / construct.

[0088] A qPCR assay was developed and successfully used in five different mouse studies. In the GPRc5d CAR-T study, proviral copy number correlated with streaming data ( Figures 3A to 3B In these experiments, 5LTR was used against 3 (group 3 according to Table 1).

[0089] Example 4 : CAR-T Quantification of integrated transgenes in the product A quantitative real-time PCR (qPCR) assay for quantifying integration transgenes has been developed by targeting the cellular genome integration form of the HIV-derived lentiviral 5' LTR region (proviral sequence target region) in CAR-T products. An exemplary CAR-T product recognizing the target antigen BCMA has the amino acid sequence of SEQ ID NO: 18. The assay is a singleton or multiplex qPCR targeting the integrated 5' LTR region of the proviral sequence and human albumin (hALB; reference housekeeping gene). This method can be used, but is not limited to, pre-formulated frozen CAR-T cell particles to determine the transduction efficiency reporting the vector copy number (VCN) per cell.

[0090] Genomic DNA (gDNA) was extracted from pre-formulated CAR-T cell particles using the Purelink gDNA Isolation Kit. The pre-formulated cell particles were frozen at ≤-60°C prior to DNA isolation. The isolated DNA was stored at ≤-60°C and thawed later for quantification, or immediately quantified using a Qubit 4 fluorometer and the fluorescence-based Qubit dsDNA Wide Range Detection Kit. The Qubit 4 fluorometer was calibrated using two standards supplied with the Qubit dsDNA Wide Range Kit. Standards were prepared and run for each set of DNA samples to be quantified. The standards and DNA sample reactions were prepared using the same Qubit working solution. After quantification, the isolated DNA was diluted to a working concentration of 0.020 µg / µL.

[0091] qPCR reactions were prepared using an oligonucleotide mixture (containing proviral primer and probe set 4, including / / 56-FAM / TGCCTTGAG / ZEN / TGCTTCAAGTAGTGTGT / 3IABkFQ / (SEQ ID NO: 17) and hALB primer and probe set 5, according to Table 1) and qPCR TaqPath ProAmp premix solution, diluted with molecular-grade water. The final concentration of each of the three oligonucleotides in the proviral primer and probe set was 200 nM. The final concentration of each of the hALB forward and reverse primers was 50 nM, while the final concentration of the hALB probe was 200 nM.

[0092] Proviral standard No. 1 was prepared by incorporating 0.05 µg / µL PBMC DNA into a linear 5' LTR plasmid, resulting in 121,212,121 plasmid copies per 5 µL. Standard No. 1 was serially diluted with buffer. The proviral qPCR intermediate control was prepared by incorporating 0.02 µg / µL PBMC gDNA into a linear 5' LTR plasmid, resulting in 30,303,030 copies of the 5' LTR plasmid per 5 µL. The proviral qPCR low control was prepared by diluting the intermediate control volume 1:10 using 0.02 µg / µL PBMC gDNA as a diluent. This corresponds to a nominal vector copy number of 2.00 VCN / cell for the intermediate control and 0.20 VCN / cell for the low control per 5 µL, but the actual VCN / cell values ​​for each batch of intermediate and low controls were determined during reagent identification.

[0093] Load the premixed solution into the designated wells of the qPCR plate. Load each diluted standard spot, proviral intermediate control, proviral low control, test product DNA, and three copies of low EDTA TE buffer (i.e., template-free control or NTC) into the designated wells of the qPCR plate containing the premixed solution. Load the qPCR plate into a real-time PCR instrument to perform the qPCR reaction.

[0094] The measurement threshold cycle (Ct) for each target (provirus and hALB) was determined using a qPCR instrument. The Ct and logarithmic concentration values ​​of the standards were used to generate a standard curve via linear regression. The standard curve was used to calculate the copy number of each target in each control and test product replica.

[0095] The VCN / cell ratio for each sample, intermediate control, and low-control replica is calculated as follows.

[0096] Where x is each value from the population, x̅ is the mean of the dataset, and N is the population size, then the mean, standard deviation, and %CV of the triplicate VCN / cell values ​​for each sample, intermediate control, and low control are calculated as follows: .

[0097] The following test acceptance criteria are used to produce valid tests: • Correlation coefficient (R²) between provirus and hALB standard curves 2 The value must be ≥0.97.

[0098] • The slope of the standard curve must be between -3.585 and -3.104 (equivalent to 90.08%-109.97% PCR efficiency). • For both proviral and hALB targets, all Ct replicas of NTC must be “undetermined”.

[0099] • For both proviral and hALB targets, the average Ct of standard number 1 must be ≤22.0, and the Ct replicas of either standard cannot be "undetermined".

[0100] • For both proviral and hALB targets, the Ct SD of each standard must be ≤0.60.

[0101] • The mean hALB copy number for the intermediate and low control groups must be 30,303.030 copies + / - 30% (expected range: 21,212.121-39,393.939 copies).

[0102] • The mean VCN / cell result for the intermediate and low control must be ≥-30% and ≤+30% of the qualifying VCN / cell value for each control.

[0103] • The VCN / %CV of intermediate and low control replicates must be ≤20%.

[0104] Any sample that does not meet all of the following acceptance criteria is considered invalid: • For both proviral and hALB targets, the Ct SD for each sample must be ≤0.60. For samples determined to be below or above the assay range, the Ct SD was not accessed.

[0105] • The average hALB copy number for each sample must be 30,303.030 copies ± 30% (expected range: 21,212.121 to 39,393.939 copies).

[0106] • If the concentration of sample gDNA is <0.02µg / µL, the expected hALB copy number of the sample is calculated based on the amount of DNA actually loaded into the reaction.

[0107] • The average proviral target copy value for each sample must be equal to or within the validation range of the assay in order to calculate the VCN / cell result.

[0108] • If the average previral copy number of a sample is >121,212,121, then the sample is considered to be above the detection range.

[0109] • If the average previral copy number of a sample is <193.939 copies, the sample is considered to be below the detection range.

[0110] • The %CV of the sample VCN / cell replicate must be ≤20%. %CV was not accessed for samples determined to be below or above the assay range or samples that did not meet the hALB acceptance criteria.

[0111] • For both proviral and hALB targets, the Ct SD for each sample must be ≤0.60. %CV was not accessed for samples determined to be below or above the assay range or that did not meet the hALB acceptance criteria.

[0112] For any sample that meets all sample acceptance criteria, transduction efficiency is reported as the average VCN / cell result, accurate to two decimal places.

[0113] BCMA CAR-Ts tested using the method of this invention produced lower integrated copy numbers than the target vector packaging signal (PSI) assay, which cannot distinguish between integrated and unintegrated copy numbers. The results confirm that the PSI method may pick out plasmid contamination in the tested BCMA CAR-T batches (Table 2).

[0114] This method has also been used for other CAR-T products, such as the exemplary GPRc5d and KLK2 CAR-T products under development. The amino acid sequences of the CARs contain SEQ ID NO: 20 and 22, respectively. Comparison of these results with the PSI method does not show any differences in VCN between these methods (Table 3). The results confirm that there was no plasmid contamination during the batch production of GPRc5d CAR-T and KLK2 CAR-T.

[0115] Example 5 :other CAR-T Quantification of integrated transgenes in the product The quantitative real-time PCR (qPCR) assay used for quantifying integrated transgenes can also be used for other CAR-T products, such as those targeting GPRC5D. The method in Example 3 was used to determine CAR-T cell kinetics in mouse studies as the average VCN / cell ratio or transgene copies / µg gDNA of the GPRc5d CAR-T product in mouse blood.

[0116] Example 6 : CAR-T Quantitative analysis of integrated transgenes in the product followed by transgene identification. While proviral qPCR can be used to accurately estimate the batch VCN / cell ratio of cells whose genomes are integrated with CAR-T transgene sequences, qPCR using primers that hybridize specifically to nucleotide sequences within the transgene can still be used to confirm CAR transgene identity. This is because proviral qPCR is general rather than transgene-specific.

[0117] The design and execution of the transgenic qPCR method are identical to those of the proviral qPCR method, differing only in the sequences and optional concentrations of the forward, reverse, and probe sequences for the transgenic target. In the case where the transgenic target encodes a CAR in the BCMACAR-T product, which recognizes the target antigen BCMA, contains the amino acid sequence of SEQ ID NO: 18, and is encoded by the nucleic acid sequence of SEQ ID NO: 19, we use a primer / probe set specific to the CD137(4-1BB) and CD3ζ-derived sequences of the CAR transgene. For each of the forward and reverse primers for the transgene, we use a concentration of 100 nM. For both methods, the human albumin (hALB) housekeeping gene oligonucleotides are identical, but the concentrations of the hALB forward and reverse primers in the transgenic method are 75 nM.

[0118] In summary, to perform the transgenic qPCR method, five-spot serial dilutions of simulated transduced lymphocyte cell lines were used to generate standard curves for both the transfer plasmid and human albumin (hALB). Genomic DNA isolated from the collected samples was analyzed in triplicate. The copies of both the transgene and hALB in each of the three DNA samples were interpolated from the corresponding standard curves. The average hALB copy number was used to estimate the number of cells from which the sample DNA was derived. The average copy number of the transgene present in the sample DNA was divided by the estimated number of cells from which the sample DNA was derived to determine the vector copy number per cell in the sample.

[0119] Table 1—Sequence Table 2—BCMA CAR-T production using the method of this invention shows a higher signal intensity than the target vector packaging signal (PSI). The assay measured lower integrated copy numbers, but this assay could not distinguish between integrated and unintegrated copy numbers. Table 3—Comparison of VCNs of CAR T products targeting KLK2 and GPRC5D using the method of this invention and the PSI method. References 1. Sadelain M, Brentjens R, Riviere I (2013) The Basic Principles ofChimerica Antigen Receptor (CAR) Design. CancerDiscov 3(4): 388-398. 2. Titov A, Valiullina A, Zmievskaya E, Zaikova E, Petukhov A,Miftakhova R, Bulatov E, Rizvamov (2020) AdvancingCAR T-Cell Therapy forSolid Tumors: Lessons Learned from Lymphoma Treatment. Cancer 12: 125-146. 3. Charrier S, Ferrand M, Zerbato M, Precigout G, Viornery A, Bucher-Laurent S,Benkheilifa-Ziyyat S, Merten OW, Perea J, Galy A (2011)Quantification of Lentiviral Vector Copy Numbers in Individual HematopoieticColony-Forming CellsShows Vector Dose-Dependent Effects on the Frequency andLevel of Transduction. Gene Therapy 18: 479-487. 4. Lizee G, Aerts JL, Gonzales MI, Chinnasamy N, Morgan RA, TopalianSL (2003) Real-TimeQuantitative Reverse Transcriptase-Polymerase ChainReaction As A Method For Determining Lentiviral Vector Titers And MeasuringTransgene Expression. HumGene Ther. 14(6): 497-507. 5. Siegel R, Naishadham D, Jemal A (2012) Cancer statistics, 2012.CA: a cancer journal for clinicians 62: 10-29. Specific embodiments of the present invention are described in the following numbered paragraphs: 1. A method for quantitatively integrating recombinant vector nucleic acids into the cell genome, the method comprising: (a) Provide biological samples containing the host cell genome; (b) Amplifying the genomic DNA of the biological sample using a primer pair comprising a first oligonucleotide primer and a second oligonucleotide primer using a quantitative amplification technique, wherein at least one oligonucleotide primer of the primer pair specifically hybridizes to the integrated recombinant vector polynucleotide sequence; and (c) Detect and / or quantify the genomic nucleic acids amplified in step (b).

[0120] 2. The method according to paragraph 1, wherein the recombinant vector contains a transgene.

[0121] 3. The method according to paragraph 2, wherein the transgene encodes a chimeric antigen receptor.

[0122] 4. The method according to any one of paragraphs 1 to 3, wherein the recombinant vector is a gene therapy vector.

[0123] 5. The method according to paragraph 4, wherein the gene therapy vector is a viral vector.

[0124] 6. The method according to any one of paragraphs 1 to 5, wherein the biological sample is a cell sample or a tissue sample.

[0125] 7. The method according to paragraph 6, wherein the tissue sample is blood, plasma, serum, saliva or a tissue biopsy.

[0126] 8. The method according to any one of paragraphs 1 to 7, wherein the sample is derived from a subject.

[0127] 9. The method described in paragraph 8, wherein the subject is a human being.

[0128] 10. The method according to any one of paragraphs 1 to 9, wherein the recombinant vector is a retroviral vector.

[0129] 11. The method according to paragraph 10, wherein the retroviral vector is a lentiviral vector.

[0130] 12. According to the method described in paragraph 11, the lentivirus on which the lentiviral vector is based is human immunodeficiency virus 1 (HIV-1) or human immunodeficiency virus 2 (HIV-2).

[0131] 13. The method according to any one of paragraphs 1 to 12, wherein the oligonucleotide primer that specifically hybridizes with the integrated recombinant vector polynucleotide sequence specifically hybridizes with the LTR sequence of the integrated recombinant vector sequence.

[0132] 14. The method according to any one of paragraphs 1 to 13, wherein the oligonucleotide primer that specifically hybridizes with the integrated recombinant vector polynucleotide sequence in step (b) comprises the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 5 or SEQ ID NO: 14.

[0133] 15. The method according to paragraph 14, wherein the second oligonucleotide primer used in step (b) for amplifying the recombinant vector nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 2, SEQ ID NO: 6 or SEQ ID NO: 15.

[0134] 16. The method according to paragraph 11, wherein the oligonucleotide primer used in step (b) specifically hybridizes with the integrated lentiviral vector polynucleotide sequence and specifically hybridizes with the U3 region of the 5'LTR of the lentiviral vector nucleic acid sequence.

[0135] 17. The method according to paragraph 11, wherein the oligonucleotide primer used in step (b) specifically hybridizes with the integrated lentiviral vector polynucleotide sequence and specifically hybridizes with the U3 and R regions of the 5'LTR of the lentiviral vector nucleic acid sequence.

[0136] 18. The method according to paragraph 11, wherein the oligonucleotide primer used in step (b) specifically hybridizes with the integrated lentiviral vector polynucleotide sequence and specifically hybridizes with the PBS region of the 5'LTR of the lentiviral vector nucleic acid sequence.

[0137] 19. The method according to paragraph 11, wherein the oligonucleotide primer used in step (b) that specifically hybridizes with the integrated lentiviral vector polynucleotide sequence hybridizes specifically with the psi(Ψ) packaging signal.

[0138] 20. The method according to any one of paragraphs 1 to 19, wherein the quantitative amplification technique is qPCR.

[0139] 21. The method according to any one of paragraphs 1 to 19, wherein the quantitative amplification technique is dPCR or ddPCR.

[0140] 22. The method according to any one of paragraphs 1 to 19, wherein the quantitative amplification technique is endpoint PCR.

[0141] 23. The method according to any one of paragraphs 1 to 22, wherein step (b) utilizes a detectable nucleic acid probe that specifically hybridizes with the nucleic acid of the amplified recombinant vector.

[0142] 24. The method according to paragraph 23, wherein the recombinant vector nucleic acid is a lentiviral vector.

[0143] 25. The method according to paragraph 23 or paragraph 24, wherein the probe for the integrated recombinant vector nucleic acid specifically hybridizes with the LTR sequence of the integrated recombinant vector sequence.

[0144] 26. The method according to paragraph 24, wherein the probe used in step (b) for the lentiviral vector nucleic acid specifically hybridizes with the U3 and R regions of the 5'LTR of the lentiviral vector nucleic acid sequence.

[0145] 27. The method according to paragraph 24, wherein the probe used in step (b) for the lentiviral vector nucleic acid specifically hybridizes with the U5 region and PBS region of the 5'LTR of the lentiviral vector nucleic acid sequence.

[0146] 28. The method according to paragraph 24, wherein the probe used in step (b) for the lentiviral vector nucleic acid specifically hybridizes with the PBS region of the 5'LTR of the lentiviral vector nucleic acid sequence.

[0147] 29. The method according to paragraph 24, wherein the probe used in step (b) for the lentiviral vector nucleic acid specifically hybridizes with the R and U5 regions of the 5'LTR of the lentiviral vector nucleic acid sequence.

[0148] 30. The method according to any one of paragraphs 23 to 25, wherein the probe that specifically hybridizes with the nucleic acid of the recombinant vector comprises the nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7 or SEQ ID NO: 16.

[0149] 31. A method for monitoring the transduction efficiency of recombinant vector nucleic acids, the method comprising: a) Provides one or more biological samples containing genomic DNA transduced from a recombinant vector nucleic acid, wherein a portion of the recombinant vector nucleic acid is integrated into the genomic DNA; and b) Quantitatively integrate the recombinant vector nucleic acid into the host cell genome according to the method described in any one of paragraphs 1 to 30.

[0150] 32. The method according to any one of paragraphs 1 to 30, wherein step (c) further comprises comparing the copy number of the integrated recombinant vector sequence of the biological sample with a reference polynucleotide sequence.

[0151] 33. The method according to paragraph 32, wherein the reference polynucleotide sequence encodes a housekeeping protein.

[0152] 34. The method according to paragraph 33, wherein the housekeeping protein is human albumin.

[0153] 35. The method according to any one of paragraphs 1 to 34, the method further comprising at least one pair of oligonucleotide primers for specifically amplifying a reference polynucleotide sequence.

[0154] 36. The method according to paragraph 31, the method further comprising comparing the copy number of the integrated recombinant vector sequence of the biological sample with a reference.

[0155] 37. A method for batch release testing of cell products transduced by a recombinant vector, the method comprising: a) Provide one or more biological samples from each batch of cell products containing genomic DNA transduced by recombinant vectors; b) Quantitatively integrate the recombinant vector nucleic acid into the host cell genome in each biological sample according to the method described in any one of paragraphs 1 to 35; c) Compare the copy number of the integrated recombinant vector sequence quantified for the biological sample in step (b) with a reference; and d) Release the integrated recombinant vector sequence copy number by a predetermined standard batch.

[0156] 38. A method for quantifying the integration of lentiviral vector nucleic acid into the cellular genome, the method comprising: (a) Provide biological samples containing the host cell genome; (b) Amplifying the genomic DNA of the biological sample using a primer pair comprising a first oligonucleotide primer and a second oligonucleotide primer using a quantitative amplification technique, wherein at least one oligonucleotide primer of the primer pair specifically hybridizes to the integrated lentiviral vector polynucleotide sequence; and (c) Quantifying the genomic nucleic acids amplified in step (b), wherein quantifying the lentiviral vector nucleic acids integrated into the host cell genome includes comparing the ratio of amplified lentiviral vector nucleic acids to a reference. The oligonucleotide primer pairs described therein comprise the nucleic acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2, respectively, or comprise the nucleic acid sequences of SEQ ID NO: 5 and SEQ ID NO: 6, respectively, or comprise the nucleic acid sequences of SEQ ID NO: 14 and SEQ ID NO: 15, respectively.

[0157] 39. The method according to any one of paragraphs 1 to 38, wherein step (b) utilizes an embedded dye.

[0158] 40. The method according to paragraph 39, wherein the embedded dye is SYBR green.

[0159] 41. The method according to any one of paragraphs 32 to 35, wherein the copy number of the integrated recombinant vector sequence and the copy number of the reference polynucleotide sequence are measured in multiple ways.

[0160] 42. The method according to any one of paragraphs 32 to 35, wherein the copy number of the integrated recombinant vector sequence and the copy number of the reference polynucleotide sequence are measured in a single-weighted manner.

[0161] 43. The method according to paragraph 3, wherein the chimeric antigen receptor comprises the amino acid sequence of SEQ ID NO: 18, 20 or 22.

[0162] 44. The method according to paragraph 3, wherein the chimeric antigen receptor recognizes BCMA, KLK2, or GPRC5D.

[0163] 45. The method according to paragraph 2, the method further comprising a method for identifying the transgene.

[0164] 46. ​​The method according to paragraph 45, wherein the method for identifying the transgenic material comprises: (a) Provide biological samples containing the host cell genome; (b) Amplifying the genomic DNA of a biological sample using a primer pair comprising a first oligonucleotide primer and a second oligonucleotide primer, wherein at least one oligonucleotide primer of the primer pair specifically hybridizes to the transgene; and (c) Detect and / or quantify the genomic nucleic acids amplified in step (b).

[0165] 47. The method according to any one of paragraphs 32 to 35, wherein the method further comprises evaluating the effectiveness of the assay for quantifying the integration of recombinant vector nucleic acids by assessing one or more assay acceptance criteria selected from the group consisting of: (a) In all copies of the control containing no template DNA, the threshold cycling of both the provirus and the reference polynucleotide sequence was indeterminate; (b) The correlation coefficient of the standard curves of the provirus and the reference polynucleotide sequence generated by linear regression using standard samples is greater than or equal to 0.97; (c) The copy values ​​of the provirus and reference polynucleotide sequences estimated from the slope of the standard curve indicate that the PCR efficiency is between 90% and 110%. (d) In a copy of either of the standard samples, the threshold cycle for neither the provirus nor the reference polynucleotide sequence can be determined; (e) The average threshold cycle of both the provirus and the reference polynucleotide sequence in the baseline standard sample is less than or equal to 22.0; (f) The standard deviation of the threshold cycle for both the provirus and the reference polynucleotide sequence in each standard sample is less than or equal to 0.60; (g) The average measured copy number of the reference polynucleotide sequence in the one or more positive control samples is within 30% of the nominal expected value; (h) The measured mean VCN / cell value of the one or more positive control samples is within 30% of the nominal expected VCN / cell value for each control; and (i) The coefficient of variation of the VCN / cell value of the one or more positive control samples is less than or equal to 20%.

[0166] 48. The method according to any one of paragraphs 32 to 35, wherein the method further comprises evaluating the effectiveness of the integration of the recombinant vector nucleic acid of the quantitative sample by assessing one or more sample acceptance criteria selected from the group consisting of: (a) The average copy value of the reference polynucleotide sequence in the sample is within 30% of the expected value of 30,303.030 copies; (b) If the concentration of genomic DNA (gDNA) in the sample is less than 0.02 µg / µL, the expected copy of the reference polynucleotide sequence of the sample shall be calculated based on the amount of DNA actually loaded into the reaction; (c) The average pre-target viral copy number in the sample is within the validation range of the measured copy number; (d) The average pre-target viral copy number in the sample was between 121, 212.121 and 193.939 copies; (e) The coefficient of variation of the VCN / cell value of the target sample replica is less than or equal to 20%; and (f) The standard deviation of the cycle thresholds of both the pre-target virus and the target reference polynucleotide sequence in the sample is less than or equal to 0.60.

[0167] 49. The method according to paragraph 43 or 44, wherein the chimeric antigen receptor is a polypeptide encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 19 or 21.

[0168] 50. The method according to paragraph 45 or 46, wherein the transgene is a polypeptide encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 19 or 21.

[0169] 51. An oligonucleotide comprising the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16.

[0170] 52. A kit for measuring the copy number of an integrated recombinant vector nucleic acid sequence, the kit comprising: Primers of a primer pair, wherein the primers of the primer pair specifically hybridize with the integrated recombinant vector nucleic acid, wherein the primer pair specifically amplifies a portion of the integrated recombinant vector nucleic acid; and A detectable nucleic acid probe that specifically hybridizes with the amplified recombinant vector nucleic acid.

[0171] 53. A kit for measuring the copy number of an integrated recombinant vector nucleic acid sequence, the kit comprising: A forward primer that specifically hybridizes with the nucleic acid of a recombinant vector integrated with the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 5, or SEQ ID NO: 14; A reverse primer, said reverse primer specifically hybridizing with the nucleic acid of a recombinant vector integrated with the nucleic acid sequence of SEQ ID NO: 2, SEQ ID NO: 6, or SEQ ID NO: 15; and A detectable probe comprising a nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7 or SEQ ID NO: 16.

[0172] Incorporated by reference All publications and patents mentioned herein are incorporated herein by reference in their entirety, as if each individual publication or patent were explicitly and independently cited.

[0173] While specific embodiments of this disclosure have been discussed, the above description is illustrative and not restrictive. Many variations of this disclosure will become apparent to those skilled in the art upon review of this specification and the following claims. The full scope of this disclosure should be determined by reference to the full scope of the claims and their equivalents, and the description and such variations.

Claims

1. A method for quantitatively integrating recombinant vector nucleic acids into the cell genome, the method comprising: (a) Provide biological samples containing the host cell genome; (b) Amplifying the genomic DNA of the biological sample using a primer pair containing a first oligonucleotide primer and a second oligonucleotide primer using a quantitative amplification technique, wherein at least one oligonucleotide primer of the primer pair specifically hybridizes to the integrated recombinant vector polynucleotide sequence. as well as (c) Detect and / or quantify the genomic nucleic acids amplified in step (b).

2. The method according to claim 1, wherein the recombinant vector comprises a transgene.

3. The method according to claim 2, wherein the transgene encodes a chimeric antigen receptor.

4. The method according to claim 1, wherein the recombinant vector is a gene therapy vector.

5. The method according to claim 4, wherein the gene therapy vector is a viral vector.

6. The method according to claim 1, wherein the recombinant vector is a retroviral vector.

7. The method according to claim 6, wherein the retroviral vector is a lentiviral vector.

8. The method of claim 7, wherein the lentivirus on which the lentiviral vector is based is human immunodeficiency virus 1 (HIV-1) or human immunodeficiency virus 2 (HIV-2).

9. The method of claim 1, wherein the oligonucleotide primer that specifically hybridizes with the integrated recombinant vector polynucleotide sequence in step (b) comprises the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 5 or SEQ ID NO:

14.

10. The method of claim 9, wherein the second oligonucleotide primer used in step (b) for amplifying the recombinant vector nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 2, SEQ ID NO: 6 or SEQ ID NO:

15.

11. The method according to claim 1, wherein the biological sample is a cell sample or a tissue sample.

12. The method of claim 11, wherein the tissue sample is blood, plasma, serum, saliva, or a tissue biopsy.

13. The method of claim 1, wherein the oligonucleotide primer that specifically hybridizes with the integrated recombinant vector polynucleotide sequence specifically hybridizes with the LTR sequence of the integrated recombinant vector sequence.

14. The method of claim 7, wherein the oligonucleotide primer used in step (b) specifically hybridizes with the integrated lentiviral vector polynucleotide sequence and specifically hybridizes with the U3 region of the 5'LTR of the lentiviral vector nucleic acid sequence.

15. The method of claim 7, wherein the oligonucleotide primer used in step (b) specifically hybridizes with the integrated lentiviral vector polynucleotide sequence and specifically hybridizes with the U3 and R regions of the 5'LTR of the lentiviral vector nucleic acid sequence.

16. The method of claim 7, wherein the oligonucleotide primer used in step (b) specifically hybridizes with the integrated lentiviral vector polynucleotide sequence and specifically hybridizes with the PBS region of the 5'LTR of the lentiviral vector nucleic acid sequence.

17. The method of claim 1, wherein step (c) further comprises comparing the copy number of the integrated recombinant vector sequence of the biological sample with a reference polynucleotide sequence.

18. The method according to claim 1, wherein the quantitative amplification technique is qPCR.

19. The method of claim 1, wherein the quantitative amplification technique is dPCR or ddPCR.

20. The method of claim 17, wherein the reference polynucleotide sequence encodes a housekeeping protein.

21. The method of claim 20, wherein the housekeeping protein is human albumin.

22. The method of claim 1, wherein step (b) utilizes a detectable nucleic acid probe that specifically hybridizes with the nucleic acid of the amplified recombinant vector.

23. The method according to claim 22, wherein the recombinant vector nucleic acid is a lentiviral vector.

24. The method of claim 22, wherein the probe that specifically hybridizes with the nucleic acid of the recombinant vector comprises the nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7 or SEQ ID NO:

16.

25. The method of claim 22, wherein the probe for the integrated recombinant vector nucleic acid specifically hybridizes with the LTR sequence of the integrated recombinant vector sequence.

26. The method of claim 23, wherein the probe used in step (b) for the lentiviral vector nucleic acid specifically hybridizes with the U3 and R regions of the 5'LTR of the lentiviral vector nucleic acid sequence.

27. The method of claim 23, wherein the probe used in step (b) for the lentiviral vector nucleic acid specifically hybridizes with the U5 region and PBS region of the 5'LTR of the lentiviral vector nucleic acid sequence.

28. The method of claim 23, wherein the probe used in step (b) for the lentiviral vector nucleic acid specifically hybridizes with the PBS region of the 5'LTR of the lentiviral vector nucleic acid sequence.

29. The method of claim 7, wherein the oligonucleotide primer used in step (b) that specifically hybridizes with the integrated lentiviral vector polynucleotide sequence hybridizes specifically with the psi(Ψ) packaging signal.

30. The method of claim 23, wherein the probe used in step (b) for the lentiviral vector nucleic acid specifically hybridizes with the R and U5 regions of the 5'LTR of the lentiviral vector nucleic acid sequence.

31. The method of claim 1, wherein the biological sample is derived from a subject.

32. The method of claim 31, wherein the subject is a human being.

33. The method of claim 1, further comprising at least one pair of oligonucleotide primers for specifically amplifying a reference polynucleotide sequence.

34. A method for monitoring the transduction efficiency of recombinant vector nucleic acids, the method comprising: a) Provides one or more biological samples containing genomic DNA transduced from a recombinant vector nucleic acid, wherein a portion of the recombinant vector nucleic acid is integrated into the genomic DNA; as well as b) The recombinant vector nucleic acid, quantitatively integrated into the host cell genome using the method of claim 1.

35. The method of claim 34, the method further comprising comparing the copy number of the integrated recombinant vector sequence of the biological sample with a reference.

36. A method for batch release testing of cell products transduced by a recombinant vector, the method comprising: a) Provide one or more biological samples from each batch of cell products containing genomic DNA transduced by recombinant vectors; b) The method of claim 1 is used to quantify the recombinant vector nucleic acid integrated into the host cell genome in each biological sample; c) Compare the copy number of the integrated recombinant vector sequence quantified for the biological sample in step (b) with a reference; as well as d) Release the integrated recombinant vector sequence copy number by a predetermined standard batch.

37. A method for quantifying the integration of lentiviral vector nucleic acid into the cell genome, the method comprising: (a) Provide biological samples containing the host cell genome; (b) Amplify the genomic DNA of the biological sample using a primer pair containing a first oligonucleotide primer and a second oligonucleotide primer using a quantitative amplification technique, wherein at least one oligonucleotide primer of the primer pair specifically hybridizes to the integrated lentiviral vector polynucleotide sequence. as well as (c) Quantifying the genomic nucleic acids amplified in step (b), wherein quantifying the lentiviral vector nucleic acids integrated into the host cell genome includes comparing the ratio of amplified lentiviral vector nucleic acids to a reference. The oligonucleotide primer pairs described therein comprise the nucleic acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2, respectively, or comprise the nucleic acid sequences of SEQ ID NO: 5 and SEQ ID NO: 6, respectively, or comprise the nucleic acid sequences of SEQ ID NO: 14 and SEQ ID NO: 15, respectively.

38. An oligonucleotide comprising the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO:

16.

39. A kit for measuring the copy number of an integrated recombinant vector nucleic acid sequence, the kit comprising: The primers of the primer pair specifically hybridize with the integrated recombinant vector nucleic acid, wherein the primer pair specifically amplifies a portion of the integrated recombinant vector nucleic acid; and A detectable nucleic acid probe that specifically hybridizes with the amplified recombinant vector nucleic acid.

40. A kit for measuring the copy number of an integrated recombinant vector nucleic acid sequence, the kit comprising: A forward primer that specifically hybridizes with the nucleic acid of a recombinant vector integrated with the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 5, or SEQ ID NO: 14; A reverse primer that specifically hybridizes with the nucleic acid of a recombinant vector containing the nucleic acid sequence of SEQ ID NO: 2, SEQ ID NO: 6, or SEQ ID NO: 15; and A detectable probe comprising a nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7 or SEQ ID NO:

16.

41. The method of claim 3, wherein the chimeric antigen receptor comprises the amino acid sequence of SEQ ID NO: 18, 20 or 22.

42. The method of claim 3, wherein the chimeric antigen receptor recognizes BCMA, KLK2, or GPRC5D.

43. The method according to claim 1, wherein the quantitative amplification technique is endpoint PCR.

44. The method of claim 1, wherein step (b) utilizes an embedded dye.

45. The method of claim 44, wherein the embedded dye is SYBR Green.

46. ​​The method of claim 17, wherein the copy number of the integrated recombinant vector sequence and the copy number of the reference polynucleotide sequence are measured in multiple ways.

47. The method of claim 17, wherein the copy number of the integrated recombinant vector sequence and the copy number of the reference polynucleotide sequence are measured in a single-weighted manner.

48. The method of claim 2, further comprising a method for identifying the transgene.

49. The method of claim 48, wherein the method for identifying the transgenic material comprises: (a) Provide biological samples containing the host cell genome; (b) Amplifying the genomic DNA of the biological sample using a primer pair comprising a first oligonucleotide primer and a second oligonucleotide primer using a quantitative amplification technique, wherein at least one oligonucleotide primer of the primer pair specifically hybridizes with the transgene; as well as (c) Detect and / or quantify the genomic nucleic acid amplified in step (b).

50. The method of claim 17, wherein the method further comprises evaluating the effectiveness of the assay for quantifying recombinant vector nucleic acid integration by assessing one or more assay acceptance criteria selected from the group consisting of: (a) In all copies of the control containing no template DNA, the threshold cycling of both the provirus and the reference polynucleotide sequence was not determined; (b) The correlation coefficient of the standard curves of the provirus and the reference polynucleotide sequence generated by linear regression using standard samples is greater than or equal to 0.97; (c) The copy values ​​of the provirus and reference polynucleotide sequences estimated from the slope of the standard curve indicate that the PCR efficiency is between 90% and 110%. (d) In a copy of either of the standard samples, the threshold cycle for neither the provirus nor the reference polynucleotide sequence can be determined; (e) The average threshold cycle of both the provirus and the reference polynucleotide sequence in the baseline standard sample is less than or equal to 22.0; (f) The standard deviation of the threshold cycle for both the provirus and the reference polynucleotide sequence in each standard sample is less than or equal to 0.60; (g) The average measured copy number of the reference polynucleotide sequence in the one or more positive control samples is within 30% of the nominal expected value; (h) The measured mean VCN / cell value of the one or more positive control samples is within 30% of the nominal expected VCN / cell value for each control; and (i) The coefficient of variation of the VCN / cell value of the one or more positive control samples is less than or equal to 20%.

51. The method of claim 17, wherein the method further comprises evaluating the effectiveness of the integration of the recombinant vector nucleic acid of the quantitative sample by assessing one or more sample acceptance criteria selected from the group consisting of: (a) The average copy value of the reference polynucleotide sequence in the sample is within 30% of the expected value of 30,303.030 copies; (b) If the concentration of genomic DNA (gDNA) in the sample is less than 0.02 µg / µL, the expected copy of the reference polynucleotide sequence of the sample shall be calculated based on the amount of DNA actually loaded into the reaction; (c) The average pre-target viral copy number in the sample is within the validation range of the measured copy number; (d) The average pre-target viral copy number in the sample was between 121, 212.121 and 193.939 copies; (e) The coefficient of variation of the VCN / cell value of the target sample replica is less than or equal to 20%; and (f) The standard deviation of the cycle thresholds of both the pre-target virus and the target reference polynucleotide sequence in the sample is less than or equal to 0.

60.

52. The method of claim 41, wherein the chimeric antigen receptor is a polypeptide encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 19 or 21.

53. The method of claim 48, wherein the transgene is a polypeptide encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 19 or 21.

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