Methods for determining viral titers

Abstract

The present disclosure relates to a method for determining the viral titer of a biological sample, preferably from a mammalian cell sample, comprising mechanical disruption of the cells followed by determining the viral titer using droplet digital polymerase chain reaction (ddPCR). The mechanical disruption method preferably comprises the use of glass beads.

Description

[Technical Field]

[0001] The present disclosure relates to a method for determining the viral titer of a biological sample, preferably from a mammalian cell sample, comprising mechanical disruption of the cells followed by determining the viral titer using droplet digital polymerase chain reaction (ddPCR). The mechanical disruption method preferably comprises the use of glass beads. [Background technology]

[0002] Lentiviruses (LVs) are one of the most popular delivery vehicles for cell and gene therapy. Similarly, adeno-associated viruses (AAVs) have also found use as gene therapy vehicles. Accurate measurement of infectious titers is an absolute requirement in the processes of viral vector production, purification, and application. Traditional assay methods for measuring viral titers, such as flow cytometry or quantitative polymerase chain reaction (qPCR), have several major drawbacks. These assays require reporters or specific antibodies for measuring infectious titers. In addition, primers, probes, and standards must be optimized before being used in a qPCR assay, which is a very tedious process.

[0003] Droplet digital polymerase chain reaction (ddPCR) has emerged as a reliable, state-of-the-art technique for quantifying the absolute copy number of any gene of interest without the use of a standard curve. The RNA genome of LV is first reverse-transcribed into its cDNA before integration into the host chromosome. Therefore, the infectious titer of LV can be determined using ddPCR by measuring the integration frequency of the transgene into the chromosome of target cells. AAV viral vectors can also be measured using ddPCR. However, current methods for determining viral titer by ddPCR are limited in speed by the laborious process of genomic DNA isolation, which involves extracting chromosomal DNA from a large number of virus-transduced cells.

[0004] Therefore, what is needed is a high-throughput method that eliminates genomic DNA extraction during sample preparation for ddPCR applications and also eliminates the use of various potentially contaminating buffers and solutions. The present invention meets these needs. Summary of the Invention

[0005] In some embodiments, provided herein are methods for determining viral titer in a biological sample, comprising obtaining a biological sample containing virally-transduced cells, mechanically disrupting the virally-transduced cells of the biological sample, performing droplet digital polymerase chain reaction (ddPCR) on nucleic acid molecules removed from the disrupted virally-transduced cells, and calculating the viral titer.

[0006] In additional embodiments, provided herein are methods for determining viral titer in a biological sample, consisting essentially of obtaining a biological sample containing virally-transduced cells, mechanically disrupting the virally-transduced cells of the biological sample using glass beads, performing droplet digital polymerase chain reaction (ddPCR) on nucleic acid molecules removed from the disrupted virally-transduced cells, and calculating the viral titer. [Brief explanation of the drawings]

[0007] [Figure 1] 1 shows a lentiviral titer comparison between the three methods described herein. DETAILED DESCRIPTION OF THE INVENTION

[0008] The use of the word "a" or "an," when used in conjunction with the term "comprising" in the claims and / or specification, can mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

[0009] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the method / device being employed to determine the value. Typically, the term is intended to encompass a variation of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% or less, depending on the context.

[0010] Although the use of the term "or" in the claims is used to mean "and / or" unless expressly indicated to refer to alternatives only or unless the alternatives are mutually exclusive, the present disclosure supports a definition that refers to alternatives only, and "and / or."

[0011] As used in the specification and claims, the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed herein can be implemented with respect to any method, system, host cell, expression vector, and / or composition of the invention. Furthermore, the compositions, systems, cells, and / or nucleic acids of the invention can be used to achieve any of the methods as described herein.

[0012] As used herein, "nucleic acid," "nucleic acid molecule," or "oligonucleotide" refers to a polymeric compound containing covalently linked nucleotides. The term "nucleic acid" includes polyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA), both of which can be single-stranded or double-stranded. DNA includes, but is not limited to, complementary DNA (cDNA), genomic DNA, plasmid or vector DNA, and synthetic DNA. RNA includes, but is not limited to, mRNA, tRNA, rRNA, snRNA, microRNA, miRNA, or miRNA.

[0013] As used herein, "gene" refers to an assembly of nucleotides that encodes a polypeptide, including cDNA and genomic DNA nucleic acid molecules. "Gene" also refers to a nucleic acid fragment that can act as a regulatory sequence before (5' non-coding sequence) and after (3' non-coding sequence) the coding sequence. In some embodiments, the gene is integrated with multiple copies. In some embodiments, the gene is integrated in a predetermined copy number.

[0014] Methods for determining viral titers In exemplary embodiments, the present invention provides a method for determining viral titer in biological samples.As used herein, " viral titer " generally refers to the numerical expression of the amount of virus in a given volume, expressed as viral particles, transduction units, or infectious particles per milliliter (mL).Therefore, the method described herein for determining viral titer is quantitative in that it determines the actual number of viral particles, rather than simply being a qualitative measurement.

[0015] As used herein, a "biological sample" refers to a solution or suspension of cells or tissues that may contain a viral vector, or a solution or suspension that has been dried prior to reconstitution. Preferably, the biological sample is a cell solution containing at least one virally transduced cell.

[0016] As used herein, a "virally transduced cell" is a cell into which a viral vector has been inserted, either transiently integrated (inserted without integration into the genome) or genomically integrated (inserted into the genome of the cell). As used herein, a "vector" or "expression vector" is a replicon, such as a plasmid, phage, virus, or cosmid, to which a nucleic acid molecule can be attached and which can result in replication and / or expression of the attached nucleic acid molecule in the cell. "Vector" includes episomal (e.g., plasmid) and non-episomal vectors. The term "vector" includes both viral and non-viral means for introducing a nucleic acid molecule into a cell in vitro, in vivo, or ex vivo. The term vector may include synthetic vectors. Vectors can be introduced into desired cells by well-known methods, including, but not limited to, transfection, transduction, cell fusion, and lipofection. Vectors can include various regulatory elements, including promoters.

[0017] As used herein, "transduction" refers to the introduction of exogenous nucleic acid molecules, including vectors, into cells, and includes transfection (e.g., the use of lipid- or polymer-based carriers, as well as mechanical transfection, electroporation) and viral transduction. A "transfected" cell contains an exogenous nucleic acid molecule inside the cell, and a "transformed" cell is one in which the exogenous nucleic acid molecule inside the cell induces a phenotypic change in the cell. The transfected nucleic acid molecule can be integrated into the genomic DNA of the host cell and / or can be maintained by the cell temporarily or extrachromosomally (transiently) for long periods of time. A host cell or organism that expresses an exogenous nucleic acid molecule or fragment is referred to as a "recombinant," "transformed," or "transgenic" organism. Several transfection techniques are commonly known in the art. See, for example, Graham et al., Virology, 52:456 (1973), Sambrook et al., Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York (1989), Davis et al., Basic Methods in Molecular Biology, Elsevier (1986), and Chu et al., Gene 13:197 (1981), the disclosures of each of which are incorporated herein by reference in their entireties. Preferably, transfection of mammalian cells with one or more vectors utilizes a transfection agent, such as polyethyleneimine (PEI) or other suitable agent, including various lipids and polymers, to integrate the nucleic acids into the genomic DNA of the host cell.

[0018] The method for determining viral titer includes obtaining a biological sample containing virally transduced cells. The biological sample can be obtained from a laboratory setting, or a large-scale batch process, or other suitable setting, and includes samples that are prepared and then measured as described herein, as well as biological samples that are prepared, stored, and potentially shipped in other settings or areas and then measured using the methods described herein.

[0019] The method further comprises mechanically disrupting the virally transduced cells of the biological sample. As used herein, "mechanical disruption" or "mechanical disruption" refers to the application of a force to the biological sample that is effective to disintegrate or lyse cells contained therein that are not native to the sample. Exemplary mechanical disruption techniques include the use of glass beads for disruption, sonication (including the use of a sonication bath and a sonication tip / probe or ultrasonic tip / probe), high-power vortexing or mixing, the application of shear force through a glass or plastic plate, grinding, mixing, the use of a mechanical homogenizer, etc.

[0020] In a preferred embodiment, mechanical disruption occurs via disruption using glass beads. In such methods, a biological sample containing virally transduced cells is contacted with a solution of glass beads, vortexed for about 1 minute, and then revortexed three to five additional times, each for about 1 minute. Additional times and repetitions of vortexing can also be used. Glass beads for use in the methods described herein include silica beads from COLE-PARMER® (Vernon Hills, IL), preferably having diameters of about 100 mm to 1 mm, more preferably about 100 mm, about 500 mm, or about 1 mm. Beads of other materials, such as zirconium beads, can also be utilized. Prior to use with a biological sample, the glass beads are preferably soaked in an acidic solution (e.g., HCl), rinsed thoroughly with deionized water, and then baked at above 150°C for 12 to 24 hours to completely dry them. The beads are then cooled at 4°C or on ice for at least about 30 minutes before use to allow them to cool completely. The acid wash and heat treatment can also be eliminated if the beads are purchased pretreated and preferably nuclease-free.

[0021] As described herein, the present methods preferably exclude the use of detergents or lysis buffers to lyse virally transduced cells of a biological sample. As described herein, the use of such detergents and lysis buffers is not required, and it has been determined that their elimination can all reduce costs, sample preparation, and analysis time, and can also reduce or eliminate contamination from by-products, unwanted debris, or bacteria, as well as potential nucleases in the buffer.

[0022] Following mechanical disruption of virally transduced cells, droplet digital polymerase chain reaction (ddPCR) is performed on the nucleic acid molecules removed from the disrupted cells. As used herein, nucleic acid molecules are "removed" from the disrupted cells simply by the action of cell lysis or degradation. Preferably, no further action is required to isolate nucleic acid molecules, including DNA, from the disrupted cells, and the crude lysate (disrupted product) is directly applied to a ddPCR assay. As described herein, ddPCR performs digital PCR based on water-oil emulsion droplet technology. The sample is fractionated into 20,000 droplets, and PCR amplification of template molecules (DNA) occurs in each individual droplet. ddPCR technology uses reagents and workflows similar to those used in most standard TaqMan probe-based assays. Exemplary ddPCR analysis kits and assays are readily available, for example, from BIO-RAD® (Hercules, CA). In embodiments, an additional step of cell counting prior to ddPCR may be included. Methods for performing ddPCR to determine viral titer can be found, for example, in Dobnik et al., "Accurate Quantification and Characterization of Adeno-Associated Viral Vectors," Frontiers in Microbiology 10:Article 1570 (2019), and Abachin et al., "Comparison of reverse-transcriptase qPCR and droplet digital PCR for the quantification of dengue virus nucleic acid," Biologicals 52:49-54 (2018), the disclosures of each of which are incorporated herein by reference in their entirety, particularly with respect to the ddPCR methods disclosed therein.

[0023] Based on the ddPCR analysis, the viral titer is then calculated. Calculation of viral titer is easily performed from the ddPCR analysis and result output. The infectious viral titer from ddPCR can be calculated by using the formula: TU / mL = F x C x D / V, where TU / mL is transducing units / mL, F is the fraction of transduced cells, C is the number of cells input into the assay at the time of transduction, D is the dilution factor of the viral inoculum, and V is the volume (mL) of the viral inoculum input into the assay.

[0024] For example, assume that 20% of cells were transduced in an assay where 1,000 cells were seeded at inoculation. The virus was diluted 100-fold before being added to the assay, and 0.1 mL was added to the assay. Then, TU / mL = 20 x 0.01 x 1,000 x 100 / 0.1 = 2.0E+05. To determine the fraction of transduced cells (F), the total copy number of the viral genome integrated into the chromosome, as determined from the ddPCR results, is divided by the total number of cells at harvest.

[0025] As described herein, preferably, virally transduced cells containing viral vectors are mammalian cells. As used herein, the term "mammalian cells" includes cells derived from any member of the mammalian order, such as, for example, human cells, mouse cells, rat cells, monkey cells, hamster cells, and the like. In some embodiments, the cells are mouse cells, human cells, Chinese hamster ovary (CHO) cells, CHOK1 cells, CHO-DXB11 cells, CHO-DG44 cells, CHOK1SV cells, including all variants (e.g., POTELLIGENT®, Lonza, Slough, UK), and CHOK1SV GS-KO (glutamine synthetase knockout) cells, including all variants (e.g., XCEED™, Lonza, Slough, UK). Exemplary human cells include human embryonic kidney (HEK) cells, such as HEK293, HeLa, or HT1080 cells.

[0026] Mammalian cells include mammalian cell cultures, which can be either adherent or suspension cultures. Adherent cultures refer to cells grown on a substrate surface, such as a plastic plate, dish, or other suitable cell culture growth platform, and can be anchorage-dependent. Suspension cultures refer to cells that can be maintained, for example, in culture flasks or large suspension tanks, which allow for a large surface area for gas and nutrient exchange. Suspension cell cultures often utilize a stirring or agitation mechanism to provide adequate mixing. Media and conditions for maintaining cells in suspension are generally known in the art. Exemplary suspension cell cultures include human HEK293 clonal cells.

[0027] As described herein, exemplary viral vector titers that can be determined using the provided methods include lentiviral viral titers and adeno-associated viral (AAV) viral titers, as well as other viral vector titers.

[0028] Lentiviral vectors (LVs) are a well-studied vector system based on the human immunodeficiency virus (HIV-1). Other lentiviral systems, including HIV-2, simian immunodeficiency virus, non-primate lentiviruses, feline immunodeficiency virus, and bovine immunodeficiency virus, have also been developed as gene transfer systems. Driven by safety concerns due to the pathogenicity of HIV-1 in humans, the most widely used lentiviral system for clinical and research and development purposes is based on a four-plasmid system expressing the following: 1) Lentivirus group-specific antigen (GAG) genes and lentivirus polymerase (POL) proteins 2) envelope protein (usually vesicular stomatitis virus glycoprotein (VSV-G)) 3) HIV regulators of virion protein (Rev) protein expression, and 4) Transfer vector (TV) containing the gene of interest (GOI)

[0029] Lentiviral vectors are generally produced with a gene of interest to be introduced into desired cells for therapy and disease treatment, including immune deficiencies and neurodegenerative diseases.

[0030] As used herein, the term "adeno-associated virus (AAV)" refers to a small, replication-deficient, non-enveloped virus containing single-stranded DNA belonging to the Parvoviridae and Dependoparvovirus families. To date, more than 10 adeno-associated virus serotypes have been identified, with serotype AAV2 being the most well-characterized. Other non-limiting examples of AAV serotypes include ANC80, AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11. In addition to these serotypes, AAV pseudotypes have been developed. AAV pseudotypes contain the capsid of one serotype and the genome of a second serotype (for example, pseudotype AAV2 / 5 would correspond to an AAV with the genome of serotype AAV2 and the capsid of AAV5).

[0031] As used herein, the term "adenovirus" refers to a non-enveloped virus with an icosahedral nucleocapsid containing double-stranded DNA of the Adenoviridae family. More than 50 adenovirus subtypes have been isolated from humans, and many additional subtypes have been isolated from other mammals and birds. Birds. See, for example, Ishibashi et al., "Adenoviruses of Animals," In The Adenoviruses, Ginsberg, ed., Plenum Press, New York, NY, pp. 497-562 (1984); Strauss, "Adenovirus Infections in Humans," In The Adenoviruses, Ginsberg, ed., Plenum Press, New York, NY, pp. 451-596 (1984). These subtypes belong to the Adenoviridae family, which is currently divided into two genera: mastadenovirus and aviadenovirus. All adenoviruses are morphologically and structurally similar. However, in humans, adenoviruses exhibit different immunological properties and are therefore divided into serotypes. Two human serotypes of adenovirus, AV2 and AV5, have been intensively studied and provide most of the general information about adenoviruses.

[0032] In a further embodiment, provided herein is a method for determining viral titer in a biological sample, consisting essentially of obtaining a biological sample containing virally-transduced cells, mechanically disrupting the virally-transduced cells of the biological sample using glass beads, performing droplet digital polymerase chain reaction (ddPCR) on nucleic acid molecules removed from the disrupted virally-transduced cells, and calculating the viral titer.

[0033] Methods described herein that "consist essentially of" the recited steps exclude steps that use lysis buffers, detergents, or detergent or lysis steps; such steps are considered material alterations to a method consisting essentially of the recited steps and are therefore specifically excluded from such methods. Preferably, column purification steps are also excluded from methods consisting essentially of the recited steps.

[0034] Methods for producing virally transduced cells that can be measured using the methods described herein can be produced in any suitable reactor, including, but not limited to, stirred tank, air-lift, fiber, microfiber, hollow fiber, ceramic matrix, fluidized bed, fixed bed, and / or entrained-flow bioreactors. As used herein, a "reactor" can include a fermenter or fermentation unit, or any other reaction vessel, and the term "reactor" is used synonymously with "fermentor." The terms fermenter or fermentation refer to both microbial and mammalian cultures. For example, in some embodiments, an exemplary bioreactor unit can perform one or more or all of the following: feeding nutrients and / or carbon sources; injecting a suitable gas (e.g., oxygen); inflow and outflow of fermentation or cell culture media; separating gas and liquid phases; maintaining temperature; maintaining oxygen and CO2 levels; maintaining pH levels; agitation (e.g., stirring); and / or cleaning / sterilization. Exemplary reactor units, such as fermentation units, may contain multiple reactors within the unit; for example, a unit may have 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 or more bioreactors within each unit, and / or a facility may contain multiple units with single or multiple reactors within the facility. In various embodiments, the bioreactor may be suitable for batch, semi-fed-batch, fed-batch, perfusion, and / or continuous fermentation processes. Any suitable reactor diameter may be used. In embodiments, the bioreactor may have a volume of from about 100 mL to about 50,000 L.Non-limiting examples include 100 mL, 250 mL, 500 mL, 750 mL, 1 liter, 2 liters, 3 liters, 4 liters, 5 liters, 6 liters, 7 liters, 8 liters, 9 liters, 10 liters, 15 liters, 20 liters, 25 liters, 30 liters, 40 liters, 50 liters, 60 liters, 70 liters, 80 liters, 90 liters, 100 liters, 150 liters, 200 liters, 250 liters, 300 liters, 350 liters, 400 liters, 450 liters, 500 liters, and 550 liters. 1, 600 liters, 650 liters, 700 liters, 750 liters, 800 liters, 850 liters, 900 liters, 950 liters, 1000 liters, 1500 liters, 2000 liters, 2500 liters, 3000 liters, 3500 liters, 4000 liters, 4500 liters, 5000 liters, 6000 liters, 7000 liters, 8000 liters, 9000 liters, 10,000 liters, 15,000 liters, 20,000 liters, and / or 50,000 liters. Additionally, suitable reactors may be multi-use, single-use, disposable, or non-disposable and may be formed from any suitable material, including stainless steel (e.g., 316L or any other suitable stainless steel) and metal alloys such as Inconel, plastic, and / or glass.

[0035] Additional Exemplary Embodiments Embodiment 1 is a method for determining a viral titer in a biological sample, comprising obtaining a biological sample containing virally-transduced cells, mechanically disrupting the virally-transduced cells of the biological sample, performing droplet digital polymerase chain reaction (ddPCR) on nucleic acid molecules removed from the disrupted virally-transduced cells, and calculating the viral titer.

[0036] Embodiment 2 includes the method of embodiment 1, wherein the method does not include lysing the virally-transduced cells with a detergent or lysis buffer.

[0037] Embodiment 3 includes the method of embodiment 1 or 2, wherein the mechanically disrupting comprises disrupting with glass beads.

[0038] Embodiment 4 includes the method of embodiment 1 or 2, wherein the mechanically disrupting comprises sonication.

[0039] Embodiment 5 includes the method of any one of embodiments 1 to 4, wherein the virally transduced cells are mammalian cells.

[0040] Embodiment 6 includes the method of embodiment 5, wherein the mammalian cells are human cells.

[0041] Embodiment 7 includes the method of embodiment 6, wherein the human cells are human embryonic kidney (HEK) cells.

[0042] Embodiment 8 includes the method of embodiment 7, wherein the viral titer is an adeno-associated virus (AAV) viral titer.

[0043] Embodiment 9 includes the method of embodiment 7, wherein the viral titer is a lentiviral viral titer.

[0044] Embodiment 10 includes the method of embodiment 5, wherein the mammalian cells are Chinese hamster ovary (CHO) cells.

[0045] Embodiment 11 includes the method of embodiment 10, wherein the viral titer is an adeno-associated virus (AAV) viral titer.

[0046] Embodiment 12 includes the method of embodiment 10, wherein the viral titer is a lentiviral viral titer.

[0047] Embodiment 13 is a method of determining viral titer in a biological sample, consisting essentially of obtaining a biological sample containing virally-transduced cells, mechanically disrupting the virally-transduced cells of the biological sample using glass beads, performing droplet digital polymerase chain reaction (ddPCR) on nucleic acid molecules removed from the disrupted virally-transduced cells, and calculating the viral titer.

[0048] Embodiment 14 includes the method of embodiment 13, wherein the virally transduced cells are mammalian cells.

[0049] Embodiment 15 includes the method of embodiment 14, wherein the mammalian cells are human cells.

[0050] Embodiment 16 includes the method of embodiment 15, wherein the human cells are human embryonic kidney (HEK) cells.

[0051] Embodiment 17 includes the method of embodiment 16, wherein the viral titer is an adeno-associated virus (AAV) viral titer.

[0052] Embodiment 18 includes the method of embodiment 16, wherein the viral titer is a lentiviral viral titer.

[0053] Embodiment 19 includes the method of embodiment 14, wherein the mammalian cells are Chinese hamster ovary (CHO) cells.

[0054] Embodiment 20 includes the method of embodiment 19, wherein the viral titer is an adeno-associated virus (AAV) viral titer.

[0055] Embodiment 21 includes the method of embodiment 19, wherein the viral titer is a lentiviral viral titer. [Example]

[0056] Example 1: High-throughput format for measuring viral titers To avoid the laborious DNA extraction process, which typically involves detergent-mediated cell lysis and subsequent column purification of the DNA, cells are instead mechanically disrupted using glass beads.

[0057] Crude lysates prepared from cells transduced with lentivirus (LV) encoding green fluorescent protein (GFP) were directly applied to the ddPCR assay. To compare and validate this approach with conventional methods, DNA was also isolated from LV-transduced cells using a commercially available kit (QIAamp DNA Blood Mini Kit) from Qiagen. A primer probe set specific for the long terminal repeat (LTR) region of LV and the host beta-actin sequence was used to amplify the target sequence. To calculate the infectious titer, the following three methods were compared: 1. Sample DNA for ddPCR was isolated using a Qiagen kit. The cell number in the corresponding sample was calculated from the copy number of beta-actin in the same sample, and LV titer was calibrated based on this. 2. Sample DNA for ddPCR was isolated using a Qiagen kit. RNase A was included in the isolation procedure to remove any cellular RNA. The number of cells in the corresponding sample was calculated from the amount of DNA in the same sample, and the LV titer was calibrated accordingly. 3. Crude cell lysates were prepared by disrupting cells using glass beads and directly applied to ddPCR. The cell numbers in the corresponding samples were directly counted before cell disruption using ViCell, and the LV titers were calibrated based on this.

[0058] Cells in 6-well culture plates were transduced with LV-GFP and treated by the three different methods described above. Three samples for ddPCR were prepared for each method. LV titers from these samples were calculated and presented in Figure 1 as transducing units (TU) / mL. Table 1 below summarizes the results with statistical analysis. [Table 1]

[0059] The infectious LV titers calculated from the three different methods were comparable for the three samples tested, indicating that crude cell lysates prepared by bead disruption are sufficient for direct ddPCR applications. Furthermore, the coefficient of variation (CV) from the third method (bead disruption-cell count) was significantly smaller (8.2%) than the other methods, suggesting consistency and reproducibility.

[0060] It will be readily apparent to those skilled in the relevant art that other suitable modifications and adaptations to the methods and applications described herein can be made without departing from the scope of any of the embodiments.

[0061] Although particular embodiments have been illustrated and described herein, it is to be understood that the claims are not limited to the specific forms or arrangements of parts described and illustrated. Illustrative embodiments are disclosed herein, and although specific terms are employed, they are used in a generic and descriptive sense only, and not for purposes of limitation. Modifications and variations of the embodiments are possible in light of the above teachings. It is therefore to be understood that the embodiments may be practiced otherwise than as specifically described.

[0062] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. 1. A method for determining viral titer in a biological sample, comprising: a. obtaining said biological sample containing virally transduced cells; b. Mechanically disrupting the virally transduced cells of the biological sample to generate a crude lysate; c. subjecting nucleic acid molecules in the crude lysate obtained from the disrupted virally-transduced cells to droplet digital polymerase chain reaction (ddPCR) without further isolation; and d. calculating the viral titer.

2. 10. The method of claim 1, wherein the method does not include lysing the virally-transduced cells with a detergent or lysis buffer.

3. 3. The method of claim 1 or 2, wherein said mechanical disruption comprises disruption with glass beads.

4. 3. The method of claim 1 or 2, wherein the mechanical disruption comprises sonication.

5. The method of claim 1 or 2, wherein the virally transduced cells are mammalian cells.

6. 6. The method of claim 5, wherein the mammalian cell is a human cell or a Chinese hamster ovary (CHO) cell.

7. 7. The method of claim 6, wherein the human cells are human embryonic kidney (HEK) cells.

8. 8. The method of claim 6 or 7, wherein the viral titer is an adeno-associated virus (AAV) viral titer or a lentivirus viral titer.

9. 1. A method for determining viral titer in a biological sample, comprising: a. obtaining said biological sample containing virally transduced cells; b. mechanically disrupting the virally-transduced cells of the biological sample using glass beads to generate a crude lysate; c. subjecting nucleic acid molecules in the crude lysate obtained from the disrupted virally-transduced cells to droplet digital polymerase chain reaction (ddPCR) without further isolation; and d. calculating said viral titer.

10. 10. The method of claim 9, wherein the virally transduced cell is a mammalian cell.

11. 11. The method of claim 10, wherein the mammalian cell is a human cell or a Chinese hamster ovary (CHO) cell.

12. 12. The method of claim 11, wherein the human cells are human embryonic kidney (HEK) cells.

13. 13. The method of claim 11 or 12, wherein the viral titer is an adeno-associated virus (AAV) viral titer or a lentiviral viral titer.

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