Analysis of viral particles by digital assays

Abstract

Methods, compositions, and kits for analyzing viral particles. In an exemplary method of analyzing a viral particle, the capsid of the viral particle may be labeled with a label. A subsample comprising a sample of viral particles may be formed. Only a subset of the sub-samples may each comprise at least one of the viral particles. One or more targets may be amplified from the genome of the viral particle. Tag-related data and amplification data for one or more targets may be collected from the sub-sample. In some examples, the collected data may be used to determine the capsid occupancy of the viral particle in a calibration-free method without quantifying the viral particle or its capsid.

Description

Cross-reference to prior applications

[0001] This application is based upon and claims the benefit of 35 U.S.C. §119(e) prior to filing U.S. Provisional Patent Application Serial No. 63 / 327,727, filed on April 5, 2022. Each of these applications is incorporated herein by reference in its entirety for all purposes. introduction

[0002] Adeno-associated virus (AAV) is a non-pathogenic human virus, and its recombinant form (rAAV) is used for gene therapy. In the rAAV production process, the desired product is a real virus particle comprising a complete genome. However, defective virus particles with empty capsids (not containing genomes) or partially real capsids (comprising partial genomes) are also produced as product-related impurities. These defective virus particles lack therapeutic benefits and may cause unwanted immunotoxicity. Therefore, due to the clinical importance of capsid occupancy, the ratio of real capsids, partially real capsids and empty capsids in AAV gene therapy products is considered to be a key quality attribute (CQA).

[0003] For a number of reasons, existing methods available to quantify this ratio are inadequate and can result in significant differences in percent capsid occupancy values ​​for the same sample. There is currently no single "gold standard" method recommended by regulatory agencies to quantify capsid occupancy. Summary of the invention

[0004] The present disclosure provides methods, compositions and kits for analyzing viral particles. In an example method for analyzing viral particles, the capsid of the viral particles can be tagged with a label. Subsamples of a sample containing viral particles can be formed. Each subsample of only a subset of the subsamples can contain at least one viral particle. One or more targets can be amplified from the genome of the viral particle. Tag-related data and amplification data of one or more targets can be collected from the subsamples. In some examples, the collected data can be used to determine the capsid occupancy of the viral particle in a calibration-free method without quantifying the viral particle or its capsid. BRIEF DESCRIPTION OF THE DRAWINGS

[0005] Figure 1 is a flow chart listing exemplary steps that may be performed in a method of analyzing viral particles.

[0006] Figure 2 is another flow chart listing exemplary steps that may be performed in a method of analyzing viral particles.

[0007] Figure 3 is a flow chart schematically illustrating selected aspects of an exemplary method for analyzing capsid occupancy of viral particles by digital assay using proximity ligation assay (PLA) probe pairs.

[0008] Figure 4 yes Figure 3 Schematic diagram of a PLA probe pair and linker in the flowchart of FIG, taken before the linker is annealed to the PLA probe and the PLA probes are ligated to each other to create a template.

[0009] Figure 5 yes Figure 3 Another schematic diagram of a PLA probe pair and linker, taken after the template is created, and showing amplification of the first target from the template in the presence of the target-specific amplification probe.

[0010] Figure 6 yes Figure 3 Schematic representation of the viral genome in the flow chart and illustrating amplification of a second target in the viral genome in the presence of a target-specific amplification probe.

[0011] Figure 7 yes Figure 3 Zoomed-in, more detailed view of the scatter plot of amplification data collected in the flow chart.

[0012] Figure 8 is a schematic diagram of a recombinant adeno-associated virus ("GFP-AAV2") constructed to express a green fluorescent protein (GFP) transgene.

[0013] Fig. 9 is a table listing capsid occupancy values ​​for various predefined ratios of "full" GFP-AAV2 particles to "empty" AAV2 particles as determined by transmission electron microscopy (TEM).

[0014] Fig.10 is a table showing a two-dimensional array of dilutions and solid: empty ratios for comparing the exemplary digital experiments of the present disclosure with those obtained by different techniques. Fig. 9 The capsid occupancy values ​​of

[0015] Fig.11 yes Fig.10 An expanded version of a table of , wherein the cells of the table contain experimental values ​​obtained using an exemplary digital assay, TEM or qPCR (quantitative PCR) / ELISA (enzyme-linked immunosorbent assay) of the present disclosure.

[0016] Fig.12 is a series of tables showing the Fig.11 Figure 1. Exemplary calculation of capsid occupancy using experimental data obtained from experiments.

[0017] Fig.13 is a graph comparing different methods of calculating the percentage of capsids that contain the genome (ie, % solid capsids).

[0018] Fig.14 and 15 It is a graph showing the relationship between the copy number / μL of PLA and GC (GFP) and the capsid concentration at various capsid occupancy rates.

[0019] Fig.16 is a flow chart schematically illustrating selected aspects of an exemplary method for analyzing capsid occupancy of viral particles by a digital assay using beads and spiked probes including premade full-length templates.

[0020] Fig.17 is a flow chart schematically illustrating selected aspects of an exemplary method for analyzing capsid occupancy of viral particles by a digital assay using labeled spiked probes to detect capsids.

[0021] Fig.18 yes Fig.17 Magnified view of the labeled spike-in probe. DETAILED DESCRIPTION

[0022] Various aspects and examples of methods, compositions and kits for analyzing viral particles are described below and shown in the relevant drawings. Unless otherwise stated, methods, compositions and kits may (but not necessarily) include at least one of the structures, components, functions and / or changes described, shown and / or incorporated herein. In addition, unless specifically excluded, the processing steps, structures, components, functions and / or changes described, shown and / or incorporated herein may be included in other similar methods, compositions and kits, including being interchangeable between the disclosed examples. The description of various examples below is essentially illustrative and is in no way intended to limit the examples, their applications or their uses. In addition, the advantages provided by the examples described below are illustrative in nature, and not all examples provide the same advantages or the same degree of advantages.

[0023] The present disclosure provides methods, compositions and kits for analyzing viral particles. In an example method for analyzing viral particles, the capsid of the viral particles can be tagged with a label. Subsamples of a sample containing viral particles can be formed. Each subsample of only one subset of the subsamples may not contain viral particles, and each subset of another subset of the subsamples may contain at least one (or only one) viral particle. One or more targets can be amplified from the genome of the viral particles. Tag-related data and amplification data of one or more targets can be collected from the subsamples. In some examples, the collected data can be used to determine the capsid occupancy of the viral particles in a calibration-free method without quantifying the viral particles or their capsids.

[0024] The data (tag-related data and amplification data) can be used to determine one or more values ​​of any suitable one or more characteristics of the virus particle. In some examples, the data can be processed to quantify the capsid and the viral genome separately. However, since specific binding agents (such as antibodies) can be used to achieve capsid addition to detect the capsid, capsid quantification is not absolute in the absence of calibration, because the addition efficiency of the specific binding agent is less than 100% and depends on the affinity of the specific binding agent to its partner. Therefore, capsid quantification can involve comparing the tag-related data or one or more values ​​derived therefrom with a calibration curve generated separately using a known absolute amount / concentration of the capsid. This comparison allows the relative / approximate capsid quantification from the above-mentioned tag-related data to be converted into the absolute amount / concentration of the viral particle capsid. Differently, due to the inherent high efficiency of target amplification, the viral genome can be absolutely quantified with sufficient accuracy based on the amplification data without reference to a separate calibration curve. In this calibration-dependent method, the capsid and the viral genome are absolutely quantified (for example, as a concentration), and the accurate capsid occupancy (for example, the percentage of the real capsid) can be calculated based on the absolute viral genome concentration and the absolute concentration of the capsid as a ratio.

[0025] The present disclosure also provides a calibration-free method for quantifying capsid occupancy, which does not require a separate calibration curve. The calibration-free method can determine the value of the capsid occupancy with sufficient accuracy without quantitative capsids (or viral particles). In this method, the ratio of subsample / partition counts from two or more subsample / partition populations can be calculated to determine the capsid occupancy. The ratio utilizes the relative amount / concentration of the following two viral particles: (i) viral particles containing capsids but not containing copies of the viral genome (or at least not complete copies) (i.e., the capsid is empty) and (ii) viral particles containing capsids and viral genome copies (i.e., the capsid is real). Whether it is a real capsid or an empty capsid, the same (usually unknown) spike efficiency of the capsid is inherently present in both the numerator and the denominator of the ratio, so the spike efficiency of the capsid does not affect the ratio or does not need to be measured. If necessary, the absolute quantification of the viral genome and the capsid occupancy value can then be used to calculate the capsid concentration.

[0026] Other aspects of the disclosure are described in the following subsections: (I) Definitions, (II) Overview, (III) Examples, Components, and Alternatives, (IV) Illustrative Combinations and Additional Examples, (V) Advantages and Benefits, and (VI) Conclusion.

[0027] The features, functions, and advantages can be achieved independently in various examples of the present disclosure or may be combined in yet other examples, further details of which can be seen with reference to the following description and drawings. I. definition

[0028] The technical terms used in the present disclosure have meanings recognized by those skilled in the art. However, the following terms may be further defined as follows.

[0029] "Amplicon" is the product of an amplification reaction. A copy of the amplicon can be generated by amplifying the target sequence so that the amplicon corresponds to the target sequence (i.e., matches and / or is complementary to the target sequence). However, the sequence of the amplicon, such as the sequence at the primer binding site, may not completely match and / or be incompletely complementary to the target sequence.

[0030] "Amplification" is a process of preparing multiple copies of an amplicon by matching, complementing and / or otherwise corresponding to a target sequence. The process is interchangeably referred to as an amplification reaction. As the amplification proceeds, the amplification may produce an exponential increase in the number of copies. Typical amplification may increase the number of copies of the amplicon by more than 100 times or 1,000 times. An exemplary amplification reaction of the method disclosed herein may include a polymerase chain reaction (PCR) or a ligase chain reaction (LCR), each of which is driven by a thermal cycle. The method may also use or alternatively use other amplification reactions, which may be carried out under isothermal conditions, such as branched probe DNA detection, cascade RCA, helicase-dependent amplification, loop-mediated isothermal amplification (LAMP), nucleic acid-based amplification (NASBA), nickase amplification reaction (NEAR), PAN-AC, Q-β replicase amplification, rolling circle replication (RCA), self-sustaining sequence replication, strand displacement amplification and / or similar means. Amplification may utilize a linear or circular template.

[0031] An "amplifier" is any reagent that promotes the generation of an amplicon by amplifying a target sequence. The reagent may include any combination of: at least one primer or primer pair for amplifying at least one target sequence, at least one marker for detecting the amplification of the at least one target sequence (e.g., at least one probe including a fluorophore and / or an intercalating dye as a marker), at least one polymerase and / or ligase (which may be thermostable), and nucleoside triphosphates (dNTPs and / or NTPs), etc.

[0032] As used herein, "capsid" (also referred to as "viral capsid") is the outer shell of a virus that contains and surrounds viral genetic material. Therefore, a viral particle that does not contain viral genetic material is a capsid (i.e., an empty capsid). The outer shell of a virus has a shell that surrounds the viral genome. In most or all viruses, the shell is a protein shell composed of capsid proteins. In some cases, such as typical non-enveloped viruses, the outer shell of the virus is formed entirely by the shell. In these cases, the shell provides the inside and outside of the viral shell. In other cases, such as enveloped viruses, the outer shell of the virus has other components located outside the shell. Other components can wrap around the shell and form one or more layers, such as an envelope and a matrix, wherein the matrix is ​​located between the shell and the envelope. The envelope includes a lipid bilayer and typically also includes proteins that are integrated with or otherwise associated with the lipid bilayer. Any protein of the envelope can be virally encoded or host-encoded.

[0033] Each capsid of a collection of viral particles can be described as "full" or "empty" for the viral genome and / or one or more nucleic acid sequences thereof, which means that the viral genome or one or more sequences are present or absent (or deemed to be present or absent) within the capsid, respectively. The viral genome can be a natural genome of a natural virus, or a recombinant genome comprising a transgenic or other non-natural / engineered sequence. The "occupancy rate" of a collection of viral particles (or their capsids) is a numerical description of the genome / target content in the viral particles (or their capsids). Exemplary units of capsid occupancy rate include the percentage / fraction of a full capsid, the percentage / fraction of a capsid that is only partially full, the percentage / fraction of an empty capsid, the ratio of a full capsid to an empty capsid, or the ratio of an empty capsid to a full capsid, etc.

[0034] A "carrier fluid" is a fluid that contacts the partitions, optionally surrounding the partitions. The fluid may be a liquid or a gas. The carrier fluid may be described as a continuous phase, and wherein the partitions are described as dispersed phases. The carrier fluid may be immiscible with the partitions and encapsulate the partitions. In some instances, the carrier fluid may be an oil, such as, for example, a fluorocarbon oil or a silicone oil.

[0035] "Complementary" means the rule of base pairing. If the first nucleic acid polymer or its region can hybridize with the second nucleic acid polymer in an antiparallel manner by forming a continuous (uninterrupted) or almost continuous base pair series (for example, at least 5, 6, 7, 8, 9 or 10 continuous base pairs), the first nucleic acid polymer or its region is "complementary" to the second nucleic acid polymer. If the hybridization of the first nucleic acid polymer (or its region) with the second nucleic acid polymer forms a continuous base pair series with each nucleotide in the first nucleic acid polymer or its region, the first nucleic acid polymer (or its region) is referred to as "completely complementary" to the second nucleic acid polymer. The "complement" of the first nucleic acid polymer or its region is the second nucleic acid polymer or its region that is completely complementary to the first nucleic acid polymer or its region. The "complementarity" between the first nucleic acid polymer (or its region) and the second nucleic acid polymer (or its region) refers to the number or percentage of base pairs that can be formed when the first nucleic acid polymer (or its region) is optimally arranged to hybridize with the second nucleic acid polymer (or its region) in an antiparallel manner. A first nucleic acid polymer or region thereof that is complementary to a second nucleic acid polymer or region thereof typically has a complementarity of at least 80%, 90%, 95% or 100%.

[0036] “Comprising,” “including,” and “having” (and variations thereof) are used interchangeably and mean including but not necessarily limited to, and are open-ended terms that are not intended to exclude additional, unrecited elements or method steps.

[0037] A "linker" is one or more oligonucleotides configured to base pair with the sequence of a PLA probe pair. In some instances, such base pairing can position the 5' and 3' ends of the PLA probe pair together so that the 5' and 3' ends are connected to each other, thereby creating a template. In other instances, such base pairing can position the 5' and 3' ends of the linker together so that the 5' and 3' ends are connected to each other, thereby creating a template, optionally by circularizing the linker.

[0038] "Coupled" refers to a relationship whereby the properties of one affect the properties of another, and may include permanent or releasable connection, whether direct or indirect through intermediate components, and is not necessarily limited to physical connections.

[0039] A "digital test" is one or more research procedures capable of detecting a single copy of an analyte in a collection of subsamples or partitions, wherein each subsample / partition of only a subset of the subsamples / partitions contains one or more copies of the analyte. A "digital amplification test" is a digital test that utilizes an amplification reaction to facilitate the detection of a single copy of a target. A digital test may be performed with any suitable number of subsamples / partitions that give statistically significant results, such as at least twenty, one hundred, one thousand, or ten thousand, etc.

[0040] A "droplet" is a small volume of liquid surrounded by an immiscible liquid (e.g., encapsulated by an immiscible liquid that may form a continuous phase of an emulsion). The immiscible liquid may include oil and / or may consist primarily of oil. The droplets disclosed herein may, for example, have an average volume of less than about 500 nL, 100 nL, 10 nL, or 1 nL, etc.

[0041] An "enumerated value" is any value generated by enumerating a subset of a tag / target definition of a set of subsamples / partitions in a digital assay. For example, an enumerated value may represent the number of subsamples / partitions in a set of subsamples / partitions that are positive for the presence of a given tag / target or two or more given tags / targets, negative for the presence of a given tag / target or two or more given tags / targets, positive for only a specified subset of one or more tags / targets in a set of tags / targets, negative for only a specified subset of one or more tags / targets in a set of tags / targets, etc.

[0042] “Exemplary” means “illustrative” or “serving as an example.” Likewise, the term “example” (or “exemplary”) means “illustrative.” Neither word implies desirability or superiority.

[0043] "First," "second," "alpha," "beta," and similar terms are used to distinguish or identify members of a group, etc., in the order in which they are introduced in a particular context, and are not intended to indicate an order or numerical limitation.

[0044] "Fluorescence" is optical radiation emitted in response to the absorption of light. As used herein, fluorescence is intended to encompass any form of photoluminescence in which the absorption of one or more photons promotes an electron to an excited state and results in the subsequent emission of new photons, whether from a singlet, triplet, or other state. The excited state resulting from the absorption may have any suitable lifetime.

[0045] "Fluorophore" refers to any atom, functional group, molecule or substance that is capable of fluorescing.

[0046] The "genome" of a viral particle or collection of viral particles is the genetic material (nucleic acid, DNA and / or RNA) contained in the capsid of the viral particle or collection of viral particles. The genome can be a recombinant / engineered genome or a natural genome constructed at least in part by human activity. A collection of viral particles can be equivalently described as comprising a genome, or comprising a complete / partial copy of a genome.

[0047] "Label" is any detectable marker or identifier associated with a member, structure or substance, such as any detectable marker or identifier associated with a primer, probe, amplicon, capsid, subsample, partition, etc. The marker can be covalently associated with a member, structure or substance (e.g., the marker is coupled to an oligonucleotide or antibody) or non-covalently associated (e.g., by embedding, hydrogen bonding, electrostatic interaction, encapsulation, etc.). Exemplary markers include optical markers, radioactive markers, magnetic markers, electrical markers, epitopes, enzymes, antibodies, oligonucleotides, etc. Optical markers can be optically detected by their interaction with light. Exemplary optical markers that may be suitable include fluorophores and quenchers, etc.

[0048] "Labeling" is any action, process or procedure that links or associates a label with a specified structure, member or substance. Labeling can result in non-covalent linking or association, or covalent attachment of the label to a structure, member or substance.

[0049] A "no particle control" (NPC) is an assay or procedure performed in the absence of the viral particle of interest. One or more values ​​obtained from a no particle control can be used to adjust one or more values ​​of a corresponding assay or procedure performed in the presence of the viral particle of interest.

[0050] "Nucleic acid polymers" are molecules or molecular duplexes of any length comprising naturally occurring nucleotides (e.g., the polymer is DNA and / or RNA), or synthetically produced compounds that can hybridize with DNA or RNA in a sequence-specific manner similar to two naturally occurring nucleic acids, for example, can participate in Watson-Crick base pairing interactions. Nucleic acid polymers can contain any suitable number (such as at least about 5, 10, 100, or 1000, etc.) of nucleotides. The term "nucleic acid" refers to one or more nucleic acid polymers.

[0051] Nucleic acid polymers can have natural or artificial structures, or a combination of the two. Nucleic acid polymers with natural structures, i.e., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), generally have a backbone of alternating pentose groups and phosphate groups. Each pentose group is connected to a core base (e.g., purine (e.g., adenine (A) or guanine (T)) or a pyrimidine (e.g., cytosine (C), thymine (G) or uracil (U))). Nucleic acid polymers with artificial structures are analogs of natural nucleic acids, and can, for example, be produced by changing the pentose and / or phosphate groups and / or one or more core bases of the natural backbone. Exemplary artificial nucleic acid polymers include glycol nucleic acids (GNA), peptide nucleic acids (PNA), locked nucleic acids (LNA), threose nucleic acids (TNA), heterologous nucleic acids (XNA), etc.

[0052] The sequence of a nucleic acid polymer is determined by the order in which the nucleobases are arranged along the backbone. This order generally determines the ability of a nucleic acid polymer to hybridize with another nucleic acid through hydrogen bonding. Specifically, adenine pairs with thymine (or uracil), and guanine pairs with cytosine.

[0053] "Oligonucleotide" is a relatively short and / or chemically synthesized nucleic acid polymer. For example, the length of the oligonucleotide can be 3 to 1000 nucleotides, etc. In some cases, the oligonucleotide can be labeled with at least one marker, which can be coupled to any suitable structure of the oligonucleotide. The at least one marker can include at least one fluorophore, and therefore can be a fluorescent marker. Each marker can be coupled to the oligonucleotide at any suitable position, including 5' end, 3' end or middle 5' and 3' end.

[0054] "Optical radiation" means electromagnetic radiation in the spectrum, namely ultraviolet light, visible light and / or infrared light. The term "light" without modification has the same meaning as optical radiation.

[0055] A "particle" is a small, discrete mass or portion of a substance. A particle can be substantially solid (i.e., solid phase particle), liquid (i.e., liquid phase particle), or a combination thereof, among others. Exemplary particles include droplets, beads, biological cells, and viral particles. Exemplary sizes (e.g., length or diameter) of particles include less than 2000, 1000, 500, 200, or 100 microns, and / or at least 5, 10, or 20 nanometers, among others.

[0056] "Partitions" are discrete volumes of fluid (i.e., fluid volumes) that are separated from one another (also referred to as separation volumes). Each partition in a set of partitions may contain portions of the same sample. Partitions may be separated from one another by fluids (e.g., oil or air), walls of a device, or a combination thereof, etc. Thus, a partition may be a droplet of an emulsion, or a volume held by a well, chamber (e.g., a nanochamber with a capacity of less than 1 μL), tube (e.g., a microtube with a diameter of less than 1 mm), or microfluidic device, etc. Partitions may have the same diameter as one another and / or may contain the same amount of fluid as one another.

[0057] A "partition count" (or "subsample count") is a value for the number of partitions (or subsamples) in a specified group, e.g., a population of partitions (or subsamples) having a specified tag / target content. A partition count or subsample count may be an enumerated value. A partition / subsample count may be determined by counting and / or calculation. A partition / subsample count (or its value) may be "uncalibrated," meaning that the count or value has not been adjusted, read out, or otherwise derived using a capsid quantitation calibration curve.

[0058] When used to indicate the tag / target content of a viral particle, partition, subsample, or a population of partitions / subsamples, the term "positive" indicates that the viral particle, partition / subsample, or each partition / subsample of a population contains (or at least appears and / or is considered to contain) at least one copy of a given tag / target or each tag / target of a given tag / target set. When used to indicate the target content of a viral particle, partition / subsample, or a population of partitions / subsamples, the term "negative" indicates that the viral particle, partition / subsample, or each partition / subsample of a population does not contain (or at least appears and / or is considered not to contain) at least one copy of a given tag / target or each tag / target of a given set of two or more tags / targets.

[0059] "Primer" is an oligonucleotide (DNA / RNA or its analogue) that can serve as a starting point for template-directed nucleic acid synthesis or ligation under appropriate reaction conditions (e.g., in the presence of a template to which the primers are annealed, a nucleoside triphosphate, and a polymerizing agent (such as a DNA or RNA polymerase or ligase, or a reverse transcriptase), in an appropriate buffer and at an appropriate temperature). Primers can have any suitable length, such as 5 to 500 nucleotides, etc. The primers can be members of a "primer pair" or "primer set", which includes a "forward primer" and a "reverse primer", which define the ends of the amplicon produced in the amplification reaction. (The adjectives "forward" and "reverse" are arbitrarily specified relative to each other). The forward primer hybridizes with a complementary sequence to the 5' end region of the template sequence to be amplified, and the reverse primer hybridizes with a 3' end region of the template sequence. The term "primer binding site" refers to the portion of the template (or its complement) to which the primers are annealed. The full-length sequence of the primer does not need to be completely complementary to the primer binding site, as long as there is sufficient complementarity under the reaction conditions to anneal. Thus, a primer may have a 3' terminal region that is complementary to the primer binding site and a 5' terminal region that is not complementary to the primer binding site (and forms a "5'-tail").

[0060] "Probe" is any substance that is configured to facilitate or is capable of testing, identifying and / or detecting an analyte (e.g., an analyte such as a viral particle, a viral capsid, a viral genome, a tag and / or a target) among other analytes. For example, a probe can be a spiked probe or an amplification probe. In some instances, either type of probe can include an oligonucleotide. A spiked probe is any probe that includes at least a portion of a label. An amplification probe is configured to be capable of detecting the occurrence of an amplification reaction and / or the amplicon formed by the amplification reaction. In some instances, an amplification probe can be a fluorescent probe that includes an oligonucleotide labeled with a fluorophore. An amplification probe can be configured to hybridize with at least a portion of the amplicon produced by amplification. For example, an amplification probe can be a hydrolysis probe, a molecular beacon probe, a strand displacement probe, or a labeled primer, etc.

[0061] A "proximity assay" (PA) is a test or procedure in which a template is created in a proximity-dependent manner in the presence of a viral particle being tested. Copies of the template are created when copies of separate portions of the template are brought into proximity with each other on these same individual viral particles by binding to the individual viral particles. The template can be created by one or more oligonucleotides by any suitable reaction or reactions, such as ligation (in a proximity ligation assay (PLA)) or extension (in a proximity extension assay (PEA)). A PA probe is a probe comprising an oligonucleotide that includes only a portion of the template to be created, or is complementary to at least a portion of the template to be created. A PA probe may include a specific binding agent, such as for direct binding to a capsid, or for indirect binding to a capsid via a capsid-specific binding agent. The specific binding agent of the oligonucleotide and the PA probe may be covalently coupled or otherwise attached to one another. In some instances, the PA probe may be a PLA probe for a proximity ligation assay, or a PEA probe for a proximity extension assay.

[0062] A "specific binding agent" is any molecule, complex, substance, or member that specifically binds to another substance. Specific binding may be characterized by a dissociation constant of less than about 10 -4 , 10 -6 , 10 -8 or 10 -10 M. Exemplary pairs of specific binding agents include biotin and avidin / streptavidin, sense nucleic acid and complementary antisense nucleic acid (e.g., amplification probe and amplicon), primer and its target, antibody and corresponding antigen, receptor and its ligand, aptamer and corresponding antigen, etc.

[0063] A "subsample" is a smaller sample provided by a bulk sample or sample-containing fluid, containing only a portion of the bulk sample or sample-containing fluid. A collection of subsamples may be a collection of partitions, and vice versa.

[0064] "Substantially" means conforming primarily to the particular size, range, shape, concept or other aspect modified by the term, such that the feature or component does not need to conform exactly as long as it is suitable for its intended purpose or function. For example, a "substantially cylindrical" object means that the object resembles a cylinder, but may have one or more deviations from a true cylinder.

[0065] A "tag" is any identifier or marker that is or will be associated with a member, structure, substance, or complex. A tag can be covalently coupled to a member, structure, substance, or complex, such as a tag coupled to an antibody, or can be non-covalently and / or indirectly associated (e.g., by hydrogen bonding, electrostatic interactions, encapsulation, etc.). Exemplary tags include antibodies, antigens, aptamers, beads, epitopes, enzymes, nucleotide sequences, oligonucleotides, etc. A tag can contain or be attached to a label, either covalently or non-covalently, so that the tag can be detected by the label.

[0066] "Tagging" is any process or collection of processes that results in the association of a tag with a specified member, structure, substance or complex. The association can be a direct attachment (eg, covalent attachment or direct binding) or an indirect connection.

[0067] "Target / target" (also referred to as "target sequence / target sequence") is a nucleic acid polymer sequence (DNA and / or RNA) of any suitable length to be amplified in an amplification reaction. Exemplary target sequences are about 20-1000 nucleotides, or about 30-500 nucleotides, etc.

[0068] A "template" is a nucleic acid polymer that includes a target sequence and / or its complementary sequence. II. Overview

[0069] This section summarizes the methods, compositions, and kits described herein.

[0070] A method for analyzing viral particles is provided. In the method, the capsid of the viral particles can be tagged with a tag. Subsamples of a sample containing viral particles can be formed. Each subsample of only a subset of the subsamples can contain at least one of the viral particles. One or more targets can be amplified from the genome of the viral particles. Tag-related data and amplification data of one or more targets can be collected from the subsamples.

[0071] Another method for analyzing viral particles is provided. In this method, the capsid of the viral particles can be spiked with a template. Sample partitions containing viral particles can be formed so that only a subset of the partitions contain one of the viral particles. A first target can be amplified from the template in the partition, and one or more other targets can be amplified from the viral particle genome in the partition. Amplification data for each of the first target and the one or more other targets can be collected from the partition.

[0072] A composition for analyzing viral particles is provided. The composition may include a partition of a bulk sample containing viral particles. The partition may include a single viral particle in the viral particle. The capsid of a single viral particle may be labeled with a label. If a genome is present in the partition, the partition may include reagents sufficient to amplify a target from the genome of the viral particle. III. III. Examples, components and alternatives

[0073] The following subsections A to J describe selected aspects of exemplary methods, compositions, and kits for analyzing viral particles. The examples in these subsections are intended to illustrate and should not be construed as limiting the full scope of the disclosure. Each subsection may include one or more different examples and / or contexts or related information, functions, structures, and / or processes. A. Method for analyzing viral particle capsid occupancy by digital experiment

[0074] This subsection describes an exemplary method for analyzing viral particles by digital assays to quantify viral particle capsid occupancy; see Figure 1 and 2 20, 120. Each method may include any suitable combination of the steps listed in flowcharts 20 and 120, performed in any suitable order, and omitting or performing any step any suitable number of times, and using or including any features, aspects, reagents, configurations, additional steps or modifications described elsewhere in this disclosure.

[0075] In the selection step 21 or 121 of flowcharts 20 and 120, a sample containing virus particles is selected (see Figure 1 and 2 ). A viral particle may include a capsid (also referred to as a copy of the capsid) and a copy of the viral genome located within the capsid (also referred to as a viral genome). Each capsid may be a "real" capsid (or viral particle) comprising a complete copy of the viral genome (and / or one or more predefined targets therein), a partially real capsid (or viral particle) comprising only a partial copy of the viral genome (and / or a subset (at least one, but less than all) of one or more predefined targets), or an "empty capsid" (or viral particle) that does not contain a copy of the viral genome (and / or does not contain one or more predefined targets).

[0076] The viral particles may be a collection of viral particles that have structural similarity / identity to each other. At least the majority of the viral particles may have the same capsid and / or for the majority of a subset of viral particles containing the genome, have the same copy of the genome.

[0077] The sample may be from any suitable source and may be prepared in any suitable manner. In some examples, the sample may be treated, for example, with a nuclease (e.g., DNase) to degrade uncoated copies of the viral genome and / or other nucleic acids located outside the viral particles. The nuclease may then be inactivated by adding an inhibitor (e.g., EDTA), removing / inactivating an activator or a required cofactor, or by heating, etc.

[0078] The sample may contain any suitable type of viral particles. For example, illustrative viral particles may correspond to adeno-associated viruses, adenoviruses, herpes viruses, lentiviruses, retroviruses, etc. Thus, the viral genome may be composed of DNA or RNA, and the viral particles may or may not have a corresponding viral envelope surrounding the capsid.

[0079] In the labeling step 22 of flow chart 20, the virus particles are labeled with a label (see Figure 1). The spike-in 22 is independent of the viral genome of the viral particles, meaning that individual viral particles are spiked regardless of whether they contain a copy of the viral genome, typically with very similar or identical spike-in efficiencies. The spike-in process may result in the tag (at least one copy thereof) being attached to each viral particle (and / or each capsid) in a plurality of viral particles (and / or their plurality of capsids) so that the viral particles (and / or capsids) and at least one copy of the tag are later co-localized to the same subsample (and / or partition) (see below). The at least one copy of the tag may be attached to the viral particles and / or capsids covalently or non-covalently, directly or indirectly.

[0080] In some instances, it is not necessary to spike every viral particle. For example, if a specific binding agent is involved in the spiking, the spiking efficiency may be significantly less than 100%, depending on the affinity of the specific binding agent. As described elsewhere herein, in some instances, even if not all capsids are spiked, characteristics of viral particles, such as capsid occupancy, can be quantified without knowing or measuring the spiking efficiency or calibrating separately to account for the spiking efficiency.

[0081] The spike-in can include merging the viral particles (and / or the sample) and at least one spike-in probe with each other. Each spike-in probe can include a specific binding agent (e.g., an antibody, an antibody fragment, or an aptamer) and at least a portion of a label. The specific binding agent and at least a portion of the label can be covalently coupled to each other, or can be non-covalently connected to each other. In some instances, the specific binding agent itself can provide the label.

[0082] Specific binding agents can have any suitable recognition and binding properties. In some instances, specific binding agents can recognize and directly specifically bind to a viral particle structure (i.e., a viral capsid, such as its shell, matrix, or envelope) that is separated from the viral genome. In other instances, specific binding agents can be indirectly bound to a viral particle structure through at least one other specific binding agent (e.g., a primary antibody), which in turn directly binds to the viral particle structure.

[0083] The label can have any suitable characteristics to facilitate its detection. In some instances, the label or its portion can include a detectable marker (e.g., see subsections I and J). In some instances, the label can be configured to be connected to a detectable marker during the method. In some instances, the label can indirectly change the detectability of the marker.

[0084] In the labeling step 122 of the flowchart 120, the capsid of the viral particle is labeled with a tag that provides a template (see Figure 2). As described above with respect to step 22, the efficiency of capsid addition is generally independent of the occupancy of the genome to the capsid. Empty capsids and full capsids can be added with very similar or identical efficiencies. The addition process may result in the tag / template (at least one copy thereof) being attached to each viral particle (and / or each capsid) in a plurality of viral particles (and / or their multiple capsids) so that the viral particles (and / or capsids) and at least one copy of the tag / template are later co-localized to the same subsample (and / or partition) (see below). The tag / template may be created during addition 122 in the presence of viral particles, or may be combined with viral particles as a preformed full-length tag / template. In some instances, it is not necessary to add a tag to each viral particle, only a plurality and / or at least a subset of viral particles need to be added, as explained above with respect to the addition step 22. Therefore, the efficiency of addition may be significantly less than 100%.

[0085] The addition of the label 122 can include merging the viral particles (and / or the sample) and at least one addition of the label probe. Each addition of the label probe can include a specific binding agent (e.g., an antibody, an antibody fragment and / or an aptamer) and an oligonucleotide. The oligonucleotide provides at least a portion of the label / template or is complementary to at least a portion of the label / template (e.g., a sequence comprising at least 5, 6, 7, 8, 9 or 10 nucleotides that are matched or fully complementary to a portion of the label / template). The specific binding agent and the oligonucleotide of the addition of the label probe can be covalently coupled to each other, or can be non-covalently connected to each other. In some cases, the oligonucleotide can also provide a specific binding agent.

[0086] Each spiked probe can be a proximity assay (PA) probe. Spiking 122 can include (i) combining a viral particle and a PA probe pair (e.g., a pair of PLA probes or a pair of PEA probes) with each other, and (ii) creating a tag / template, such as by ligation (e.g., using a ligase) or by extension (e.g., using a polymerase).

[0087] If the PA probe pair is a PLA probe, at least one linker can also be combined with the viral particle before creating the tag / template. At least one linker is annealed with the oligonucleotides of a pair of PLA probes to facilitate the creation of the tag / template, for example, by connecting copies of the oligonucleotide pair to each other. In some examples, the copies of the oligonucleotide pair are connected to each other and have different or identical sequences to each other.

[0088] If the PA probe pair is a PEA probe, the PEA probe can provide a pair of oligonucleotides (a first oligonucleotide and a second oligonucleotide) that are configured to base pair with each other at their 3' end regions. In other words, the first and second oligonucleotides can be complementary to each other at the 3' end regions, which allows them to form a hybrid that can be extended by a polymerase. More specifically, the 3' end region of the first oligonucleotide can be used as a primer to extend it using the second oligonucleotide as a template, and / or the 3' end region of the second oligonucleotide can be used as a primer to extend it using the first oligonucleotide as a template. In any case, extension of one or both oligonucleotides by the polymerase will create a tag / template for the viral particle and / or capsid to be labeled.

[0089] In other examples, spike 122 may include combining viral particles with spike probes comprising oligonucleotides that provide a full-length tag / template (see also subsections H and Fig.16 ). In this configuration, no connection is required.

[0090] Spiking 22 or 122 can be performed in the presence of beads (see, for example, subsections H and Fig.16 ). The viral particles can advantageously be combined with beads so that the viral particles are bound to the beads. The beads provide a solid support that can be washed after the beads and viral particles are combined with each other and / or after the beads, viral particles, one or more spiked probes and / or one or more linkers are combined with each other to remove viral particles that are not bound to the beads, and / or to remove copies of one or more spiked probes and / or one or more linkers that are not connected to the beads through the viral particles. Thus, the use of beads and one or more washing steps can reduce background in any of the methods of the present disclosure.

[0091] In the formation step 23 of the flowchart 20, a sub-sample is formed (see Figure 1 ). Subsamples may be formed from a bulk sample comprising viral particles such that each subsample comprises a portion of the bulk sample. Only a subset of subsamples comprises individual viral particles, and / or only a subset of subsamples comprises at least one of the viral particles. Subsamples may have any combination of the characteristics described for partitioning.

[0092] In the formation step 123 of the flowchart 120, a collection of partitions containing virus particles is formed (see Figure 2). Each partition in the set of partitions of only a subset may contain one (individual) or more (individual) virus particles, so another subset in the set of partitions may not contain virus particles. Multiple partitions in the set of partitions may each contain only one (individual) virus particles. In other words, the set of partitions formed may contain a set of "partially occupied" virus particles, which means that the set of virus particles co-exists in or is contained in the set of partitions of only a subset. If the virus particles have been bound to beads (as described above) before forming step 123, the set of partitions may also contain partially occupied beads. The virus particles (and beads, if present) in the set of virus particles may be randomly distributed between partitions, but the supply is limited so that each partition cannot receive at least one of the virus particles (and / or at least one of the beads). The size of the partitions may be substantially uniform and may have any of the characteristics described in Section J above. In some instances, the partitions may be substantially identical to each other except for the different contents of virus particles / beads and related molecules. Each partition in the partition set can include a portion of the same sample-containing fluid, which in addition to the sample can also contain amplification reagents (e.g., amplification primers, amplification probes, amplification enzymes, etc.) and any other suitable reagents, whose concentrations / amounts are sufficient to distribute at least multiple copies of each reagent to each partition in the partition set.

[0093] The capsid may be lysed after the subsample / partition is formed so that copies of the viral genome can be amplified. Lysis is any change in the integrity of the capsid in the subsample / partition that releases viral nucleic acid from the capsid and / or allows amplification reagents to effectively interact with the viral nucleic acid. Lysis may be driven by any suitable treatment that does not interfere with the integrity of the partition, such as heating, sonication, protease activation, exposure to detergents, and / or the like.

[0094] In the amplification step 24 of flowchart 20, one or more targets are amplified from the viral genome copies in the subsample (see Figure 1 ). One or more targets can be amplified using a corresponding number of primer sets present in the subsample. Amplification in the subsample can be performed for the partition at least substantially as described below.

[0095] In the amplification step 124 of flowchart 120, a first target can be amplified from a template, and one or more other targets including a second target can be amplified from a copy of the viral genome from a viral particle (see Figure 2). Amplification is performed in the partitions, and amplification can be driven by heating the partitions to above room temperature (e.g., to promote isothermal amplification) and / or thermal cycling the partitions (e.g., to promote polymerase chain reaction or ligase chain reaction). In some examples, each partition can include amplification primers for amplifying each of the first target and one or more additional targets, and specific amplification probes for each target.

[0096] In the collection step 25 of flowchart 20, the tag-related data and the amplification data of each target can be collected from the subsamples (see Figure 1 ). The tag-related data can be used to detect the presence of the tag (and thus, the presence of the capsid / virion) in the individual subsamples. The amplification data can be used to detect the presence of the viral genome in the individual subsamples. Detection in subsamples can be performed for partitions at least generally as described below.

[0097] In the collection step 125 of flowchart 120, amplification data may be collected from the set of partitions (see Figure 2 ). The collecting step may be interchangeably described as "reading" a partition. In some instances, collecting amplification data may include detecting light, such as fluorescence, from a partition. For example, a partition may contain target-specific amplification probes for each target, and fluorescence (e.g., fluorescence intensity) may be detected from a marker (fluorophore) for each target-specific amplification probe, optionally in a different detection channel for each marker. Each detection channel may be defined based on one or more detected fluorescence wavelengths, one or more excitation light wavelengths, fluorescence lifetime, etc.

[0098] The tag-related data and / or amplification data can be compared to one or more positive / negative thresholds to designate individual subsamples / partitions as positive or negative for a tag and / or individual amplification targets. Individual populations of subsamples / partitions having the same tag / target content can be identified (e.g., see Figure 3 , 7 , 16 and 17). The tag / target content can be defined by positive / negative relative to each tag / target.

[0099] In the enumeration steps 26, 126 of flowcharts 20, 120, each of the two or more subsample / partition populations in the subsample partition set can be enumerated to obtain a numerical value of the subsample / partition count from each of the two or more populations (see Figure 1 and 2). This numerical value represents the number of partitions present in the population. Each population has a different designated label / target content (positive / negative). Each subsample / partition count can be adjusted. Adjustment may include standardizing the subsample / partition count by scaling up or down the subsample / partition count according to the ratio of the predefined or standard subsample / partition total set count to the actual subsample / partition total set count. For example, if the subsample / partition count of a given population is 100 out of 1000 total set counts of the subsamples / partitions tested, the subsample / partition count standardized to the total set count of 2000 subsamples / partitions will be 100*(2000 / 1000)=200. Adjustment may also or alternatively include subtracting a particle-free control (NPC) value (with or without standardization) from the subsample / partition count to remove background contributions to the subsample / partition count that are not related to viral particles.

[0100] In the determination steps 27 and 127 of the flowcharts 20 and 120, the occupancy of the viral particles by the genome is determined (see Figure 1 and 2 ). The occupancy can be the capsid occupancy of at least a portion of the viral genome to the viral particle. The capsid occupancy can be a numerical value that quantifies the fraction or percentage of viral particles that contain a portion of the viral genome corresponding to one or more targets amplified from the viral genome. B. Homogeneous digital assay for quantifying capsid occupancy

[0101] This subsection describes exemplary assay reagents and exemplary assay configurations produced by performing the illustrative methods according to subsection A, wherein the methods include a homogenous digital assay for quantifying capsid occupancy and using a PLA probe pair to spike capsids; see Figure 3-7 .

[0102] Figure 3 A flow chart 130 is shown schematically illustrating selected aspects of a homogenous digital assay. A sample 132 containing viral particles 134 is shown in the upper left corner of the flow chart 130. The viral particles 134 include a viral capsid 136 that surrounds a copy of a viral genome 138. However, not every capsid 136 surrounds a copy of the genome 138. Instead, the viral particles 134 include solid particles 140 that contain a complete genome 138 in the capsid 136, and empty particles 142 that lack the genome 138 (or at least a portion thereof) in the capsid 136 and are therefore defective. The purpose of the homogenous digital analysis is to quantify the occupation of the capsid 136 by the genome 138 in the viral particles 134.

[0103] Sample 132 (with virus particles 134), spiked probe pairs 144a, 144b, and linker 146 are combined with one another, as shown at 148 (see Figure 3and 4 ). In this example, the labeled probes 144a, 144b are PLA probes, respectively including specific binding agents 150a, 150b to directly or indirectly bind to the capsid 136, and oligonucleotides 152a, 152b to connect to each other. The specific binding agent binds to the real capsid and the empty capsid in a similar manner, and has very similar or identical efficiency. Each specific binding agent 150a, 150b includes an antibody, as shown here, although any type of specific binding agent is applicable. If one or both specific binding agents 150a, 150b are configured to indirectly bind to the capsid 136, then the merging can include merging one or more other specific binding agents with the sample 132 and the labeled probes 144a, 144b to provide at least one bridge for connecting the capsid 136 to one or both labeled probes 144a, 144b. The members of each pair of spiked probes 144a, 144b, specific binding agents 150a, 150b and / or oligonucleotides 152a, 152b may be structurally different from one another or identical to one another.

[0104] Figure 4 An enlarged view of the labeled probes 144a, 144b and the linker 146 taken from before the connection without the capsid 136 is shown. Each specific binding agent 150a, 150b is coupled to one of the oligonucleotides 152a, 152b. In the example shown, the oligonucleotides 152a, 152b are coupled to the specific binding agents 150a, 150b via their 5' and 3' ends, respectively. In other examples, each oligonucleotide can be coupled or non-covalently attached to one of the specific binding agents 150a, 150b at any position of the oligonucleotide.

[0105] Connexon 146 is configured to base pair with 3' end region 156 of oligonucleotide 152a and 5' end region 157 of oligonucleotide 152b, as shown in dotted arrows 154a, 154b. In other words, the first sequence portion 158 of connexon 146 is complementary to 3' end region 156, and the second sequence portion 159 of connexon 146 is complementary to 5' end region 157. The hybridization of connexon 146 and 3' end region 156 and 5' end region 157 arranges 3' end and 5' end of oligonucleotide 152a, 152b together, thereby can effectively connect at the end. In the illustrated example, connexon 146 is provided by the oligonucleotide separated from oligonucleotide 152a, 152b. In other examples, connexon 146 can be provided by the connexon sequence present in one of oligonucleotide 152a, 152b.

[0106] The merging 148 produces a mixture including the viral particles 134, the spiked probes 144a, 144b and the linker 146 (see Figure 3), and can be implemented in any suitable order. For example, the viral particles 134 and the spiked probes 144a, 144b can be combined with each other to create a first mixture. The first mixture can be incubated at a controlled temperature to allow the spiked probes to bind to the capsid 136, and then diluted as needed. The first mixture can then be combined with a DNA ligase and a linker 146 to produce a ligation mixture 160, wherein the linker 146 hybridizes with copies of the oligonucleotides 152a, 152b to achieve ligation. The ligation mixture is incubated to allow the spiked probes 144a, 144b to ligate to each other to produce a tag 162 (see Figure 3-5 More specifically, copies of oligonucleotides 152a, 152b of the labeled probes 144a, 144b are ligated to each other to generate a label 162, which provides a template 164 (see Figure 4 and 5 ). Template 164 includes corresponding template portions 166a, 166b from oligonucleotides 152a, 152b. However, the addition efficiency is not 100%, so some capsids 136 are not added with template 164, such as Figure 3 The connection mixture 160 is shown.

[0107] The ligation mixture 160 is then combined with amplification reagents (designated 167) (see Figure 3 The amplification reagents include a first primer pair 168a, 168b for amplifying a first target 170 from a template 164, and a first amplification probe 171 for detecting the amplification of the first target 170 (see Figure 5 The amplification reagents also include a second primer pair 172a, 172b for amplifying a second target 174 from the genome 138, and a second amplification probe 175 for detecting the amplification of the second target 174 (see Figure 6 ). In the example shown, only one pair of primers 172a, 172b is used to amplify one target in the transgene 176 of the genome 138. In other examples, one or more additional primer sets (and one or more probes) can be used to amplify one or more other targets in the genome 138 to better distinguish between real viral particles and other viral particles that lack at least a portion of the genome.

[0108] After merging 167, a partition set 178 is formed, as shown in 179 (see Figure 3). Partitions 178 include capsid+tag partitions (configuration 180a, including capsid 136 and tag 162, but without genome 138), capsid+tag+genome partitions (configuration 180b, including capsid 136, genome 138 and tag 162), particle-only partitions (configuration 180c, including capsid 136 and genome 138, but without tag 162), and target-negative partitions (configurations 180d and 180e, both of which do not include genome 138 or tag 162).

[0109] Partition 178 is then treated (e.g., heated) to promote capsid cleavage, as shown at 181. Capsid cleavage brings the amplification reagents and copies of the genome 138 into contact with each other for subsequent amplification, and may cause the nucleic acid duplex to melt into single-stranded form.

[0110] Amplification of each target is then performed in the partitions, as shown in 182 (see Figure 3 , 5 and 6). Target amplification hydrolyzes oligonucleotide 183a (first target) or oligonucleotide 183b (second target) of amplification probes 171, 175, thereby separating the probe's label, fluorophore 184a, 184b from quencher 185, resulting in an increase in fluorescence 186a, 186b (see Figure 5 and 6 ).

[0111] Amplification data (as shown in 187) is collected by detecting fluorescence from each partition (if any) in at least two detection channels (channel 1 and channel 2) (see Figure 3 ). Fluorescence 186a from the fluorophore 184a of the first amplification probe 171 is detected in channel 1, which represents the amplification of the first target 170 (see also Figure 5 ). Fluorescence 186b from the fluorophore 184b of the second amplification probe 175 is detected in channel 2, which represents the amplification of the second target 174 (see also Figure 6 ).

[0112] The scatter plot 188 of the amplification data uses corresponding points to represent each partition, and the points are positioned within the plot according to the magnitude (eg, intensity) of the channel 1 and channel 2 fluorescence detected from the corresponding partition (see Figure 3 and 7 ). The points / partitions are clustered into four groups 189a-189d (in Figure 7 Each partition can be assigned to one of the populations by comparing the partition amplitude in each detection channel (1 and 2) to the corresponding threshold.

[0113] The partitions 178 present in each group 189a-189d are superimposed on Figure 7180a-180e. Additional partition configurations 180f and 180g were also identified as being present in populations 189a and 189b, respectively. Configurations 180f and 180g were positive for the first target, independent of the presence / absence of capsid 136. In other words, in the absence of capsid 136 (configuration 180f) or in the absence of one or both of the spiked probes 144a, 144b bound to capsid 136 (configuration 180g), ligation of the spiked probes 144a, 144b occurred to create template 164 (see also Figure 3-5 ). C1. Quantification of capsid occupancy using partitioned population counts

[0114] This subsection provides an overview of the first approach to quantifying capsid occupancy, specifically, using partitioned population counts. This subsection provides illustrative equations and calculations, and references the partition counts AD and D* (from populations 189a-189e, respectively) and the partition configurations 180a-180g of subsection B (see Figure 3-7 ).

[0115] Equation 1 states that the capsid occupancy percentage (% real capsid) is equal to the ratio of the sum of the partition counts of populations B and C (i.e., each partition containing the second target) to the sum of the partition counts of populations A, B, C, and D* (i.e., each partition containing one or both of the first and second targets), multiplied by 100: D is negative for both the first and second targets. D* is a subset of D that contains empty capsids that were not labeled in the assay (see Figure 7 However, the partition count value of D* can be calculated based on the capsid spiking efficiency (i.e., "PLA efficiency").

[0116] Equation 2 states that PLA efficiency is equal to the ratio of the sum of the partition counts of populations A and B (i.e., each partition that is positive for the first target) to the sum of the partition counts of populations A, B, C, and D* (i.e., each partition that contains one or both of the first and second targets), multiplied by 100:

[0117] It is assumed that PLA efficiency is independent of the presence or absence of the second target. The inventors have demonstrated that this assumption is empirically valid. According to this assumption, PLA efficiency should be the same for populations A, B, C, and D* in general, with only two populations A and D* being negative for the second target and only two populations B and C being positive for the second target, as shown in Equation 3:

[0118] The ratio of the middle and right sides of Equation 3 can be used to solve for D*, as shown in Equation 4: Substituting Equation 4 into D* in Equation 1, we simplify to obtain Equation 5:

[0119] Populations A and B each include a corresponding portion of the background signal generated by the partitions in which the first target template was generated spontaneously and independently of the capsid (see Figure 7 The corresponding parts of the background signals of populations A and B can be subtracted to obtain A sub and B sub , which is used to calculate capsid occupancy according to Equation 6:

[0120] The background signal was measured in a no particle control (NPC) assay, which was performed using the same reagents and conditions, but using a control (NPC) sample that did not contain any viral particles. The background signal was determined from population A generated from the NPC sample (population B would not be generated when no viral particles were present). However, in samples containing virus, this background signal was split between populations A and B, so the corresponding portion of the background signal should be subtracted from both populations.

[0121] Each time a test is performed using a sample containing a virus or an NPC sample, the test may generate a different total number of accepted partitions (total partition counts). For example, in some embodiments, the total partition count may vary between 12,000 and 20,000. In order to correctly subtract the NPC background signal from the A and B populations generated by the virus-containing samples, the partition counts of each population (containing one or more virus samples and one or more NPC samples) can be standardized relative to the same value of the total partition count (e.g., 20,000). For example, for the A population of test "x", based on a total partition count of 20,000, A x To A N The normalization of the partition counts is given by Equation 7:

[0122] In samples containing viral particles, the background signal is unevenly distributed between populations A and B, and its distribution pattern depends on the relative pool sizes of the genome-negative partitions (A+D) and the genome-positive partitions (B+C).

[0123] The fraction of background signal distributed to population A can be calculated using Equation 8:

[0124] The fraction of background signal distributed to population B can be calculated using Equation 9:

[0125] When multiple NPC samples were tested, the normalized mean NPC population A, A NPC The corresponding NPC scores were subtracted from the normalized populations A and B of the virus-containing samples according to equations 10 and 11, respectively: A sub =A N -(A NPC ×D fr ) (10) B sub =B N -(A NPC ×C fr ) (11) C2. Quantification of capsid occupancy using partitioned population counts and junction concentration

[0126] This subsection provides an overview of a second approach to quantifying capsid occupancy, specifically, using partitioned population counts and junction concentrations. This subsection provides illustrative equations, like the previous subsections, and refers directly or indirectly to partition counts AD and D* (from populations 189a-189e, respectively) and partition configurations 180a-180g of subsection B (see Figure 3-7 ). However, here, population 180g includes partitions having both capsids 136 with genome 138 and capsids 136 without genome 138, capsids 136 with genome 138 not labeled with tag 162 (e.g., as shown in configuration 180c), and capsids 136 without genome 138 labeled with tag 162 (e.g., as shown in configuration 180a). In this case, the expression of real capsid % can be expressed in terms of partition population counts and junction concentrations as follows: Here, B 连接 is the junction concentration of double-positive partitions (i.e., partitions positive for both capsid and genome), (A+B) sub is the concentration of labeled capsids after subtracting the NPC. It is noteworthy that Equation 12 can provide higher accuracy than Equation 6, especially at high capsid concentrations. D. Experimental testing and verification with AA V particles (Part 1 point)

[0127] This section describes exemplary test data obtained using the experimental protocols of Sections B and G, the equation of Section C1, and samples containing commercially available empty and full AAV2 viral particles; see Figure 8-11 .

[0128] Two AAV standard samples were obtained from Vigene Biosciences. The "real GFP-AAV2" sample contained AAV2 viral particles containing a genome 238 with a GFP transgene 276 under the control of a CMV promoter (see Figure 8 ). The “empty AAV2” sample contained empty AAV2 viral particles.

[0129] Table 1 lists the capsid occupancy data (real %) reported by the vendors, as determined by three different techniques, namely TEM (transmission electron microscopy), AUC (analytical ultracentrifugation), and qPCR / ELISA (quantitative polymerase chain reaction / enzyme-linked immunosorbent assay). The capsid occupancy values ​​given by these three techniques vary widely. Table 1

[0130] The real and empty AAV2 samples were mixed with each other at three different ratios to provide a total of five samples for testing (see Fig. 9 ). Fig. 9 The capsid occupancy measured by TEM is given for each sample, as listed or calculated in the supplier's Certificate of Analysis (CoA). No particle control (NPC) samples were also tested using the same protocol in order to subtract the NPC background signal from samples containing viral particles (see also subsection C1).

[0131] Each sample was tested at a dilution series according to Section B (see Fig.10 and 11 ). The partitions formed are droplets. Target amplification is performed in the droplets by PCR. Primers for the GFP transgene are used to quantify genome copies (GC) in the droplets. Capsid occupancy values ​​measured according to the protocol of subsection B are consistent across a range of sample dilutions and are highly consistent with values ​​determined by TEM. E. Exemplary capsid occupancy calculation

[0132] This subsection describes an exemplary calculation of capsid occupancy using the equation of subsection D and the values ​​obtained using droplets as partitions in the scheme of subsections B and G; see Fig.12 . F. Experimental testing and validation with AAV particles (Part N2 point)

[0133] This section describes and compares exemplary test data obtained using the experimental protocols of Sections B and G, the equations of Sections C1 and C2 (with particular emphasis on Equations 6 and 12), and samples containing commercially available AAV2 viral particles having various occupancies; see Figure 13-15 .

[0134] Fig.13It is a graph comparing different methods for calculating the percentage of capsids containing genomes (i.e., real capsid %). Specifically, the figure shows the values ​​of real capsid % calculated using equation 6 (inverted triangle; "partition population count" method) and equation 12 (equilateral triangle; "partition population count and connection concentration" method). The figure also shows the concentration of PLA capsids (circles) and genome copy number (asterisks) for comparison. Real capsid % is independent of the concentration of capsids. Therefore, the calculated value of real capsid % should remain unchanged at different measured capsid concentrations. In other words, the graph of real capsid % should be horizontal. The value of real capsid % calculated using equation 6 is actually very sensitive to capsid concentration. Specifically, at higher capsid concentrations, the value of real capsid % increases with the increase of capsid concentration, essentially tracking the capsid concentration. This may reflect the random co-partitioning of capsids and genome content. In contrast, the value of real capsid % calculated using equation 12 is relatively constant and consistent over a wide range of capsid concentrations. Furthermore, the calculated value of % solid capsid of approximately 60% agreed well with the value of 70% measured by transmission electron microscopy (TEM).

[0135] Fig.14 and 15 Figure 1 is a graph obtained by experimenting with a wide range of capsid concentrations to assess the appropriate range for PLA capsid quantification, ddPCR GC quantification, and real capsid % calculation. All samples were tested in duplicate. Two replicates of the "real" sample were used to generate the PLA standard curve. Significantly, the recoveries for most samples were within the acceptable range of 70-130%. The PLA and GC capsid concentration ranges do not completely overlap and depend on the GC content of the sample. Overall, GC can be detected and quantified over a wider range of capsid concentrations. Here, the PLA capsid range was 2.9×10 9 to>2.4×10 11 Capsids / mL( Fig.13 ), GC ddPCR capsid range is 1.1×10 8 to 2.4×10 11 Capsids / mL( Fig.14 ). G. Exemplary Reagents and Workflow

[0136] This section describes exemplary reagents and an exemplary workflow that is substantially based on the experimental protocol of sections A and B. The protocol utilizes droplets as partitions in which PCR reactions are performed to detect capsids and genomes of AAV particles. However, the protocol can be followed for any suitable type of partitions, viral particles, and amplification methods.

[0137] The present disclosure presents a workflow that allows quantification of % actual AAV capsid using a single assay. The assay is based on droplet digital PCR (ddPCR) and does not require a calibration curve to calculate % actual capsid. The workflow can be adapted for other viral vectors.

[0138] In the experimental protocol, AAV genome copies were detected by ddPCR reaction and measured using one detection channel of the ddPCR system. In parallel, AAV capsids were detected using antibody-mediated proximity ligation assay (PLA) and detected in the second detection channel. This measurement generates four different droplet populations. The real capsid % was calculated by the ratio between these populations.

[0139] The following steps can be completed. 1) Treat the AAV sample with DNA enzyme to digest any uncoated DNA. 2) Dilute the sample to reach the ddPCR dynamic range. In addition to one or more AAV samples, each experimental run also includes one or more control samples that do not contain AAV particles, thereby providing a particle-free control (NPC). The signal of the NPC is used to subtract the background. 3) Perform a proximity ligation assay (PLA) reaction. The AAV sample is incubated with two PLA probes. The PLA reaction begins with the binding interaction between the AAV particles of the sample and the two PLA probes. Each probe consists of a specific antibody for the complete AAV particles, and each antibody is coupled to an oligonucleotide. The ligation reaction of the PLA reaction can then be performed. When both PLA probes are bound to the capsid and are close to each other, the oligonucleotide portion of the PLA probe can form a new DNA template through a ligation reaction mediated by a short linker sequence. 4) Prepare the ddPCR reaction. The connected samples (virus and control) are combined with ddPCR supermix, hydrolysis probes and primers. 5) The sample is divided into droplets. 6) The sample undergoes thermal cycling. Capsid cleavage occurs in the first step of the thermal cycle at 95°C, so both the ligated template and the viral genome are available for amplification. 7) Read the droplets. Two amplified targets are measured by two different detection channels, one from the PLA template and the other from the viral genome.

[0140] The workflow described generates four different populations of droplets (AD). Droplets containing empty viral capsids will be positive for PLA, while droplets containing full capsids will be positive for both PLA and genome copies (GC) (see also Figure 7 ). H. Capsid spiking using premade full-length template

[0141] This subsection describes exemplary assay reagents according to the illustrative method of subsection A, and exemplary assay configurations produced by practicing the illustrative method, wherein the method utilizes (i) a spiked probe comprising a premade full-length template to provide a first target for amplification, and (ii) beads as a solid support to enable one or more wash steps to remove unbound material; see Fig.16 .

[0142] Flowchart 330 illustrates various experimental configurations. A sample 332 is selected that contains a viral particle 334. The viral particle 334 includes a capsid 336 that surrounds a copy of a genome 338.

[0143] Sample 332 is combined with beads 331, as shown in 333. Beads 331 have beads 335, which may be magnetic, and specific binding agents 337 attached to each other. Specific binding agents 337 are configured to specifically bind to structures of viral particles 334, such as their capsids 336. Any suitable number of copies of specific binding agents 337 may be attached to each bead 335. However, the number of beads 331 may exceed the number of viral particles 334, such that most beads 331 do not bind to any viral particles or bind to a single viral particle 334, as shown in mixture 339. After viral particles 334 bind to beads 331, the beads may be washed to remove unbound material.

[0144] Beads 331 and viral particles 334 are combined with spiked probes 344, as shown at 348. The spiked probes include a specific binding agent 350 and a label 362. The specific binding agent 350 allows the spiked probe 344 to bind directly or indirectly to the capsid 336 of the viral particle 334. The label 362 is covalently or non-covalently attached to the specific binding agent 350 and provides a template 364. After binding and washing, as shown at 341, the viral particles 334 are spiked and bound to the beads, as shown at 343. The viral particles 334, beads 331, and spiked probes 344 can be combined with each other in any suitable order.

[0145] The spiked, bead-bound viral particles 334 are combined with an amplification reagent, as shown at 367, and partitions 378 are formed, as shown at 379. The partitions 378 may receive, on average, less than one bead 331 per partition to reduce interference from partitions 378 that receive more than one viral particle 334. After the partitions 378 are formed, the capsids 336 are lysed and amplified, as shown at 382. A first target is amplified from the template 364, and at least one other target is amplified from a copy of the genome 338. Amplification data can be collected from the partitions 378, as shown at 387. The amplification data can be visualized in a scatter plot 388. The partition populations 389a-389d (AD) can be assigned based on target content (see also subsection B of Figure 7 ). I. Illustrative capsid markers

[0146] This subsection describes exemplary assay reagents for use in accordance with the illustrative method of subsection A and exemplary assay configurations produced by performing the illustrative method, wherein the method utilizes a labeled probe comprising an optically detectable label to render the capsid detectable; see Fig.17 and 18 .

[0147] Flowchart 430 illustrates various experimental configurations of the method (see Fig.17 ). A sample 434 containing a virus particle 432 is selected. The virus particle 434 includes a capsid 436 that surrounds a copy of a genome 438. The capsid 436 includes a real capsid 440 and an empty capsid 442.

[0148] The viral particle 434 is combined with a spike probe 444, as shown at 448. The spike probe 444 includes a specific binding agent 450 linked to one or more copies of a label 490. The specific binding agent 450 is directly or indirectly bound to a capsid 436, which is labeled with the label 490, as shown at configuration 491. Each capsid 436 can have multiple recognition sites for the specific binding agent 450, so that multiple copies of the spike probe 444 can be bound to the same capsid 436 to increase the detectable signal from the label 490 for a given spiked viral particle 434.

[0149] Virus particles 434 are combined with amplification reagents, as shown at 467, and formed into partitions 478, as shown at 479. The amplification reagents are sufficient to amplify one or more targets from genome 438.

[0150] Capsid 436 is lysed to produce configuration 492. Amplification is then performed, as shown at 482, and tag-associated data and amplification data are collected from partitions 478, as shown at 487. Tag-associated data (detected from marker 490 in channel 1) and amplification data (detected from at least one amplification probe for at least one genomic target in channel 2) can be visualized in a scatter plot 488. Partition populations 489a-489d (AD) can be assigned based on tag / target content (see also subsection B of FIG. Figure 7 ). J. Compositions and methods for detecting viral components

[0151] This subsection describes other aspects of compositions and methods for analyzing viral particles. The ability to study, detect, quantify, and process viruses is a major focus of human health, and methods and / or techniques for quantifying the viruses contribute to this field. Improved compositions and methods for detecting and quantifying viral components and functional viruses are needed.

[0152] Provided herein are compositions and methods for analyzing viral samples. In certain embodiments, compositions and subsequent use of reagents are provided to quantify viral titers in unknown samples, and in certain cases quantify functional titers. In certain embodiments, provided herein are compositions and uses of compositions for simultaneous detection of viral DNA and viral capsids, from which, for example, viral integrity, infectivity, and infection titers can be inferred. Compositions and methods can be used together with appropriate instruments to assist in analysis. In some embodiments, the instrument is a digital analytical instrument.

[0153] Viruses are infectious agents that require living cells to replicate. Typically, they consist of a protein shell (or capsid) and a nucleic acid genome. The genome can include single-stranded or double-stranded DNA or RNA. In addition, depending on the category, viruses can be enveloped or non-enveloped. The nucleic acid genomes of viruses are extremely diverse. As a group, viruses have more structural genome diversity than any other kingdom of life.

[0154] The capsid is the protein shell that surrounds the viral genetic material. There are many capsid types, which can generally be classified based on their structure (icosahedral, prolate, or helical), and then further classified based on the underlying protein sequence of the capsid component.

[0155] The protein shell of enveloped viruses is covered with a phospholipid bilayer as a result of budding from the cytoplasmic membrane after viral replication. All viruses, regardless of envelope status, display specific proteins on their surface that facilitate host cell attachment and internalization.

[0156] The single-stranded viral genome lacking the complementary strand may be positive-sense (plus or minus strand), depending on the virus species and the downstream replication machinery after the host is infected.

[0157] Furthermore, the viral genome can exist in either linear or circular form in macroscopic structure.

[0158] Furthermore, viral genomes vary in size, and therefore the number of proteins they encode. One of the smallest known viruses contains a 2kB genome that encodes just two proteins, while one of the largest known viruses has a 2MB genome that encodes about 2,500 proteins.

[0159] In addition, RNA viruses often exhibit higher mutation and evolution rates as they reproduce. This is due to the inherent lack of fidelity of RNA-dependent polymerases compared to typical DNA-dependent polymerases. As a result, these viruses also tend to have smaller genomes due to the strong balancing selection between the accumulation of beneficial and deleterious mutations during replication.

[0160] Due to these different properties and / or characteristics of viruses, methods, reagents and techniques for virus analysis can be targeted.

[0161] Naturally occurring viruses often cause disease in the host in which they replicate. For humans, such examples include the common cold, influenza, chickenpox, HIV, rabies, Ebola, etc. The ability of a virus to cause disease is called virulence. Viruses have different mechanisms for producing disease in host organisms, which may be related to the mechanism of viral replication. Lytic replication refers to the cell bursting (or lysing) when the virus replicates to a sufficient state and triggers release, which can lead to immediate death in unicellular organisms, or disease in multicellular organisms after a sufficient number of cells are damaged.

[0162] Some viruses may lie dormant or latent in their hosts, where they remain relatively harmless within the organism. Other times, viruses may cause chronic infections where the host is unable to eliminate the virus.

[0163] Additionally, because viruses have an innate ability to transfer genetic material to cells, researchers have used viruses as delivery vehicles in the hope of developing targeted gene therapies.

[0164] Viral particles can be engineered to deliver genetic material to the cytoplasm and nucleus, and the engineered vehicles are called viral vectors. Therapies that alter a patient's genome in vivo and ex vivo by replacing or repairing mutant genes or redirecting the body's response to diseased cells are emerging as the next generation of therapeutics. Unfortunately, there is a lack of consensus on viral vector characterization tests that are critical to the safety and efficacy of such therapies. Patient dosing requires accurate quantification of viral titers or determination of viral counts in a given volume. Interrogation of the active fraction, or the proportion of capsids containing the viral genome, provides insight into changes in immunogenicity risk and gene transfer efficiency. Imperfect sample processing can result in empty capsids or partial capsids that only incorporate a portion of the viral genome (vg). Empty and partial capsids are unable to infect and therefore cannot successfully transfer genes, limiting the "active" fraction in the sample. Therefore, empty and partial capsids are considered sample impurities and must be closely monitored batch by batch.

[0165] A single workflow that enables simultaneous assessment of viral titer and viability fraction could have a positive impact on vector and plasmid design, manufacturing process development and scale-up, and clinical batch characterization. In addition, such an assay could have a beneficial impact on a wide range of efforts in academic and industrial R&D, medicine, public health, and manufacturing, including vaccines, antiviral therapeutics, recombinant protein production using viral vectors, viral load management, and viral outbreak monitoring.

[0166] Viral titers can be described as physical titers or functional titers. Physical titers typically involve quantification of viral nucleic acids, proteins, or physical particles. Functional titers, also called infectious titers, are a measure of viral particles that can successfully infect cells. Because not all detectable viral components in a sample are properly assembled into infectious units, infectious titers are often lower than physical titers and are considered the gold standard for dosing in patients.

[0167] Common tests for functional titer testing include plaque assay and 50% tissue culture infectious dose (TCID 50 ) test. The plaque assay involves infecting a monolayer of cells with a dilution of virus, then sealing with a top layer of agar to prevent the virus from spreading beyond neighboring cells, and counting the number of visible viral infections, or plaques, after a period of incubation. TCID 50 The assay also treats cells with a range of virus concentrations, but is performed in microplates. Visual inspection then determines the concentration of virus at which 50% of the wells in the microplate show evidence of a cytopathic effect, regardless of the amount of events within the well. While functional titer assays are highly sensitive, produce absolute measurements of infectious units, and do not require specific reagents, they are labor intensive, require extended protocols (usually more than 5 days), and can be subject to user subjectivity.

[0168] Physical titers can be quantified using methods such as quantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA). qPCR is a similar assay in which the concentration of a target nucleic acid in a bulk sample is determined relative to a standard curve prepared using reference materials. Reference-related quantification increases the potential for variability across assays, users, and laboratories, and limits assays to well-characterized standards. In addition, poor amplification efficiency of either the target or reference nucleic acid can lead to over- or underestimation of target concentrations. ELISA measures viral proteins through specific antigen capture. First, the antigen is immobilized on a microplate, either directly or indirectly (i.e., bound by a capture antibody), and then the antigen is bound by a detection antibody, facilitating an enzymatic or fluorescent readout. These methods enable faster results and reduced labor while maintaining acceptable levels of sensitivity. However, these benefits come at the expense of relative (rather than absolute) quantification, the need for specific reagents, and a lack of insight into infectious units.

[0169] Physical titers can also be determined by directly counting viral particles using techniques such as flow cytometry (FCM), transmission electron microscopy (TEM), and liquid chromatography (LC). FCM can provide absolute quantification quickly with low labor costs and, depending on the protocol, can quantify infectious units, but it has reduced sensitivity compared to the methods discussed previously. The most common FCM protocols involve staining vgs with fluorescent intercalating dyes, or detecting virus-infected cells through reporter genes or viral proteins. TEM stains viral capsids to enable direct counting, viral morphology studies, and the designation of empty vs. full capsids. LC requires that the sample be prepared in a solvent or liquid phase and then pumped into a stationary phase made of an adsorbent material. The interaction of the sample with the liquid and the stationary phase affects the time required for elution or its retention time, which can be used to identify analytes in heterogeneous mixtures. LC is seen as having advantages in manufacturing processes because results are rapid and can be generated using samples from any stage of the production process (i.e., crude samples). Methods that interrogate viral particles by their physical properties are generally lower throughput and have more specific use cases.

[0170] Several of the methods previously discussed for quantification of viral titers can be adapted for assessment of the active fraction, such as qPCR, ELISA, TEM, and LC. The active fraction is the percentage of virus in a sample that is able to introduce its genetic material into target cells and is typically interpreted as the proportion of capsids containing the viral genome. qPCR and ELISA can be used in combination to estimate the ratio of vg to viral capsids as a readout of the active fraction; however, while this approach can be easily incorporated into existing workflows, it lacks accuracy and precision. TEM enables visual determination of empty and full particles. While data acquisition from TEM is relatively fast, interpreting the data is often very time-consuming and requires skilled technicians. High-performance liquid chromatography (HPLC) relies on a pump rather than gravity to generate the pressure required to move the sample through a phase column, which separates particles by size. A method not previously discussed, analytical ultracentrifugation (AUC), relies on density-based sedimentation to distinguish subpopulations in heterogeneous samples. AUC provides high resolution and eliminates the need for reference materials, however, it does require large amounts of sample compared to the production scale that many researchers are working on.

[0171] Digital testing has become an important tool for quantifying chemical or biochemical substances in scientific research and clinical diagnosis. In digital testing, the analytical targets in a sample are divided into multiple subsamples according to a known probability distribution (in many cases, a Poisson distribution). A method is applied to determine whether each subsample contains one or more analytical targets of interest. By measuring the total number of subsamples and the number of subsamples that contain or do not contain any targets, and comparing them to the probability distribution, the user can infer the amount of each analytical target in the original sample. The method is called "digital" because only discrete measurements are made for each subsample, that is, whether the subsample contains each analytical target.

[0172] Digital assays offer a number of key advantages over traditional methods for quantifying the amount or concentration of an analytical target. First, because the method directly counts the number of subsamples that are occupied or unoccupied by the analytical target and compares it to a probability distribution, the original amount of the analytical target can be directly measured without reference to a calibration standard. This advantage is particularly valuable when such standards are difficult to repeat, difficult to accurately, or simply impossible to construct (no reference material available). Second, because only the presence or absence of the analytical target in each subsample needs to be measured, small or moderate changes or noise in the detection signal will not change the final determination of the presence (and thus the amount / concentration) of the target in the sample. Third, because the volume of each subsample is inherently smaller than the original sample, the relative concentration of the analytical target in each subsample containing the target is higher than that in the bulk sample. In the case of a large number of subsamples and a small amount of analytical target, the increase in concentration can reach multiple orders of magnitude, thereby increasing the concentration-dependent sensitivity.

[0173] In digital PCR (dPCR), if RNA is to be quantified, then the one or more analytical targets of interest are DNA or cDNA sequences. Nucleic acid molecules containing the sequence of interest are distributed in subsamples, which are then subjected to PCR thermal cycling. Individual subsamples are optically interrogated to determine whether the sequence of interest was amplified, and the original amount of DNA molecules with the one or more sequences of interest can be inferred from the probability of the Poisson distribution. Note that this quantification is a discrete measurement that relies on endpoint amplification, and is therefore less susceptible to amplification inhibitors than qPCR. In addition, the quantification is independent of the reference standard, which reduces the inter-assay variability compared to qPCR.

[0174] With proper assay design and optimization, PCR can provide quantification of physical viral titer and, in some cases, even infectious units. However, researchers seeking more detailed information, such as fractional viability, will find a single assay that can quantify both vg and capsid concentration useful. While qPCR can provide a solution for parallel sample processing of either vg or capsid concentration, dPCR allows for simultaneous interrogation of both genome and capsid in compartmentalized samples, enabling absolute quantification of both viral titer and fractional viability in a single assay.

[0175] Parallel sample processing utilizes the external physical properties of the capsid to quantify the capsid concentration in an untreated sample and to quantify the vg concentration in a second treated sample in which the capsid has been broken down to release the vg. Capsids can be broken down using a variety of techniques including, but not limited to, heat treatment, enzymatic digestion (i.e., proteinase K, trypsin, cathepsin B), sonication, surfactants, acids, and bases (i.e., Tween 20, guanidine hydrochloride). Examples of PCR vg quantification methods include, but are not limited to, intercalating dyes, hydrolysis probes, and molecular beacons. These methods can be used in parallel with PCR-based capsid quantification techniques (e.g., quantification of oligonucleotide binding molecule conjugates) and PCR-compatible techniques (e.g., direct and indirect fluorophore-labeled binding molecules).

[0176] Concurrent sample processing allows for quantification of both capsid and vg in the same sample, seeking to identify subsamples that are positive for both capsid and vg to confirm correct assembly / loading and subsequently confirm the active fraction. Most vg quantification techniques identified for qPCR can be converted to concurrent sample processing in a dPCR format with some optimization. Concurrent dPCR sample processing typically employs the following workflow: labeling capsids in the bulk sample, partitioning the sample into a sufficient number of subsamples, disassembling capsids while retaining the ability to detect capsid markers, and finally detecting vg and capsids in the subsamples. Additional insight into the efficiency of the manufacturing process is provided in the form of vg-only or capsid-only subsample readouts. A higher level of precision can be introduced by using multiple sets of PCR primers to detect the viral genome, each set of primers being specific for a different portion of the genome in the same partition; if these primer sets are differentially labeled, the complete copy number of the viral genome can be estimated by counting partitions containing a detectable marker in all primer sets, and the more primer sets included, the more accurate the assessment of completeness (and, potentially, infectivity) will be. In cases where the viral genome is relatively small, such as AAV, two sets of primers (e.g., for distal portions of the genome) may be sufficient. In other cases, more sets of primers (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 sets) may be sufficient. In some cases, for example, where a process is known to produce a mostly or fully intact copy of the viral genome, a single set of primers may be sufficient.

[0177] In general, the compositions and methods provided herein are intended to analyze partitions (i.e., relatively small portions of a parent sample) that may contain viral nucleic acid; capsid; both viral nucleic acid and capsid; or contain neither viral nucleic acid nor capsid. The analysis will detect which class the partition belongs to. In certain embodiments, methods and compositions for preparing partitions are also provided.

[0178] A sample containing viral particles containing both capsids and viral nucleic acids, and possibly the viral particles also containing capsids without viral nucleic acids and / or viral nucleic acids without capsids, if the sample is subdivided into partitions, a single partition may contain no viral components; may contain only capsids; may contain only viral nucleic acids; may contain viral capsids and viral nucleic acids from a single viral particle; and may contain viral capsids and / or nucleic acids from multiple viral particles. The latter will be uncommon if a sufficient number of partitions are generated from the sample. In one analysis, if the sample is subdivided into multiple partitions, each partition is classified as containing no viral components; only capsids; only viral nucleic acids; or viral capsids and viral nucleic acids (which may be from a single or multiple viral particles, but if the sample is subdivided into a sufficient number of samples, each partition is almost entirely from a single viral particle), then the proportion of samples containing real capsids (i.e., capsids and viral nucleic acids) or empty capsids (only capsids) can be determined. The initial concentration of each in the original sample can also be determined based on the number of partitions in each category and, in some cases, the volume of the partitions. In another analysis, for example, where multiple sets of primer pairs are used, the proportion of samples containing partial capsids (i.e., capsids and incomplete viral nucleic acid); the proportion of samples containing solid capsids (i.e., capsids and complete viral nucleic acid) and empty capsids (capsids only) can be determined. The initial concentration of each in the original sample can also be determined based on the number of partitions for each class and, in some cases, the volume of the partitions. In some cases, solid viral particles can be considered infectious and the activity fraction determined. 1. Capsid inspection Test and Quantification

[0179] Typically, the viral capsid (protein) or a component associated with the capsid (lipid bilayer) is spiked with a component that is sufficiently specific for the particular assay being used. The spiked component is bound to the viral capsid or lipid bilayer and can be detected directly or indirectly. The directly detectable spiked component can be labeled with a marker that is detectable alone or after certain modifications (see, e.g., FRET, described elsewhere herein). The marker can be an intrinsic part of the spiked component or can be bound to the spiked component; the binding of the marker can occur before the spiked component is bound to the capsid or lipid bilayer (e.g., a fluorescently labeled antibody) or after the spiked component is bound to the capsid or lipid bilayer (e.g., a labeled antibody specific for the spiked component, or a fluorescent marker associated with PCR of a spiked oligonucleotide). In addition, the binding of the spiked component to the capsid or lipid bilayer can be covalent or non-covalent.

[0180] In some embodiments, the viral capsid is marked in the bulk sample, i.e., before forming a partition. The sample is then divided into a plurality of partitions, some of which will contain capsids. Then, as described elsewhere herein, the capsid is processed to fully destroy the integrity of the capsid so that the nucleic acid (if any) present in the capsid is fully exposed to amplify (if used) and detect reactions in each partition. Before forming a partition, there are reagents sufficient to carry out the desired reaction so that the partition contains reagents individually. Alternatively, the partition containing reagents or other appropriate components can be fused with the sample partition before reaction or other operations. In the case of marking capsids with a component containing an oligonucleotide to be amplified, the partition will contain a suitable primer set and PCR reagents, such as a master mix. As described elsewhere, a set of primers specific for viral nucleic acids can be used, or more than one set of primers can be used, each set of primers specific for different viral nucleic acid sequences. In addition, in the embodiment of marking capsids with a component containing an oligonucleotide to be amplified, one or more sets of primers specific for capsid marking oligonucleotides will be present in the partition. Thus, compositions are provided herein in some embodiments, comprising a subsample (e.g., partition) of a larger bulk sample containing viral particles, wherein the subsample (e.g., partition) comprises a spiked capsid component of the virus (e.g., a spiked capsid protein or lipid bilayer as described herein), and reagents sufficient for polymerase chain reaction of viral nucleic acid (if present). In certain embodiments, the subsample will also comprise viral nucleic acid. Reagents sufficient for PCR of viral nucleic acid will include at least one set of primers specific for viral nucleic acid sequences; in certain embodiments, reagents sufficient for PCR of viral nucleic acid will include at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 different sets of primers, each set of primers being specific to a different sequence of viral nucleic acid. In certain embodiments, the capsid tag comprises an oligonucleotide to be amplified, and the subsample further comprises at least one set of primers specific for the oligonucleotide. Depending on the specific experiment, a suitable spike component may also be present. In some embodiments, at least 10, 50, 100, 200, 500, 1000, 2000, 5000, 10,000, 20,000, or 50,000 subsamples (e.g., partitions) of the original sample are present in the composition. The volume of the subsample can be any suitable volume, depending on further processing of the subsample, for example, 0.01-100 nL, or 0.1-10 nL, or 0.1-5 nL, or 0.1-1 nL. In some embodiments, the composition includes subsamples containing spiked capsids as described above, and subsamples that do not contain spiked capsids. In some embodiments, the virus is AAV.

[0181] Capsid detection and / or quantification can be based on specific or non-specific interactions with viral capsids (e.g., viral proteins or viral lipid bilayers). Specific interactions involve binding to specific portions of viral capsids; binding to non-viral capsid portions does not occur to a significant extent, for example, does not occur to the extent that it interferes with the desired analysis; specificity can be specific to the virus, specific to a class of viruses (e.g., AAV), or specific to one or more viral subclasses (e.g., AAV serotypes). Non-specific interactions involve binding to portions of viral capsids that may also be present in large quantities in non-viral material, for example, non-specific protein binding or lipid bilayer insertion. In relatively pure samples, such as samples of transgenic AAV or other types of artificially produced viral particles, most or all of the proteins and / or lipids present may be capsids and / or lipid bilayers, so non-specific interactions are sufficient to detect and quantify capsids. In samples that may contain other proteins and / or lipids other than viral particles, specific interactions are preferred, i.e., interactions involving specific binding to viral capsid components or specific binding to specific viral serotypes.

[0182] Capsid detection and / or quantification can take advantage of physical surface properties, such as exposed amino acids, antigens, or (in the case of enveloped viruses) lipid bilayers. In some cases, binding to the capsid may be non-specific. For example, compositions and methods for detecting lipid bilayers (discussed in more detail below) will typically detect the lipid bilayer that encapsulates the viral particle, and may detect other lipid bilayers that may be present. Detection molecules, such as fluorescent dyes or oligonucleotides, can be coupled directly to the capsid surface at exposed amino acid sites. For example, well-studied click chemistry reactions, such as alkyne-azide cycloadditions (i.e., DBCO-azide) can be used for coupling.

[0183] If a more specific approach is required, one or more surface capsid antigens can be used as binding moieties to achieve capsid quantification to determine total capsid counts; these can be serotype specific or ambiguous, recognizing partial or complete capsids. Potential binding molecules include, but are not limited to, antibodies, peptides, single-chain variable fragments (scFv), nanobodies, aptamers, lipids, and nucleic acids. The choice of an appropriate binding molecule may depend on the virus type, serotype, and assay commercialization requirements. Given the interest in developing a single assay for vector characterization in the field of gene and cell therapy (GCT), the binding molecules highlighted below focus on AAV-specific molecules. 2. Examples of AAV binding molecules

[0184] AAV receptor (AAVR): The type I transmembrane protein KIAA0319L, also known as AAVR, has been identified as a direct binder of AAV serotype 2 (AAV2) and is essential for disease susceptibility. Genetic ablation of AAVR protects cells from AAV2 infection. In addition, other studies have shown that AAVR is a key host factor for AAV serotypes 1, 3B, 5, 6, 8, and 9, making it an ideal binding molecule for nonspecific detection of most AAV capsid serotypes, i.e., specific for AAV but not for AAV serotype. AAVR contains five polycystic kidney disease (PKD) domains that mediate cell binding, but only PKD 1-2 appear to be required for infection. The use of shortened PKD 1-2AAVR constructs (occupying positions 309-400 and 408-497, respectively) may have advantages in terms of binding capacity and supply chain logistics. The shortened construct may prove to have further advantages because it has a higher binding capacity than the full-length AAVR; studies have shown that the binding capacity increased by at least 14 times, depending on the model adjustment, as shown by cryo-EM results. Therefore, in certain embodiments, the compositions and methods provided herein may include an AAVR or a fragment thereof, such as a shortened PKD 1-2AAVR construct, as long as the AAVR or fragment binds to the AAV capsid with sufficient specificity and tightness to be used in the methods provided herein. Such a portion can be used to label the capsid with the desired specificity. In certain embodiments, the portion can be linked to a detectable marker. In certain embodiments, the portion can be linked to an oligonucleotide, which can be amplified by PCR by itself or in combination with other components.

[0185] A20 monoclonal antibody (mAb): A20 mAb is the most extensively studied mAb against AAV2. A20 mAb is favored because it is specific only for complete assembled capsids because it recognizes a conformational epitope defined by the tertiary structure of the protein. It is a neutralizing mAb, which means that it renders infectious particles ineffective upon binding. Although A20 mAb is known to bind AAV3B in addition to AAV2, it does not bind to other serotypes and can therefore be used to characterize heterogeneous samples. In certain embodiments, the compositions and methods provided herein may include A20 mAb, or a fragment or aptamer thereof, or a combination of these, as long as the antibody, fragment or aptamer binds to the AAV capsid with sufficient specificity and tightness for use in the methods provided herein. Such a portion can be used to spike capsids with the desired specificity. In certain embodiments, the portion can be linked to a detectable marker. In certain embodiments, the portion can be linked to an oligonucleotide that can be amplified by PCR by itself or in combination with other components.

[0186] PCR-compatible fluorescent readouts can be used to nonspecifically or specifically label capsid proteins. Nonspecific protein labeling (e.g., using Capsid labeling can be performed with fluorophore Orange (Life Technologies). Specific fluorescent protein labeling can be achieved by direct or indirect association of the fluorophore with a surface binding molecule. Directly labeled conjugates can be prepared using random or site-specific coupling strategies. Indirect labeling methods include fluorescently labeled secondary binding molecules (specific for the primary binding molecule) or proximity readout. After sufficient binding to the primary or secondary fluorophore conjugate, thorough washing can be performed to remove unbound fluorophores to prevent false positive results. Alternatively, a proximity-dependent fluorescence readout can be used, such as Forster resonance energy transfer (FRET) or PerkinElmer's AlphaScreen TM , without washing. In the proximity readout, a pair of indirectly labeled molecules must be in suitable proximity to produce a positive signal; free, unbound pairs in the reaction solution have a low probability of producing a positive signal. After binding and any necessary washing steps, the sample can be divided into subsamples and intact capsids quantified.

[0187] In cases where the positive readout depends on the successful binding of different targets, a multiplexed approach should be employed. For example, one can directly label the molecule with different fluorophores, each corresponding to a specific surface target or a unique FRET pair. The choice of the exact fluorescence profile will vary depending on the potential fluorometer limitations of the instrument and, for both FRET and AlphaScreen TM , will vary depending on the expected distance between bound molecules. In either case, determination of a positive signal will depend on successful binding of all targets.

[0188] Preparation of conjugates: Proteins (including antibodies) can be labeled using random or site-specific strategies. The most mature protein labeling technology is the random labeling of primary amines on lysine groups. An example is the copper-free click chemistry strategy, in which primary amines are labeled with DBCO and then reacted with azides to form covalent bonds. Since there may be many primary lysines exposed on the protein surface, the degree of labeling is difficult to control and the final product is a heterogeneous substance. For homogeneous products, site-specific strategies are generally preferred. These generally involve chemical or enzymatic modifications to produce or expose reactive amino acids. Non-exhaustive examples include the expression of genetically modified active sites (i.e., HaloTag), site-directed mutagenesis of cysteine ​​introduced by active thiols, subsequent coupling with maleimide, and enzymatic formation of isopeptide bonds between the amine and amide groups of lysine and glutamine residues, respectively. The formation of these isopeptide bonds is catalyzed by the enzyme transglutaminase, and generally requires the insertion or exposure of glutamine residues.

[0189] Washing steps: The choice of washing method depends primarily on the desired throughput and sample volume. Potential methods include spin columns, magnetic bead capture, and coated plate capture. Spin column methods are able to process larger sample volumes compared to plate-based methods, but this comes at the expense of reduced throughput for most commercial kits. It should be noted that for all washing methods, false positives may occur if the molecules required for capture and detection are the same.

[0190] Spin columns typically separate samples by molecular weight or sample-resin interaction. Columns that use a molecular weight cutoff allow all molecules below a given molecular weight to pass through while non-specifically retaining all molecules above a given cutoff. Spin columns that exploit specific sample-resin interactions utilize sample affinity tags. Affinity tags, such as glutathione S-transferase (GST), histidine (HIS), and biotin, are commonly used for sample purification during manufacturing. The spiked molecules are captured within the spin column resin while the rest of the sample is washed away. The captured molecules are then eluted or released from the resin using an appropriate buffer or competitive displacement. Affinity resins include glutathione, metal ions, and avidin for GST, HIS, and biotin, respectively.

[0191] Magnetic bead capture can be used to separate samples based on the binding of an affinity tag, similar to a spin column, or the binding of a specific antigen to coated magnetic microbeads. When placed next to a strong magnet, the magnetic beads "pull down" sample bound to their surface. This allows unbound molecules to be washed away while retaining bound molecules. Magnetic bead capture can be performed in a tube-based or plate-based approach.

[0192] Coated plate-based capture can use coated microplates to bind and capture affinity tags and surface antigens. Unbound molecules can then be washed away. Captured molecules can be eluted from the plate using an appropriate buffer or competitive displacement, similar to a spin column.

[0193] While many of the capture method interactions discussed above are strong, capsid binding molecule interactions may not be strong enough to withstand washing. Therefore, crosslinking can be performed to ensure retention of the capsid during the wash steps.

[0194] Given that the biotin-avidin interaction is the strongest known non-covalent interaction between a protein and a ligand, one could consider tailoring the interaction to facilitate milder elution conditions (i.e., desthiobiotin or monomeric avidin).

[0195] Proximity fluorescence readout: FRET occurs when energy is transferred from a donor fluorophore to an acceptor molecule (fluorophore or quencher) with spectral overlap. Therefore, FRET pairs can be used as an indirect readout of molecules bound to the capsid surface as long as the target is close enough (1-10 nm).

[0196] AlphaScreen TM An alternative homogeneous proximity assay with a larger physical range than FRET, up to 200nm, it assumes the same assay design - unbound molecules are sufficiently distant to prevent signal generation. AlphaScreen TM Fluorescence excitation of the donor bead results in the emission of singlet oxygen, which triggers a chemiluminescent reaction on the surface of a nearby acceptor bead and ultimately produces measurable fluorescence emission. FRET and AlphaScreen TM Both technologies enable simplified, no-wash assay protocols for detecting capsid-specific surface molecules.

[0197] Oligonucleotides coupled to surface-bound molecules provide a PCR-based readout for capsid quantification by creating or protecting intact primer binding sites. A positive readout can be determined using a fluorescent primer PCR set.

[0198] Oligonucleotide Binding Molecule Conjugates: Binding molecules labeled with an oligonucleotide at the terminus opposite to the target provide a readout, where the primer binding site has been created or protected using an appropriate ligase or nuclease, respectively.

[0199] For primer site generation, two binding molecules that are coupled to the oligonucleotide and form the primer binding site together need to be bound to the capsid surface at a sufficiently close distance to promote the nucleic acid chain to be joined by connection. According to the connection conditions, it is conceivable that the energy barrier of the connection of the unbound conjugate in the solution is higher than that of the connection of the bound conjugate, so the washing step may be unnecessary. However, if the unbound nucleic acid conjugate is easily connected, a washing step may be required. If this is the case, the capsid can be treated with a kind of oligonucleotide conjugate first, and then the unbound excess conjugate can be washed away before being treated with the second oligonucleotide conjugate required for connection. Or alternatively or in other cases, cleaning can be carried out after introducing the second oligonucleotide conjugate.

[0200] To protect the primer site, the binding molecule coupled to the oligonucleotide will serve as a target for the complementary free oligonucleotide to form a double-stranded product. The double-stranded product is protected from being cleaved by nucleases targeting single-stranded nucleic acids (e.g., S1 nuclease). If the sample is treated with an excess of oligonucleotide conjugate, a washing step is required to prevent false positive signals.

[0201] While all strategies discussed so far are applicable to any capsid with surface antigens, there are additional quantification options for enveloped viral capsids (e.g., herpes simplex virus and retroviruses, including lentiviruses) that utilize their lipid bilayers. Probes that spontaneously insert into the lipid bilayer can be quantified using a readout based on wash preference.

[0202] Lipid Bilayer Labeling Methods: Probes that spontaneously insert into lipid bilayers can be quantified using a fluorescence readout. The fluorescence readout can be the result of a stable signal (i.e., direct fluorophore conjugates) or a dynamic signal that changes based on dye proximity.

[0203] Direct fluorophore conjugates consisting of a fluorophore dye and a sterol probe (e.g., cholesterol or α-tocopherol probe) can be used to visualize intact enveloped viruses. The inserted probe will produce a signal that is independent of the amount and proximity of the incorporated probe. Any free, unbound probe must be removed by extensive washing to prevent false positive signals.

[0204] Dyes whose fluorescence signal emission depends on proximity can be used without a wash step. At the same time, there are many methods to achieve proximity-dependent emission, the formation of pyrene excimers and the self-quenching of octadecylrhodamine B are two examples. At sufficient incorporation levels, pyrene-labeled fatty acids exist in the form of excimers. The fluorescence emission of the pyrene excimer is shifted relative to the fluorescence emission of the pyrene monomer, which is the expected state of free probes in solution and can be used as a positive signal for the presence of enveloped capsids. Octadecylrhodamine B self-quenches after forming dimers within the lipid bilayer, allowing the capsid to be quantified by the change in fluorescence signal between monomers and dimers. Quantification techniques that rely on these proximity dyes can be optimized to ensure the release of the viral genome without capsid disassembly. 3. Viral Genome Detection and Quantification

[0205] For proper quantification, the viral genome must be released from the viral capsid. This can be achieved through a variety of capsid disassembly methods, some of which have been highlighted above. If only encapsulated copies of the genome are to be quantified, the sample can be treated with a DNase I pretreatment to digest any non-encapsulated nucleic acids. Given the imperfections of gene and cell therapy manufacturing processes, viral genome quantification may require confirmation not only of viral genome counts, but also of genome integrity and sample purity. This can be achieved through specific targeting and signal amplification.

[0206] Fluorescent Primer Probe Set: The polymerase chain reaction (PCR) relies on the annealing of a single-stranded oligonucleotide primer to a complementary ssDNA target sequence. The bound primer serves as a starting point for a DNA polymerase, which synthesizes a complementary strand of DNA to produce double-stranded DNA (dsDNA). The target is then amplified by multiple denaturation cycles of the dsDNA product to allow annealing of the ssDNA primer and subsequent extension by the DNA polymerase. Prior to PCR amplification, RNA samples can be reverse transcribed into cDNA.

[0207] Fluorescent dyes that intercalate into dsDNA, such as SYBR TMGreen I (Life Technologies), can be used to generate a positive / negative signal readout using the final dsDNA product; the structure of the dye changes upon binding to dsDNA, resulting in an increase in fluorescence. However, if multiple primer sequences are used, this readout does not provide information about the particle size of a specific target. Primer multiplexing may prove advantageous when examining the genomic integrity of viral genomes (i.e., confirming the presence of two distal sequences of interest) or sample purity (i.e., primers specific to the plasmid backbone).

[0208] To better understand whether the entire genome or any host nucleic acid is encapsulated, fluorescent hydrolysis probes corresponding to each target can be used. In this case, an oligonucleotide probe with a 5′ fluorescent reporter and a 3′ quencher molecule is bound downstream of the PCR primer. During chain elongation, the fluorescence of the reporter molecule increases after the DNA polymerase separates the reporter and quencher molecules. Various combinations of fluorescent reporter molecules can be used for multiplexed readouts to provide valuable feedback about manufacturing and downstream processes. If the number of desired targets exceeds the capabilities of the fluorometer, one can consider including one or more chemiluminescent readouts or any other suitable labels. By making the signal readout of the final target rely on a chain chemiluminescent reaction, the technician can assume that the putative intermediate targets are all present.

[0209] Molecular Beacons: The reporter-quencher principle of the hydrolysis probe can be applied to an alternative probe design, namely molecular beacons. Molecular beacons are oligonucleotide probes with a stem-loop structure. The target sequence is flanked by two complementary stem sequences with 3′ reporter and 5′ quencher molecules, which remain annealed to form a hairpin in the absence of the target sequence. Binding of the probe to the target sequence disrupts the hairpin structure, resulting in separation of the reporter-quencher and increased fluorescence emission. Much like the multiplexed hydrolysis probe assay described above, information on the presence of multiple specific targets can be discerned from a heterogeneous probe mixture. 4. Simultaneous detection and quantification of capsid and viral genome using dPCR method

[0210] Sample processing can be advantageously performed simultaneously, where the presence of capsids and viral genomes in a single subsample in a dPCR assay can be verified. Such simultaneous sample processing can yield more accurate quantification of activity fractions compared to parallel sample processing. Several of the above capsid and viral genome quantification methods can be combined into multiplex assays with some sample pre-treatment to allow for simultaneous quantification on a dPCR platform.

[0211] Nonspecific capsid labeling: Nonspecific protein labeling methods can be used with or without washing steps, depending on the background signal of protein impurities in the sample. Such nonspecific protein labels include, but are not limited to, fluorescent dyes such as Orange (Life Technologies) and oligonucleotides coupled using click chemistry. Although these methods will nonspecifically label all proteins in the sample and are therefore generally not compatible with biological samples, they may provide a more cost-effective way to prepare samples in a homogenous manner.

[0212] Fluorescent capsid binding molecules: Any potential binding molecule discussed above can be used to label the capsid prior to partitioning, as long as the reporting method can withstand the subsequent capsid dissociation conditions. The spiked capsid can be partitioned and decomposed to release the viral genome (e.g., by heating, sonication, etc.). Viral genome quantification can then be performed using any of the previously discussed methods that do not require sample pretreatment. While PCR-based viral genome quantification is being performed, subsamples can also be asked whether they have labeled capsids. Samples that are positive for both viral genome and capsid readouts can be considered to contain complete viral capsids carrying the genome and are part of the active score. Samples that are positive for either the viral genome or the capsid (but not both) can be considered to contain unincorporated genomes or empty capsids and can provide information about the efficiency of the manufacturing process.

[0213] Therefore, the present invention includes methods for detecting viral nucleic acid and capsid in the same partition, for example, a partition in a plurality of partitions comprises viral nucleic acid and viral capsid components. The partition is as described herein, for example, a part of an emulsion comprising a plurality of partitions, for example, a partition comprising an aqueous phase in a continuous phase, wherein the aqueous phase is insoluble in the continuous phase, such as oil. The volume of the partition can be as described herein. The partition can also comprise a surfactant, such as a fluorosurfactant. The continuous phase can be an oil, such as a fluorinated oil. The capsid components in the partition can include capsid proteins, capsid lipid bilayers, or a combination thereof. Typically, the capsid component will be spiked as described elsewhere herein, for example, by combining a component that can be directly or indirectly detected with the capsid component for spiked, also as described elsewhere herein. The method may also include, before dividing a bulk sample containing the virus into a plurality of partitions, spiked with the viral capsid therein. The method may also include dividing the sample into a plurality of subsample partitions, as described herein. The plurality of partitions may also include partitions containing only capsids and / or only viral nucleic acids, and the method may also include detecting only capsids in partitions containing only capsids and / or detecting only viral nucleic acids in partitions containing only viral nucleic acids. The method may also include changing the integrity of the capsids in the partitions to be sufficient to release the viral nucleic acids (if present) while retaining the ability to manipulate the tags associated with the capsids to produce or display markers; in some cases, the markers are already present and only need to be maintained. The method may also include amplifying the viral nucleic acids (if present) in the subsamples (e.g., partitions) by, for example, PCR. PCR may include amplifying viral nucleic acids using an appropriate number of primers, such as 1, 2, 3, 4, 5 or more than 5 different primer sets, each primer set targeting a different viral nucleic acid sequence. If the capsids are spiked with oligonucleotides to be amplified, another set or sets of primers may be used, wherein the primers are specific to the sequence of the spiked oligonucleotides. The method can also include labeling the viral capsid by binding the detection molecule to the capsid surface, for example, by coupling to exposed amino acids on the capsid surface, or by binding to one or more specific antigens on the capsid surface, for example, by binding to a portion, which comprises a detection molecule, and a binding entity comprising an antibody, a peptide, a single-chain variable fragment (scFv), a nanobody, an aptamer, a lipid or a nucleic acid. Any suitable detection molecule can be used, such as a fluorescent dye or an oligonucleotide. In the latter case, the detection oligonucleotide and / or viral nucleic acid (if present) is amplified in a single partition (i.e., the same partition). In some cases, the virus can be AAV. In this case, the binding entity can include, for example, an AAV receptor (AAVR) or a fragment thereof or an A20 monoclonal antibody (mAb) or a fragment thereof or an aptamer.In methods using multiple partitions, some of which contain viral capsids and viral nucleic acids, and some of which contain only viral capsids or only viral nucleic acids, the method may also include determining the ratio of the number of partitions containing viral capsids and viral nucleic acids to the total number of partitions containing viral components (e.g., viral capsids and / or viral nucleic acids). In some cases, it can be determined whether the viral nucleic acid is complete or partial, and the partitions can be classified as containing empty capsids (no viral nucleic acid), partial capsids (viral nucleic acid but not complete) or real capsids (complete viral nucleic acid), and the appropriate ratio (e.g., the number of partitions containing real capsids to the total number of partitions containing capsids (with or without viral nucleic acids)) can be determined. In some cases, the active fraction can be estimated based on the proportion of partitions containing real capsids. For example, partitions found to contain capsids and viral nucleic acids are considered to be part of the active fraction, while partitions found to contain capsids or viral nucleic acids but not both are considered to be part of the inactive fraction.

[0214] In certain embodiments, provided herein is a method for quantifying viral genomes and activity scores in a single assay, comprising i) spiking viral capsids in a bulk sample containing the virus; ii) dividing the sample into a plurality of subsample partitions; iii) altering the integrity of the capsids in the partitions to release viral nucleic acid (if present) while retaining the ability to detect the capsid tag; and iv) detecting viral nucleic acid in the partitions containing viral nucleic acid and detecting capsids in the partitions containing capsids, wherein in the partitions containing both viral nucleic acid and capsids, both are detected in a single partition; v) determining the activity score based on the results of step (iv). Detecting viral nucleic acid may include detecting one or more viral nucleic acid sequences indicative of functional viral nucleic acid.

[0215] In certain embodiments, provided herein is a method of including viral nucleic acid and capsid in a single viral particle, such as by treating the viral particle to tag the capsid, for example, with a detectable marker, or with a tag that can be treated to produce a detectable marker, such as an oligonucleotide that is amplified and detected. The viral nucleic acid can also be amplified.

[0216] Dual Target Nucleic Acid Binding Molecules: The "Dual Target Nucleic Acid Binding Molecules" approach for capsid quantification can be easily used for simultaneous quantification. After site generation or protection and any associated washing steps, the sample can be partitioned with new sites still bound to the capsid surface. After viral genome release, the PCR approach for viral genome quantification can be optimized to include additional capsid-associated target sites using hydrolysis or hairpin probes. Subsamples can be dually interrogated for viral genome and capsid, and the results interpreted accordingly.

[0217] Envelope vectors: In addition to the methods described above, labeling methods specific to envelope vectors can be employed. Capsids can be labeled prior to partitioning using methods previously discussed in “Lipid Bilayer Labeling Methods.” A wash step may be required to remove unbound probe. For methods that observe a shift in fluorescence upon lipid bilayer insertion, viral release conditions must be optimized to release the viral genome without disassembling the capsid. Likewise, viral genome quantification can be performed using any of the methods previously described, which can be done entirely within partitioning. s. Kit

[0218] In certain embodiments, kits are provided herein. The kit contains a set of components, such as reagents and other materials, for performing specific functions. These components are usually packaged separately, such as in separate containers, and these components are provided as a collection for use together. Appropriate packaging materials may also be included. Instructions may also be provided, such as hard copy, electronic version, or a combination thereof. In certain embodiments, kits are provided herein, which contain components for adding viral capsid components and primers for amplifying partial viral nucleic acids. The components used for adding viral capsids may be specific or non-specific, depending on the needs of the end user. For example, a user who makes relatively pure transgenic viral particles may only need non-specific labels because most or all of the materials to be analyzed that can be added will be viral capsids; in other cases, when non-capsid components are expected to be present in larger amounts, the kit may contain specific labels for viral capsids. Non-specific and specific capsid tags can be any suitable tags, such as the tags described herein. In some cases, the capsid tag is specific to a component of the AAV capsid, such as AAVR or a fragment thereof or A20 mAb, or a fragment thereof or an aptamer. The viral nucleic acid can be from any suitable virus to be analyzed; in certain embodiments, the viral nucleic acid is from AAV. The primers can be specific to universal sequences of the AAV genome, for example, sequences that are expected to be present in AAV, regardless of whether a transgene is introduced. When using such a kit, the end user can provide additional primers specific to a specific transgene or transgenic group that may have been added to the normal AAV genome, so the use of the kit will include the use of these user-provided primers. In some cases, the viral capsid spike component of the kit may include oligonucleotides to be amplified, and the kit may also include suitable primers for such amplification. The kit may also include a marker for a label of the viral capsid component. The kit may also include a master mix for a polymerase chain reaction. IV. Illustrative combinations and additional examples

[0219] This section describes additional aspects and features of the disclosed methods, compositions, and kits, presented as, but not limited to, a series of paragraphs, which, for clarity and efficiency, may be indexed alphanumeric in part or in whole. Each of these paragraphs may be combined in any suitable manner with one or more other paragraphs, and / or with the disclosure elsewhere in the application. Some of the paragraphs below explicitly mention and further limit other paragraphs, providing examples of some suitable combinations, but are not limited thereto.

[0220] A1. A method for analyzing viral particles, the method comprising: (i) tagging the capsid of the viral particles with a tag; (ii) forming subsamples of a sample containing viral particles, wherein only a subset of each subsample in the subsample contains at least one viral particle; (iii) amplifying one or more targets from the genome of the viral particles; (iv) collecting tag-related data and amplification data of one or more targets from the subsamples; and (v) enumerating a population of subsamples, defining the population based on the tag-related data and the amplification data.

[0221] A2. A method as described in paragraph A1, wherein enumeration includes obtaining subsample counts of two or more populations, and the method also includes directly using the subsample counts or (uncalibrated) count values ​​derived therefrom to determine the capsid occupancy of the genome as a ratio.

[0222] A3. A method as described in paragraph A2, wherein the count value is used as a ratio, and wherein the count value is derived from the subsample count at least in part by normalization to scale the subsample count and / or by adjustment based on one or more particle-free controls (NPCs).

[0223] A4. The method of paragraphs A2 or A3, wherein the spike has a spike efficiency, and wherein the determining inherently incorporates the same spike efficiency into the numerator and denominator of the ratio such that the spike efficiency does not affect the ratio.

[0224] A5. The method of any of paragraphs A1 to A4, wherein the tag comprises a template, wherein the amplifying comprises amplifying a first target from the template, and wherein the collecting tag-related data comprises collecting amplification data of the first target.

[0225] A6. A method as described in paragraph A5, wherein the labeling includes binding copies of a pair of proximity assay (PA) probes containing a pair of oligonucleotides to separate capsids of the viral particles, and wherein the labeling also includes using the oligonucleotide pair of the copies of the PA probe pair to perform a ligation reaction or an extension reaction to create a template, while the copies of the PA probe pair remain bound to the same separate capsid.

[0226] A7. The method of paragraph A5, wherein the tagging comprises combining a tagging probe and the viral particle with each other, wherein the tagging probe comprises an oligonucleotide that provides a pre-made full-length sequence of the template.

[0227] A8. The method of any one of paragraphs A1 to A7, further comprising: combining the beads and the virus particles with each other; binding the virus particles to the beads; and washing the beads before forming the subsamples after combining.

[0228] A9. The method of any one of paragraphs A1 to A8, further comprising treating the viral particles with a nuclease before adding the capsid and / or before forming a subsample to degrade nucleic acids located outside the viral particle capsid.

[0229] A10. The method of any of paragraphs A1 to A9, wherein the spiking comprises spiking a shell of a capsid, and wherein the shell forms an interior and an exterior of the capsid.

[0230] A11. The method of any of paragraphs A1 to A9, wherein the spiking comprises spiking an envelope of the capsid, and wherein the envelope comprises a lipid bilayer.

[0231] A12. A method as described in any of paragraphs A1 to A11, wherein the labeling includes labeling the viral particles with a marker before forming the subsample, and wherein collecting label-related data includes irradiating the marker with optical radiation and detecting light from the marker.

[0232] A13. The method of any of paragraphs A1 to A12, further comprising any limitation or combination of limitations of any of paragraphs B1 to B21, C1 to C8, D1 to D19, E1 to E5, F1 to F7, G1 to G3, and H1 to H5.

[0233] B1. A method for analyzing viral particles, the method comprising: (i) labeling the capsid of the viral particle with a template; (ii) forming partitions of a sample containing viral particles, such that each partition of only a subset of the partitions contains one of the viral particles; (iii) in the partitions, amplifying a first target from the template and one or more other targets from the viral particle genome; and (iv) collecting amplification data of each of the first target and one or more other targets from the partitions.

[0234] B2. The method described in paragraph B1 further comprises enumerating the partitioned populations according to target content based on the amplification data.

[0235] B3. The method of paragraph B2, further comprising determining the capsid occupancy of the genome to the capsid using the enumeration results.

[0236] B4. A method as described in paragraph B3, wherein the population includes at least one pair of populations, wherein each population in at least one pair of populations is positive for the first target, and wherein the determination includes calculating the percentage or fraction of partitions in at least one pair of populations that are also positive for each of the one or more other targets.

[0237] B5. A method as described in paragraph B3 or B4, wherein enumeration includes obtaining alpha partition counts for a partition population that is positive for the first target and negative for at least one of the one or more other targets, and beta partition counts for a partition population that is positive for the first target and each of the one or more other targets, and wherein a ratio representing capsid occupancy is calculated using values ​​of the alpha and beta partition counts or count values ​​derived therefrom.

[0238] B6. A method as described in paragraph B5, wherein the ratio includes the value of the β partition count or a value derived therefrom in the numerator of the ratio, and the sum of the values ​​of the α and β partition counts or values ​​derived therefrom in the denominator of the ratio.

[0239] B7. The method of paragraphs B5 or B6, wherein the spike has a spike efficiency, and wherein the calculation inherently incorporates the same spike efficiency into the numerator and denominator of the ratio such that the spike efficiency does not affect the ratio.

[0240] B8. A method as described in any of paragraphs B5 to B7, wherein the count value represents or is derived from the normalized partition count and / or the partition count from which a no particle control (NPC) value is subtracted.

[0241] B8a. A method as described in paragraph B5, wherein the enumeration includes obtaining α values ​​representing the concentration of connections between capsids and genome-positive partitions and β values ​​representing the concentration of labeled capsids from which a no particle control (NPC) value is subtracted, and wherein the determination is performed using the α values ​​and β values ​​or values ​​derived therefrom to calculate a ratio representing capsid occupancy.

[0242] B9. The method of any of paragraphs B1 to B8a, wherein the capsid comprises a shell, and wherein the spiking comprises spiking the shell.

[0243] B10. The method of any one of paragraphs B1 to B9, wherein the one or more other targets include a second target and a third target.

[0244] B11. The method of any of paragraphs B1 to B10, wherein the adding comprises connecting copies of the portions of the template to each other to create the template while the copies of the portions of the template remain connected to the same individual capsids.

[0245] B12. A method as described in any of paragraphs B1 to B11, wherein the labeling includes binding copies of a pair of proximity assay (PA) probes containing a pair of oligonucleotides to separate capsids of the viral particles, and wherein the labeling also includes using the oligonucleotide pair to perform a ligation reaction or an extension reaction to create a template while the copies of the PA probe pair remain bound to the same separate capsid.

[0246] B13. A method as described in paragraph B12, wherein each PA probe includes a specific binding agent that recognizes and binds to the capsid structure.

[0247] B14. A method as described in paragraph B12 or B13, wherein the PA probe is a proximity ligation assay (PLA) probe, and wherein labeling the capsid includes combining a PLA probe pair with a linker so that the linker hybridizes to the 3' terminal region of the oligonucleotide of one PLA probe in the PLA probe pair and the 5' terminal region of the oligonucleotide of the other PLA probe.

[0248] B15. The method of any one of paragraphs B1 to B10, wherein the tagging comprises combining a tagging probe and the viral particle with each other, wherein the tagging probe comprises a premade full-length sequence of the template.

[0249] B16. The method of any of paragraphs B1 to B15, wherein each partition, when formed, comprises primers to amplify each of the first target and one or more additional targets.

[0250] B17. A method as described in any of paragraphs B1 to B16, wherein the one or more other targets include a second target, wherein each partition when formed comprises a first amplification probe to detect amplification of the first target and a second amplification probe to detect amplification of the second target, and wherein each of the first and second amplification probes comprises a fluorescent marker.

[0251] B18. The method of any of paragraphs B1 to B17, wherein the partitions are droplets encapsulated by an immiscible continuous phase.

[0252] B19. The method of any of paragraphs B1 to B18, wherein collecting amplification data comprises detecting fluorescence from the partitions.

[0253] B20. A method as described in any of paragraphs B1 to B19, further comprising: combining the beads and viral particles with each other; binding the viral particles to the beads; and washing the beads after combining and before adding capsids and / or before forming partitions to form subsamples.

[0254] B21. The method of any of paragraphs B1 to B20, further comprising treating the viral particles with a nuclease to degrade nucleic acids located outside the capsid before forming the partitions.

[0255] B22. The method of any of paragraphs B1 to B21, further comprising any limitation or combination of limitations of any of paragraphs A1 to A12, C1 to C8, D1 to D19, E1 to E5, F1 to F7, G1 to G3, and H1 to H5.

[0256] C1. A composition comprising: a partition of a bulk sample containing viral particles, the partition containing only one of the viral particles; wherein the capsid of one of the viral particles is labeled, and wherein the partition contains reagents sufficient to amplify a target from the genome of the viral particle if the genome is present in the partition.

[0257] C2. The composition of paragraph C1, wherein the tag comprises an oligonucleotide that provides a template, and wherein the partition contains reagents sufficient to amplify the first target from the template and the second target from the genome if the genome is present in the partition.

[0258] C3. The composition of paragraph C2, wherein the template-providing oligonucleotide is a single molecule that individually attaches each specific binding agent of the pair of specific binding agents.

[0259] C4. The composition of paragraph C3, wherein the specific binding agents in the pair of specific binding agents bind to the capsid independently of each other.

[0260] C5. The composition of any of paragraphs C1 to C4, wherein the reagents include an amplification probe having a label for detecting target amplification.

[0261] C6. The composition of any of paragraphs C1 to C5, wherein the reagents include a set of primers for amplifying a target by polymerase chain reaction (PCR).

[0262] C7. The composition of any of paragraphs C1 to C6, wherein the tag comprises or is attached to a fluorophore.

[0263] C8. The composition of any of paragraphs C1 to C7, wherein the capsid comprises a genome.

[0264] C9. The composition of any of paragraphs C1 to C8, further comprising any of the limitations or combinations of limitations of any of paragraphs A1 to A12, B1 to B21, D1 to D19, E1 to E5, F1 to F7, G1 to G3, and H1 to H5.

[0265] D1. A method comprising detecting viral nucleic acid and capsid in a partition among multiple partitions containing both viral nucleic acid and viral capsid components.

[0266] D2. The method of paragraph D1, wherein the capsid component comprises a capsid protein.

[0267] D3. The method of paragraph D1 or D2, wherein the capsid component comprises a capsid lipid bilayer.

[0268] D4. The method as described in any of paragraphs D1 to D3 may also include spiking the viral capsids in the bulk sample containing the virus before dividing it into multiple partitions.

[0269] D5. A method as described in any of paragraphs D1 to D4, wherein a partition among the multiple partitions contains only capsids and / or only contains viral nucleic acids, and the method also includes detecting only capsids in partitions containing only capsids and / or detecting only viral nucleic acids in partitions containing only viral nucleic acids.

[0270] D6. The method of any of paragraphs D1 to D5, further comprising dividing the sample into a plurality of sub-sample partitions.

[0271] D7. A method as described in paragraph D6, which also includes altering the integrity of the capsid in the subsample partition sufficient to release viral nucleic acid (if present) while retaining the ability to manipulate the tag associated with the capsid to produce or display the marker.

[0272] D8. The method of any one of paragraphs D1 to D7, further comprising amplifying viral nucleic acid in the subsample, if present.

[0273] D9. The method of paragraph D8, wherein the viral nucleic acid is amplified by polymerase chain reaction (PCR).

[0274] D10. The method of any of paragraphs D1 to D9, wherein the viral capsid is labeled by binding a detection molecule to the capsid surface.

[0275] D11. The method of paragraph D10, wherein the binding is accomplished by coupling to exposed amino acids on the surface of the capsid.

[0276] D12. The method of paragraph D10, wherein the binding is to one or more specific antigens on the surface of the capsid.

[0277] D13. A method as described in paragraph D12, wherein the binding is achieved by binding to a moiety comprising a detection molecule, and a binding entity comprising an antibody, a peptide, a single-chain variable fragment (scFv), a nanobody, an aptamer, a lipid or a nucleic acid.

[0278] D14. The method of any one of paragraphs D1 to D13, wherein the detection molecule comprises a fluorescent dye or an oligonucleotide.

[0279] D15. The method of paragraph D14, wherein the detection molecule comprises an oligonucleotide.

[0280] D16. The method of paragraph D15, wherein the detection oligonucleotide and / or viral nucleic acid (if present) is amplified in a single partition of the plurality of partitions.

[0281] D17. The method of any one of paragraphs D1 to D16, wherein the virus is an adeno-associated virus (AAV) and the binding entity comprises an AAV receptor (AAVR) or a fragment thereof or an A20 monoclonal antibody (mAb) or a fragment thereof or an aptamer.

[0282] D18. A method as described in any of paragraphs D1 to D17, which also includes determining the ratio of the number of partitions containing both viral capsids and viral nucleic acids to the number of partitions containing viral nucleic acids or viral capsids based on the results of determining whether viral nucleic acids and / or viral capsids are present in the partitions.

[0283] D19. A method as described in paragraph D18, wherein partitions found to contain both capsids and viral nucleic acids are considered part of the active fraction, and partitions found to contain either capsids or viral nucleic acids but not both are considered part of the inactive fraction.

[0284] D20. The method of any of paragraphs D1 to D19, further comprising any limitation or combination of limitations of any of paragraphs A1 to A12, B1 to B21, C1 to C8, E1 to E5, F1 to F7, G1 to G3, and H1 to H5.

[0285] E1. A composition comprising: i) a subsample of a larger bulk sample containing viral particles; ii) within the subsample, spiked viral capsid components, and, if present, reagents sufficient to perform polymerase chain reaction (PCR) on viral nucleic acid.

[0286] E2. The composition of paragraph E1, further comprising viral nucleic acid.

[0287] E3. The composition of paragraph E1 or E2, wherein the reagents sufficient to perform PCR on viral nucleic acid include a set of primers specific for viral nucleic acid sequences.

[0288] E4. The composition of any of paragraphs E1 to E3, wherein the reagents sufficient to perform PCR on viral nucleic acid include at least two sets of primers for two different sequences of viral nucleic acid.

[0289] E5. The composition of any of paragraphs E1 to E4, wherein the spiked capsid component is spiked with a portion comprising an oligonucleotide and the subsample further comprises reagents sufficient to perform PCR on the tagged oligonucleotide.

[0290] E6. The composition of any of paragraphs E1 to E5, further comprising any of the limitations or combinations of limitations of any of paragraphs A1 to A12, B1 to B21, C1 to C8, D1 to D19, F1 to F7, G1 to G3, and H1 to H5.

[0291] F1. A kit comprising i) components for spiking viral capsid components; and ii) primers for amplifying partial viral nucleic acids.

[0292] F2. The kit of paragraph F2, wherein the virus is an adeno-associated virus (AAV) and the primers are specific for universal sequences in the AAV genome.

[0293] F3. The kit of paragraph F1 or F2, further comprising (iii) a master mix for polymerase chain reaction.

[0294] F4. The kit of any one of paragraphs F1 to F3, further comprising (iv) a marker for a tag of a viral capsid component.

[0295] F5. The kit of any of paragraphs F1 to F4, wherein the component used to spike the viral capsid component is non-specific to the viral capsid.

[0296] F6. The kit of any of paragraphs F1 to F5, wherein the component used to spike the viral capsid component is specific to the viral capsid.

[0297] F7. The kit of any of paragraphs F1 to F6, wherein the viral capsid comprises a capsid from AAV, and the component specific for the viral capsid comprises AAVR or a fragment thereof, or an A20 monoclonal antibody (mAb) or a fragment thereof, or an aptamer.

[0298] F8. The kit of any of paragraphs F1 to F7, further comprising any of the limitations or combinations of limitations of any of paragraphs A1 to A12, B1 to B21, C1 to C8, D1 to D19, E1 to E5, G1 to G3, and H1 to H5.

[0299] G1. A method for quantifying viral genomes and activity scores in a single assay, comprising: i) spiking viral capsids in a bulk sample containing the virus; ii) dividing the sample into multiple subsample partitions; iii) altering the integrity of the capsids in the partitions to release viral nucleic acid (if present) while retaining the ability to detect the capsid tag; and iv) detecting viral nucleic acid in partitions containing viral nucleic acid and detecting capsids in partitions containing capsids, wherein in partitions containing both viral nucleic acid and capsids, both are detected in a single partition.

[0300] G2. The method of paragraph G1, further comprising (v) determining an activity score from the results of step (iv).

[0301] G3. A method as described in paragraph G1 or G2, wherein detecting viral nucleic acid includes detecting one or more viral nucleic acid sequences indicative of functional viral nucleic acid.

[0302] G4. The kit of any of paragraphs G1 to G3, further comprising any of the limitations or combinations of limitations of any of paragraphs A1 to A12, B1 to B21, C1 to C8, D1 to D19, E1 to E5, F1 to F7, and H1 to H5.

[0303] H1. A method comprising detecting viral nucleic acid and capsid in a single viral particle.

[0304] H2. The method of paragraph H1, further comprising treating the viral particles to add viral capsids.

[0305] H3. The method of paragraph H1 or H2, further comprising labeling the spiked viral capsid with a detectable marker.

[0306] H4. A method as described in paragraph H3, wherein the marker is part of a label.

[0307] H5. The method of any of paragraphs H1 to H4, wherein viral nucleic acid is amplified.

[0308] H6. The method of any of paragraphs H1 to H5, further comprising any limitation or combination of limitations described in any of paragraphs A1 to A12, B1 to B21, C1 to C8, D1 to D19, E1 to E5, F1 to F7, and G1 to G3. V. V. Advantages and Benefits

[0309] The various embodiments of the methods, compositions, and kits for analyzing viral particles provide a variety of advantages over known solutions for analyzing viral particles. For example, the illustrative embodiments described herein allow for calibration-free quantification of capsid occupancy of a collection of viral particles. Without the need for separate calibration of capsid detection efficiency, viral particles can be analyzed faster, less expensively, more accurately, and / or with less labor.

[0310] Furthermore, the methods, compositions, and kits disclosed herein allow for the measurement of viral titer and capsid occupancy in the same assay.

[0311] There is no known system or method that can perform these functions.However, not all examples described herein provide the same advantages or the same degree of advantages. VI. in conclusion

[0312] The above disclosure may include multiple different examples with independent utility. Although various of these disclosures have been disclosed in their preferred forms, the specific examples of the invention disclosed and illustrated herein should not be considered restrictive, as multiple changes can be made. With respect to the section titles used in this disclosure, such titles are used for organizational purposes only. The subject matter of the present disclosure includes all novel and non-obvious combinations and sub-combinations of the various elements, features, functions and / or characteristics disclosed herein. The claims specifically point out certain combinations and sub-combinations that are novel and non-obvious. Other combinations and sub-combinations of features, functions, elements and / or characteristics may be claimed in applications claiming priority to this application or a related application. These claims, whether their scope is wider, narrower, the same or different than the scope of the original claims, are also deemed to be included in the subject matter of the present disclosure.

Claims

1. A method for analyzing virus particles, the method comprising: Spiking the capsid of the viral particle with the tag; forming subsamples of the sample comprising viral particles, each subsample of only a subset of the subsamples comprising at least one of the viral particles; amplifying one or more targets from the genome of the viral particle; collecting tag-associated data and amplification data of one or more targets from the subsamples; and Populations of subsamples are enumerated, the populations being defined based on the tag-associated data and the amplification data.

2. The method of claim 1, wherein enumeration comprises obtaining subsample counts of two or more populations, the method further comprising determining capsid occupancy of the genome as a ratio using the subsample counts directly or count values ​​derived therefrom.

3. A method as claimed in claim 2, wherein the count values ​​are used as ratios, and wherein the count values ​​are derived from the subsample counts at least in part by normalization to scale the subsample counts and / or by adjustment based on one or more particle-free controls (NPCs).

4. The method of claim 2, wherein the spike has a spike efficiency, and wherein the determining inherently incorporates the same spike efficiency into the numerator and denominator of the ratio such that the spike efficiency does not affect the ratio.

5. The method of claim 1, wherein the tag comprises a template, wherein the amplifying comprises amplifying a first target from the template, and wherein the collecting tag-related data comprises collecting amplification data of the first target.

6. A method as claimed in claim 5, wherein the labeling includes binding copies of a pair of proximity assay (PA) probes containing a pair of oligonucleotides to individual capsids of the viral particles, and wherein the labeling also includes using the oligonucleotide pair of the copies of the PA probe pair to perform a ligation reaction or an extension reaction to create a template while the copies of the PA probe pair remain bound to the same individual capsid.

7. The method of claim 5, wherein the spiking comprises combining (i) a spiking probe and (ii) the viral particle with one another, wherein the spiking probe comprises an oligonucleotide that provides a pre-made full-length sequence of the template.

8. The method of claim 1, further comprising: The beads and viral particles were combined with each other; binding the viral particles to beads; as well as The beads were washed after pooling and before forming subsamples.

9. The method of claim 1, further comprising treating the viral particles with a nuclease prior to spiking the capsids and / or prior to forming subsamples to degrade nucleic acids located outside the viral particle capsids.

10. The method of claim 1, wherein the spiking comprises spiking a shell of a capsid, and wherein the shell forms an interior and an exterior of the capsid.

11. The method of claim 1, wherein the spiking comprises spiking an envelope of the capsid, and wherein the envelope comprises a lipid bilayer.

12. The method of claim 1, wherein spiking comprises labeling the viral particles with a marker prior to forming the subsample, and wherein collecting label-related data comprises illuminating the marker with optical radiation and detecting light from the marker.

13. A method for analyzing viral particles, the method comprising: The capsid of the viral particle was spiked with the template; forming partitions of the sample containing viral particles such that only a subset of the partitions contain one of the viral particles; amplifying in the partitions a first target from the template and one or more other targets from the viral particle genome; as well as Amplification data for the first target and each of the one or more additional targets is collected from the partitions.

14. The method of claim 13, further comprising: Based on the amplification data, the partitioned populations are enumerated according to target content.

15. The method of claim 14, further comprising: The enumeration results were used to determine the capsid occupancy of the genome to the capsid.

16. A method as claimed in claim 15, wherein the population includes at least one pair of populations, each population in at least one pair of populations is positive for the first target, and wherein determining includes calculating the percentage or fraction of partitions in at least one pair of populations that are also positive for each of the one or more other targets.

17. A method as claimed in claim 15, wherein enumeration includes obtaining alpha partition counts for the partition population that is positive for the first target and negative for at least one of the one or more other targets, and beta partition counts for the partition population that is positive for the first target and each of the one or more other targets, and wherein it is determined to use the values ​​of the alpha and beta partition counts or count values ​​derived therefrom to calculate a ratio representing capsid occupancy.

18. The method of claim 17, wherein the ratio includes the value of the beta partition count or a value derived therefrom in the numerator of the ratio and the sum of the values ​​of the alpha and beta partition counts or values ​​derived therefrom in the denominator of the ratio.

19. The method of claim 17, wherein the spike has a spike efficiency, and wherein the calculation inherently incorporates the same spike efficiency into the numerator and denominator of the ratio such that the spike efficiency does not affect the ratio.

20. The method of claim 17, wherein the count values ​​represent or are derived from normalized compartment counts and / or compartment counts from which no particle control (NPC) values ​​have been subtracted.

21. The method of claim 15, wherein enumeration comprises obtaining α values ​​representing the concentration of connections between capsids and genome-positive partitions and β values ​​representing the concentration of labeled capsids from which a no particle control (NPC) value is subtracted, and wherein the determining is calculating a ratio representing capsid occupancy using the α and β values ​​or values ​​derived therefrom.

22. The method of claim 13, wherein the capsid comprises a shell, and wherein spiking comprises spiking the shell.

23. The method of claim 13, wherein the one or more other targets include a second target and a third target.

24. The method of claim 13, wherein labeling comprises linking copies of portions of the template to each other to create the template while the copies of portions of the template remain linked to the same individual capsids.

25. The method of claim 13, wherein the labeling comprises binding copies of a pair of proximity assay (PA) probes comprising a pair of oligonucleotides to individual capsids of the viral particle, and wherein the labeling further comprises creating a template using the oligonucleotide pair for a ligation reaction or an extension reaction while the copies of the PA probe pair remain bound to the same individual capsids.

26. The method of claim 25, wherein each PA probe comprises a specific binding agent that recognizes and binds to the capsid structure.

27. The method of claim 25, wherein the PA probe is a proximity ligation assay (PLA) probe, wherein tagging the capsid comprises combining a PLA probe pair with a linker such that the linker hybridizes to a 3' terminal region of an oligonucleotide of one PLA probe and a 5' terminal region of an oligonucleotide of the other PLA probe of the PLA probe pair.

28. The method of claim 13, wherein the spiking comprises combining (i) a spiking probe and (ii) the viral particle with one another, wherein the spiking probe comprises a premade full-length sequence of the template.

29. The method of claim 13, wherein each partition, when formed, comprises primers to amplify the first target and each of the one or more additional targets.

30. The method of claim 13, wherein the one or more other targets include a second target, wherein each partition when formed comprises a first amplification probe to detect amplification of the first target and a second amplification probe to detect amplification of the second target, and wherein each of the first and second amplification probes comprises a fluorescent marker.

31. The method of claim 13, wherein the partitions are droplets encapsulated by an immiscible continuous phase.

32. The method of claim 13, wherein collecting amplification data comprises detecting fluorescence from the partitions.

33. The method of claim 13, further comprising: The beads and viral particles were combined with each other; binding the viral particles to beads; as well as The beads were washed after pooling and before forming partitions.

34. The method of claim 13, further comprising treating the viral particles with a nuclease to degrade nucleic acids located on the exterior of the capsid prior to capsid spiking and / or prior to partitioning.

35. A composition comprising: a partition of the bulk sample containing viral particles, the partition containing only one of the viral particles; wherein the capsid of one of the viral particles is tagged with a tag, and wherein the partition contains reagents sufficient to amplify a target from the genome of the viral particle if the genome is present in the partition.

36. The composition of claim 35, wherein the tag comprises an oligonucleotide that provides a template, and wherein the partition contains reagents sufficient to amplify a first target from the template and amplify a second target from the genome if the genome is present in the partition.

37. The composition of claim 35, wherein the template-providing oligonucleotide is a single molecule that individually attaches each specific binding agent of a pair of specific binding agents.

38. The composition of claim 37, wherein the specific binding agents of the pair of specific binding agents are independently bound to the capsid.

39. The composition of claim 35, wherein the reagents include an amplification probe having a label for detecting target amplification.

40. The composition of claim 35, wherein the reagents include a set of primers for amplifying a target by polymerase chain reaction (PCR).

41. The composition of claim 35, wherein the tag comprises or is attached to a fluorophore.

42. The composition of claim 35, wherein the capsid comprises a genome.

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