Assays and methods for quality control of genome packaging for gene therapy

Abstract

The present disclosure relates assays and methods for detecting and / or measuring viral titers and genomic packaging of adeno-associated viral vectors.

Description

[0001]Docket No.103361-584WO1 ASSAYS AND METHODS FOR QUALITY CONTROL OF GENOME PACKAGING FOR GENE THERAPY CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to, and the benefit of, U.S. Provisional Patent Application No.63 / 578,375, filed August 24, 2023, which is incorporated by reference herein in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH This invention was made with Government Support under Grant No. TR003807 and TR002884 awarded by the National Institutes of Health. The Government has certain rights in the invention. FIELD The present disclosure relates assays and methods for detecting and / or measuring viral titers and genomic packaging of adeno-associated viral vectors. BACKGROUND Adeno-Associated Virus (AAV) vectors have emerged as powerful tools for gene therapy, thanks to their non-pathogenic nature and efficient human infectivity. Therefore, AAV-based gene therapy can lead to “cures” for genetic diseases following a single administration. The success of this therapy hinges upon the critical requirement for precise and reliable quantification of AAV titers, which is essential to ensure the optimal dosage and efficacy of gene therapy treatments, maximizing their therapeutic abilities. Standardized methods and quality control (QC) measurements used to quantify AAV titers and infectivity helped to improve the accuracy and reproducibility of AAV production. However, current methods do not simultaneously determine AAV titers with intact or damaged genomes – due to packaging of incomplete genomes or to cross packaging – and quantify empty AAV capsids. This leads to misestimation of the titer, which incurs serious consequences leading to administering low AAV titers with sub-optimal therapeutic efficacy or high AAV titers causing severe adverse events or death to the treated patient. At present, standardized methods used for AAV titer quantification include quantitative polymerase chain reaction (qPCR), droplet digital PCR (ddPCR), and enzyme-linked immunosorbent assay (ELISA). qPCR and ddPCR are designed to measure the single-stranded or self-complementary DNA genome of AAV (genome titer). Still, they cannot simultaneously measure the titer of intact and cross-packaged genomes and AAV empty capsids, which can be found in any AAV Docket No.103361-584WO1 preparation, and their proportion depends on the size and complexity of the AAV genome and the purification method. This critical limitation leads to adverse events (i.e., toxicity) and reduced therapeutic efficacy. Moreover, methods to measure the capsid titer (e.g., ELISA) cannot distinguish between empty and full AAV capsids (AAV packaging a genome), making them unreliable in determining an accurate titer of an efficacious and safe to administer AAV. Therefore, there is an unmet need for developing analytical methods that provide an efficient, cost-effective, and easy GMP implementation for multiparametric quantification of produced AAVs (e.g., capsid and genomic titer and AAV integrity). The assays and methods disclosed herein address these needs and more. SUMMARY The present disclosure provides assays and methods of use thereof for assessing, detecting, and / or measuring the viral titer and genomic packaging of an adeno-associated virus (AAV) for treating and / or preventing diseases (including, but not limited to Duchenne muscular dystrophy (DMD)). In some aspects, disclosed herein is a quality control (QC) assay for detecting viral titer and genomic packaging of an adeno-associated virus (AAV), wherein the QC assay comprises at least one AAV capsid probe, an AAV gene probe, and a transgene probe, wherein the QC assay simultaneously detects a viral titer of the AAV and packaging of a target nucleic acid within the AAV. In some aspects, disclosed herein is a method of detecting viral titer and genomic packaging of an adeno-associated virus (AAV), the method comprising binding at least one AAV capsid probe to an AAV, wherein the at least one AAV capsid binds to a membrane protein of the AAV and comprises a first fluorescent molecule, binding an AAV gene probe to an AAV nucleic acid within the AAV, wherein the AAV gene probe comprises a second fluorescent molecule, binding a transgene probe to a target nucleic acid within the AAV, wherein the transgene probe comprises a third fluorescent probe, and detecting one, two, three, or more fluorescent signals from any preceding fluorescent molecule, wherein detecting the first fluorescent molecule indicates a viral titer, detecting the second fluorescent molecule indicates a viral genome, and detecting the third fluorescent molecule indicates packaging of the target nucleic acid. In some aspects, disclosed herein is a method of treating a subject with Duchenne muscular dystrophy (DMD), the method comprising performing a QC assay to assess viral titer and genomic packaging of an adeno-associated virus (AAV), wherein the QC assay comprises 1) binding at least one AAV capsid probe to an AAV, wherein the at least one AAV capsid binds to a membrane Docket No.103361-584WO1 protein of the AAV and comprises a first fluorescent molecule, 2) binding an AAV gene probe to an AAV nucleic acid within the AAV, wherein the AAV gene probe comprises a second fluorescent molecule, 3) binding a transgene probe to a target nucleic acid within the AAV, wherein the transgene probe comprises a third fluorescent probe, and 4) detecting one, two, three, or more fluorescent signals from any preceding fluorescent molecule, wherein detecting the first fluorescent molecule indicates a viral titer, detecting the second fluorescent molecule indicates a viral genome, and detecting the third fluorescent molecule indicates packaging of the target nucleic acid; and subsequently administering to the subject the AAV comprising the target nucleic, wherein target nucleic acid treats or prevents DMD. In some embodiments, the at least one AAV capsid probe binds to a membrane protein of the AAV. In some embodiments, the at least one AAV capsid probe comprises an antibody, or a fragment thereof. In some embodiments, the at least one AAV capsid probe binds to a conformational epitope. In some embodiments, the at least one AAV capsid probe binds to a VP1, VP2, VP3 membrane protein, or a combination thereof. In some embodiments, the at least one AAV capsid probe comprises a first fluorescent molecule. In some embodiments, the first fluorescent molecule indicates the viral titer. In some embodiments, the AAV gene probe binds an AAV nucleic acid. In some embodiments, the AAV nucleic acid comprises a DNA or RNA. In some embodiments, the AAV gene probe comprises an antibody, a nucleic acid probe such as a capsid penetrating nucleic acid probe (CPNP), or fragments thereof. In some embodiments, the AAV gene probe comprises a second fluorescent molecule. In some embodiments, the second fluorescent molecule indicates a viral genome. In some embodiments, the transgene probe binds to a target nucleic acid within the AAV. In some embodiments, the target nucleic acid comprises a DNA or RNA. In some embodiments, the target nucleic acid comprises a non-native nucleic acid not derived from the AAV. In some embodiments, the transgene probe comprises an antibody, a capsid penetrating nucleic acid probe (CPNP), or fragments thereof. In some embodiments, the transgene probe comprises a third fluorescent molecule. In some embodiments, the third fluorescent molecule indicates the genomic packaging of the target nucleic acid. In some embodiments, the AAV comprises a single-stranded AAV (ssAAV), a self- complementary AAV (scAAV), or a recombinant AAV (rAAV). In some embodiments, the AAV comprises AAV1, AAV2, AAV3, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh74, or other serotypes thereof. In some embodiments, the QC assay and / or the method is Docket No.103361-584WO1 performed on a micropattern array, a microchip platform, a multi-well platform, or a combination thereof. In some embodiments, the QC assay or the method introduces the transgene into a subject’s genome for treatment or prevention of DMD. In some aspects, disclosed herein is a biochip for detecting viral titer and genomic packaging of an adeno-associated virus (AAV), wherein said biochip comprises the quality control (QC) assay of any preceding aspect. BRIEF DESCRIPTION OF FIGURES The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects described below. FIG. 1 shows the overview of the in situ multiparametric nano-bioassay for quality assurance and quality control of genome packaging in virus / viral vector particles engineered for gene therapy. FIG.2 shows the working principle of theAAVBiochip. Nucleic acid fluorescence probes are delivered inside AAV capsid where it will hybridize with the single strand or double strand DNA of the AAV. The nucleic acid fluorescence probes are designed to target different regions of the AAV DNA for example, common ITR region and transgene region (gene of interest). Then, the intact AAV particles are captured on the surface ofAAVBiochip by physisorption. The fluorescence antibody is added to detect the capsid protein of AAV. The fluorescence images are taken by total internal reflection fluorescence microscopy (TIRFM). The colocalization of fluorescence signal is calculated for the empty / full capsid ratio. FIGS.3A and 3B show the concept of the single EV analysis technology for protein and RNA detection. Figure 3B shows the single EVs from a glioma cell line are detected and colocalized. Figure 3C shows the TIRFM images are quantified as distributions of fluorescence intensify to depict the expression of protein and RNA at a single-EV level. FIGS.4A, 4B, 4C, and 4D show the schematic representation for the detection of protein and RNA detection in non-lysed SARS-CoV-2 virions. Figure 4B shows TIRFM images of spike glycoprotein and viral RNA detection in single virions. Figure 4C shows quantification of the intensity distribution of spike glycoprotein and RNA signals from single virions. Figure 4D shows the multiparametric detection on single virus particles by colocalization of fluorescent signals. FIGS. 5A, 5B, 5C, 5D, and 5E show the TIRFM images of AAV9s stained for capsid (VP1, VP2, and VP3(VP1-3)) protein and genomic DNA labeled with EthD-1. Figure 5B shows the histogram of fluorescent signals for protein and DNA. Figure 5C shows the colocalization of Docket No.103361-584WO1 capsid (VP1-3) protein and EthD-1 signals. Figure 5D shows the high magnification of single AAV9s with different DNA contents within the capsids. Figure 5E shows the negative stained TEM images of full (white arrows) and empty (black arrows) AAV9 capsids. FIGS.6A, 6B, and 6C show the TEM images of empty (black arrow) and full capsid (white arrow) and the AAV size characterization from TEM images. FIGS.7A, 7B, 7C, 7D, 7E, 7F, 7G, 7H, and 7I show the representative TIFRM images of AAV capsid protein (VP1-3 protein) and ITR region of DNA of AAV particles by using nucleic acid fluorescence probe 1. FIGS.8A, 8B, 8C, 8D, 8E, 8F, 8G, 8H, and 8I show the representative TIFRM images of AAV capsid protein (VP1-3 protein) and ITR region of DNA of AAV particles by using nucleic acid fluorescence probe 2. FIGS. 9A, 9B, 9C, 9D, and 9E show the working range ofAAVBiochip for the capsid protein detection for different type of AAV: empty, single strand AAV and self-complementary AAV and the representative TIRFM images of empty AAV at different concentrations. FIGS.10A, 10B, 10C, 10D, 10E, and 10F show the working range ofAAVBiochip for the gene of interest region (eGFP in this case as an example) detection for different type of AAV: empty, single strand AAV and self-complementary AAV. FIG.11 shows the representative TIRFM images of ssAAV for multiparametric detection: capsid protein, region of interest, common ITR region and the merged image of fluorescence signal. FIG. 12 shows the scatter graphs showing the single AAV signals for capsid, region of interest (eGFP) and common region ITR. FIG.13 shows theAAVBiochip provides high analytical strength through the use of specific antibodies against the AAV protein capsid and specificDNACPNPs targeting different regions of the AAV genomic DNA and transfer plasmid backbone. DETAILED DESCRIPTION The following description of the disclosure is provided as an enabling teaching of the disclosure in its best, currently known embodiment(s). To this end, those skilled in the relevant art will recognize and appreciate that many changes can be made to the various embodiments of the invention described herein, while still obtaining the beneficial results of the present disclosure. It will also be apparent that some of the desired benefits of the present disclosure can be obtained by selecting some of the features of the present disclosure without utilizing other features. Accordingly, those who work in the art will recognize that many modifications and adaptations to Docket No.103361-584WO1 the present disclosure are possible and can even be desirable in certain circumstances and are a part of the present disclosure. Thus, the following description is provided as illustrative of the principles of the present disclosure and not in limitation thereof. Reference will now be made in detail to the embodiments of the invention, examples of which are illustrated in the drawings and the examples. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Terminology Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of” and “consisting of” can be used in place of “comprising” and “including” to provide for more specific embodiments and are also disclosed. As used in this disclosure and in the appended claims, the singular forms “a”, “an”, “the”, include plural referents unless the context clearly dictates otherwise. The following definitions are provided for the full understanding of terms used in this specification. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that when a value is disclosed that “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “10” is disclosed the “less than or equal to 10”as well as “greater than or equal to 10” is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting Docket No.103361-584WO1 points, and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed. “Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. An "increase" can refer to any change that results in a greater amount of a symptom, disease, composition, condition, or activity. An increase can be any individual, median, or average increase in a condition, symptom, activity, composition in a statistically significant amount. Thus, the increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100% or more increase so long as the increase is statistically significant. A "decrease" can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity. A substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance. Also, for example, a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed. A decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount. Thus, the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100%, or more decrease so long as the decrease is statistically significant. By “prevent” or other forms of the word, such as “preventing” or “prevention,” is meant to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce. As used herein, something could be reduced but not prevented, but something that is reduced could also be prevented. Likewise, something could be prevented but not reduced, but something that is prevented could also be reduced. It is understood that where reduce or prevent are used, unless specifically indicated otherwise, the use of the other word is also expressly disclosed. The term “subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. In one aspect, the subject can be human, non-human primate, bovine, equine, porcine, canine, or feline. The subject can also be a guinea Docket No.103361-584WO1 pig, rat, hamster, rabbit, mouse, or mole. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician. "Comprising" is intended to mean that the compositions, methods, etc. include the recited elements, but do not exclude others. "Consisting essentially of'' when used to define compositions and methods, shall mean including the recited elements, but excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. "Consisting of'' shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions provided and / or claimed in this disclosure. Embodiments defined by each of these transition terms are within the scope of this disclosure. Reference also is made herein to peptides, polypeptides, proteins, and compositions comprising peptides, polypeptides, and proteins. As used herein, a polypeptide and / or protein is defined as a polymer of amino acids, typically of length≥100 amino acids (Garrett & Grisham, Biochemistry, 2nd edition, 1999, Brooks / Cole, 110). A peptide is defined as a short polymer of amino acids, of a length typically of 20 or less amino acids, and more typically of a length of 12 or less amino acids (Garrett & Grisham, Biochemistry, 2nd edition, 1999, Brooks / Cole, 110). The peptides, polypeptides, and proteins disclosed herein may be modified to include non- amino acid moieties. Modifications may include but are not limited to carboxylation (e.g., N- terminal carboxylation via addition of a di-carboxylic acid having 4-7 straight-chain or branched carbon atoms, such as glutaric acid, succinic acid, adipic acid, and 4,4-dimethylglutaric acid), amidation (e.g., C-terminal amidation via addition of an amide or substituted amide such as alkylamide or dialkylamide), PEGylation (e.g., N-terminal or C-terminal PEGylation via additional of polyethylene glycol), acylation (e.g., O-acylation (esters), N-acylation (amides), S- acylation (thioesters)), acetylation (e.g., the addition of an acetyl group, either at the N-terminus of the protein or at lysine residues), formylation lipoylation (e.g., attachment of a lipoate, a C8 functional group), myristoylation (e.g., attachment of myristate, a C14 saturated acid), palmitoylation (e.g., attachment of palmitate, a C16 saturated acid), alkylation (e.g., the addition of an alkyl group, such as an methyl at a lysine or arginine residue), isoprenylation or prenylation (e.g., the addition of an isoprenoid group such as farnesol or geranylgeraniol), amidation at C- terminus, glycosylation (e.g., the addition of a glycosyl group to either asparagine, hydroxylysine, serine, or threonine, resulting in a glycoprotein). Distinct from glycation, which is regarded as a nonenzymatic attachment of sugars, polysialylation (e.g., the addition of polysialic acid), Docket No.103361-584WO1 glypiation (e.g., glycosylphosphatidylinositol (GPI) anchor formation, hydroxylation, iodination (e.g., of thyroid hormones), and phosphorylation (e.g., the addition of a phosphate group, usually to serine, tyrosine, threonine, or histidine). The phrases “percent identity” and “% identity,” as applied to polypeptide sequences, refer to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm. Methods of polypeptide sequence alignment are well-known. Some alignment methods consider conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide. Percent identity for amino acid sequences may be determined as understood in the art. (See, e.g., U.S. Pat. No. 7,396,664, which is incorporated herein by reference in its entirety). A suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, S. F. et al. (1990) J. Mol. Biol.215:403410), which is available from several sources, including the NCBI, Bethesda, Md., at its website. The BLAST software suite includes various sequence analysis programs including “blastp,” that is used to align a known amino acid sequence with other amino acids sequences from a variety of databases. Percent identity may be measured over the length of an entire defined polypeptide sequence or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length may be used to describe a length over which percentage identity may be measured. The term “variant” or “fragment” means a polypeptide derived from a parent polypeptide by one or more (several) alteration(s), i.e., a substitution, insertion, and / or deletion, at one or more (several) positions. A substitution means a replacement of an amino acid occupying a position with a different amino acid; a deletion means removal of an amino acid occupying a position; and an insertion means adding 1 or more, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably 1-3 amino acids immediately adjacent an amino acid occupying a position. In relation to substitutions, ‘immediately adjacent’ may be to the N-side (‘upstream’) or C-side (‘downstream’) of the amino acid occupying a position (‘the named amino acid’). Therefore, for an amino acid named / numbered ‘X,’ the insertion may be at position ‘X+1’ (‘downstream’) or at position ‘X−1’ (‘upstream’). A “variant” or a “fragment” of a particular polypeptide sequence may be defined as a Docket No.103361-584WO1 polypeptide sequence having at least 50% sequence identity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the “BLAST 2 Sequences” tool available at the National Center for Biotechnology Information's website. (See Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2 sequences—a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett.174:247-250). In some embodiments a variant polypeptide may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to a reference polypeptide. A variant or fragment polypeptide may have substantially the same functional activity as a reference polypeptide. For example, a variant polypeptide may exhibit or more biological activities associated with binding a ligand and / or binding DNA at a specific binding site. The term “administer,” “administering”, or derivatives thereof refer to delivering a composition, substance, inhibitor, or medication to a subject or object by one or more the following routes: oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation or via an implanted reservoir. The term “parenteral” includes subcutaneous, intravenous, intramuscular, intra- articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injections or infusion techniques. The term “detect” or “detecting” refers to an output signal released for the purpose of sensing of physical phenomenon. An event or change in environment is sensed and signal output released in the form of light including, but not limited to fluorescent light. The term "antibody" is used in the broadest sense, and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies). Antibodies (Abs) and immunoglobulins (Igs) are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific target, immunoglobulins include both antibodies and other antibody-like molecules which lack target specificity. Native antibodies and immunoglobulins are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end. Docket No.103361-584WO1 The term "antibody fragment" refers to a portion of a full-length antibody, generally the target binding or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2and Fv fragments. The phrase "functional fragment or analog" of an antibody is a compound having qualitative biological activity in common with a full-length antibody. For example, a functional fragment or analog of an anti-IgE antibody is one which can bind to an IgE immunoglobulin in such a manner so as to prevent or substantially reduce the ability of such molecule from having the ability to bind to the high affinity receptor, FcεRI. As used herein, "functional fragment" with respect to antibodies, refers to Fv, F(ab) and F(ab')2fragments. An "Fv" fragment is the minimum antibody fragment which contains a complete target recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in a tight, non-covalent association (VH-VLdimer). It is in this configuration that the three CDRs of each variable domain interact to define a target binding site on the surface of the VH-VLdimer. Collectively, the six CDRs confer target binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for a target) has the ability to recognize and bind target, although at a lower affinity than the entire binding site. "Single-chain Fv" or "sFv" antibody fragments comprise the VHand VLdomains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VHand VLdomains which enables the sFv to form the desired structure for target binding. The term “monoclonal antibody” as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of the antibody molecules. A "gene" refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular polypeptide or protein after being transcribed and translated. Any of the polynucleotides sequences described herein may be used to identify larger fragments or full-length coding sequences of the gene with which they are associated. A "capsid penetrating nucleic acid probe" refers to an oligonucleotide that is provided as a reagent to detect a target potentially present in a sample of interest by hybridizing with the target. Usually, a capsid penetrating nucleic acid probe will comprise a label or a means by which a label can be attached, either before or subsequent to the hybridization reaction. Suitable labels include, but are not limited to radioisotopes, fluorochromes, chemiluminescent compounds, dyes, and proteins, including enzymes. Docket No.103361-584WO1 As used herein, the term “probe” refers to a molecule or group of molecules used in molecular biology or chemistry to study the properties of other molecules or structures. If some measurable property of the molecular probe used changes when it interacts with the molecule of interest, the interactions between the probe and the molecule of interest can be studied. This makes it possible to indirectly study the properties of compounds and structures which may be hard to study directly. The terms “treat,” “treating,” and grammatical variations thereof as used herein, include partially or completely delaying, alleviating, mitigating or reducing the intensity of one or more attendant symptoms of a disorder or condition and / or alleviating, mitigating or impeding one or more causes of a disorder or condition. Treatments according to the disclosure may be applied preventively, prophylactically, palliatively or remedially. Treatments are administered to a subject prior to onset (e.g., before obvious signs of disease), during early onset (e.g., upon initial signs and symptoms of disease), or after an established development of disease. A “nucleic acid” is a chemical compound that serves as the primary information-carrying molecules in cells and make up the cellular genetic material. Nucleic acids comprise nucleotides, which are the monomers made of a 5-carbon sugar (usually ribose or deoxyribose), a phosphate group, and a nitrogenous base. A nucleic acid can also be a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA). A chimeric nucleic acid comprises two or more of the same kind of nucleic acid fused together to form one compound comprising genetic material. The terms “percent identity” and “% identity,” as applied to polynucleotide sequences, refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences. Percent identity for a nucleic acid sequence may be determined as understood in the art. (See, e.g., U.S. Pat. No.7,396,664, which is incorporated herein by reference in its entirety). A suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, S. F. et al. (1990) J. Mol. Biol.215:403410), which is available from several sources, including the NCBI, Bethesda, Md., at its website. The BLAST software suite includes various sequence analysis programs including “blastn,” that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases. Also available is a tool called “BLAST 2 Sequences” that is used for direct pairwise comparison of two nucleotide sequences. “BLAST 2 Sequences” can be accessed and used interactively at the NCBI website. The “BLAST Docket No.103361-584WO1 2 Sequences” tool can be used for both blastn and blastp (discussed above). Percent identity may be measured over the length of an entire defined polynucleotide sequence or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides. Such lengths are exemplary only, and it is understood that any fragment length may be used to describe a length over which percentage identity may be measured. A “full length” nucleotide or polynucleotide sequence is one containing at least a translation initiation codon (e.g., methionine) followed by an open reading frame and a translation termination codon. A “full length” polynucleotide sequence encodes a “full length” polypeptide sequence. A “variant,” “mutant,” or “derivative” of a particular nucleic acid sequence may be defined as a nucleic acid sequence having at least 50% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the “BLAST 2 Sequences” tool available at the National Center for Biotechnology Information's website. (See Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2 sequences—a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett.174:247-250). In some embodiments a variant polynucleotide may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to a reference polynucleotide. A “genome” refers to a complete set of genes or genetic material present within a cell, tissue, or organism. A genome can be nuclear (found within the cell nucleus) or mitochondrial (found with the cell mitochondria). A “virus” is a microscopic infectious agent that replicates only inside the living cells of an organism. Viruses can infect all life forms, including mammalian and non-mammalian animals, plants, and other microorganisms. A complete virus, also known as a virion, consists of nucleic acid genetic material surrounded by a protective coat of protein called a capsid. Virus can have a lipid envelope derived from the infected host cell membrane. In general, there are five morphological virus types including helical, icosahedral, prolate, enveloped, and complex virus. A virus can either have a DNA or RNA genome, though a vast majority have RNA genomes. Irrespective of the type of nucleic acid genome, a viral genome can be either a single-stranded genome or a double-stranded genome. Docket No.103361-584WO1 As used herein, a “viral titer” refers to the lowest concentration of a virus including, but not limited to a whole virus or a partial virus, that maintains the ability to infect a host cell. A “capsid” or “viral capsid” refers to the protein shell or cage-like structure of a virus, enclosing the viral genome. The capsid can have various shapes, sizes, and protein subunits, depending on the type of virus. A “transgene” refers to a non-native gene or an artificial gene, manipulated by molecular biology techniques, that is incorporated into a vector (such as, for example a viral vector or plasmid) along with all the appropriate elements critical from gene expression. Generally, the transgene is originally derived from a different species relative to the vector species or a host species. Quality Control Assays and Methods of Use The present disclosure provides assays and methods of use thereof for assessing, detecting, and / or measuring the viral titer and genomic packaging of an adeno-associated virus (AAV) for treating and / or preventing diseases (including, but not limited to Duchenne muscular dystrophy (DMD)). In some aspects, disclosed herein is a method comprising a quality control assay for assessing, detecting, and / or measuring the viral titer and genomic packaging of an adeno- associated virus (AAV) for treating and / or preventing diseases (including, but not limited to Duchenne muscular dystrophy (DMD)). In some aspects, disclosed herein is a method comprising a quality assurance assay for assessing, detecting, and / or measuring the viral titer and genomic packaging of an adeno- associated virus (AAV) for treating and / or preventing diseases (including, but not limited to Duchenne muscular dystrophy (DMD)). As used herein, “quality control” refers to a system or method comprising a procedure or set of procedures intended to ensure that a manufactured sample adheres to a defined set of quality criteria that meets the standard requirements or functions of a control sample. As used herein, “quality assurance" refers to a systematic process of determining whether a sample meets specified requirements or functions. It should be noted that “quality assurance” can be used interchangeably with “quality control” processes. Another type of viral vector is based on an adeno-associated virus (AAV). This defective parvovirus is a preferred vector because it can infect many cell types and is nonpathogenic to humans. AAV type vectors can transport about 4 to 5 kb and wild type AAV is known to stably insert into chromosome 19. Vectors which contain this site specific integration property are Docket No.103361-584WO1 preferred. An especially preferred embodiment of this type of vector is the P4.1 C vector produced by Avigen, San Francisco, CA, which can contain the herpes simplex virus thymidine kinase gene, HSV-tk, and / or a marker gene, such as the gene encoding the green fluorescent protein, GFP. In another type of AAV virus, the AAV contains a pair of inverted terminal repeats (ITRs) which flank at least one cassette containing a promoter which directs cell-specific expression operably linked to a heterologous gene. Heterologous in this context refers to any nucleotide sequence or gene which is not native to the AAV or B19 parvovirus. Typically the AAV and B19 coding regions have been deleted, resulting in a safe, noncytotoxic vector. The AAV ITRs, or modifications thereof, confer infectivity and site-specific integration, but not cytotoxicity, and the promoter directs cell-specific expression. United states Patent No.6,261,834 is herein incorporated by reference for material related to the AAV vector. In some aspects, disclosed herein is a quality control (QC) or a quality assurance assay for detecting viral titer and genomic packaging of an adeno-associated virus (AAV), wherein the QC assay comprises at least one AAV capsid probe, an AAV gene probe, and a transgene probe, wherein the QC assay simultaneously detects a viral titer of the AAV and packaging of a target nucleic acid within the AAV. In some aspects, disclosed herein is a method of detecting viral titer and genomic packaging of an adeno-associated virus (AAV), the method comprising binding at least one AAV capsid probe to an AAV, wherein the at least one AAV capsid binds to a membrane protein of the AAV and comprises a first fluorescent molecule, binding an AAV gene probe to an AAV nucleic acid within the AAV, wherein the AAV gene probe comprises a second fluorescent molecule, binding a transgene probe to a target nucleic acid within the AAV, wherein the transgene probe comprises a third fluorescent probe, and detecting one, two, three, or more fluorescent signals from any preceding fluorescent molecule, wherein detecting the first fluorescent molecule indicates a viral titer, detecting the second fluorescent molecule indicates a viral genome, and detecting the third fluorescent molecule indicates packaging of the target nucleic acid. In some aspects, disclosed herein is a method of treating a subject with a disease or disorder, the method comprising performing a QC assay to assess viral titer and genomic packaging of an adeno-associated virus (AAV), wherein the QC assay comprises 1) binding at least one AAV capsid probe to an AAV, wherein the at least one AAV capsid binds to a membrane protein of the AAV and comprises a first fluorescent molecule, 2) binding an AAV gene probe to an AAV nucleic acid within the AAV, wherein the AAV gene probe comprises a second fluorescent molecule, 3) binding a transgene probe to a target nucleic acid within the AAV, wherein the transgene probe comprises a third fluorescent probe, and 4) detecting one, two, three, Docket No.103361-584WO1 or more fluorescent signals from any preceding fluorescent molecule, wherein detecting the first fluorescent molecule indicates a viral titer, detecting the second fluorescent molecule indicates a viral genome, and detecting the third fluorescent molecule indicates packaging of the target nucleic acid; and subsequently administering to the subject the AAV comprising the target nucleic, wherein target nucleic acid treats or prevents said disease or disorder. In some aspects, disclosed herein is a method of treating a subject with Duchenne muscular dystrophy (DMD), the method comprising performing a QC assay to assess viral titer and genomic packaging of an adeno-associated virus (AAV), wherein the QC assay comprises 1) binding at least one AAV capsid probe to an AAV, wherein the at least one AAV capsid binds to a membrane protein of the AAV and comprises a first fluorescent molecule, 2) binding an AAV gene probe to an AAV nucleic acid within the AAV, wherein the AAV gene probe comprises a second fluorescent molecule, 3) binding a transgene probe to a target nucleic acid within the AAV, wherein the transgene probe comprises a third fluorescent probe, and 4) detecting one, two, three, or more fluorescent signals from any preceding fluorescent molecule, wherein detecting the first fluorescent molecule indicates a viral titer, detecting the second fluorescent molecule indicates a viral genome, and detecting the third fluorescent molecule indicates packaging of the target nucleic acid; and subsequently administering to the subject the AAV comprising the target nucleic, wherein target nucleic acid treats or prevents DMD. In some embodiments, the at least one AAV capsid probe binds to a membrane protein of the AAV. In some embodiments, the at least one AAV capsid probe comprises an antibody, or a fragment thereof. In some embodiments, the at least one AAV capsid probe binds to a conformational epitope. In some embodiments, the at least one AAV capsid probe binds to a VP1, VP2, VP3 membrane protein, or a combination thereof. In some embodiments, the at least one AAV capsid probe comprises a first fluorescent molecule. In some embodiments, the first fluorescent molecule indicates the viral titer. In some embodiments, the method of any preceding aspect comprises one, two, three, or more AAV capsid probes. In some embodiments, the AAV gene probe binds an AAV nucleic acid. In some embodiments, the AAV nucleic acid is a native nucleic acid comprising a DNA or RNA. In some embodiments, the AAV gene probe comprises an antibody, a capsid penetrating nucleic acid probe (CPNP), or fragments thereof. In some embodiments, the AAV gene probe comprises a second fluorescent molecule. In some embodiments, the second fluorescent molecule indicates a viral genome. In some embodiments, the transgene probe binds to a target nucleic acid within the AAV. In some embodiments, the target nucleic acid comprises a DNA or RNA. In some embodiments, Docket No.103361-584WO1 the target nucleic acid comprises a non-native nucleic acid not derived from the AAV. In some embodiments, the transgene probe comprises an antibody, a capsid penetrating nucleic acid probe (CPNP), or fragments thereof. In some embodiments, the transgene probe comprises a third fluorescent molecule. In some embodiments, the third fluorescent molecule indicates the genomic packaging of the target nucleic acid. It should be noted that successful packaging of the target nucleic acid of any preceding aspect into the AAV of any preceding aspect is determined by an output signal of the first, second, and third fluorescent molecules. In some embodiments, the capsid penetrating nucleic acid probe (CPNP) of any preceding aspect includes, but is not limited to a molecular beacon. In some embodiments, the CPNP comprises a hairpin-like structure. In some embodiments, the hairpin-like structure comprises a single-strand loop, a double stranded stem, a fluorescent molecule, and / or a quencher. In some embodiments, the single stranded loop comprises 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, more, or less nucleotides. In some embodiments, the single-stranded loop is designed to recognize a specific sequence section on a target nucleic acid (such as, for example, a native or non-native nucleic acid within an AAV of any aspect disclosed herein). In some embodiments, the double-stranded stem comprises 5, 6, 7, 8, 9, 10, more, or less nucleotides. In some embodiments, the double-stranded stem is operably fused to fluorescent molecule and / or quencher of any preceding aspect. A non-limiting example of the structure of the double-stranded stem is double stranded stem with a fluorescent molecule at the 5’ end and a quencher at the 3’ end. In some embodiments, the first, second, third, or any fluorescent molecule of any preceding aspect includes, but is not limited to green fluorescent protein (GFP), yellow fluorescent protein (YFP), blue fluorescent protein (BFP), cyane fluorescent protein (CFP), monomeric red fluorescent protein (mRFP), Discosoma striata (DsRed), mCherry, mOrange, tdTomato, mSTrawberry, mPlum, photoactivatable GFP (PA-GFP), Venus, Kaede, monomeric kusabira orange (mKO), Dronpa, enhanced CFP (ECFP), Emerald, Cyan fluorescent protein for energy transfer (CyPet), super CFP (SCFP), Cerulean, photoswitchable CFP (PS-CFP2), photoactivatable RFP1 (PA-RFP1), photoactivatable mCherry (PA-mCherry), monomeric teal fluorescent protein (mTFP1), Eos fluorescent protein (EosFP), Dendra, TagBFP, TagRFP, enhanced YFP (EYFP), Topaz, Citrine, yellow fluorescent protein for energy transfer (YPet), super YFP (SYFP), enhanced GFP (EGFP), Superfolder GFP, T-Sapphire, Fucci, mKO2, mOrange2, mApple, Sirius, Azurite, EBFP, EBFP2, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 561, Alexa Fluor 568, Alexa Fluor 594, Alexa Docket No.103361-584WO1 Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, BODIPY FL, Courmarin, Cy3, TYE563, Cy5, TYE665, Cy5.5, TYE705, Fluorescein (FITC), Oregon Green, Pacific Blue, Pacific Green, Pacific Orange, PE-Cyanine7, PerCP-Cyanine5.5, Tetramethylrhodamine (TRITC), Texas Red, DAPI, Propidium Iodide, SYTO 9, SYTOX Green, TO-PRO-3, Allophycocyanin (APC), and R-Phycoerythrin (R-PE), 5' 6-FAM (Fluorescein), Int Fluorescein dT (iFluorT), Hexachlorofluorescein (HEX), JOE (6-carboxy-4',5'-dichloro-2',7'- dimethoxyfluorescein), MAX (NHS Ester), Tetrachlorofluorescein (TET), carboxy-X-rhodamine (ROX), Texas Red, TAMRA, TEX615, ATTO488, ATTO532, ATTO550, ATTO565, ATTORho101, ATTO590, ATTO633, ATTO647N, and any fluorophore-labeled nucleic acid thereof. In some embodiments, the AAV comprises a single-stranded AAV (ssAAV), a self- complementary AAV (scAAV), or a recombinant AAV (rAAV).In some embodiments, the AAV includes, but is not limited to AAV1, AAV2, AAV3, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh74, or other serotypes thereof. In some embodiments, the QC assay and / or the method is performed on a micropattern array, a microchip platform, a multi-well platform, or a combination thereof. In some embodiments, the QC assay or the method introduces the transgene into a subject’s genome for treatment or prevention of a disease or disorder including, but not limited to DMD. In some embodiments, the QC assay or the method of any preceding aspect comprises a high- throughput and multiparametric assay for assessing, detecting, and / or measuring the viral titer and genomic packaging of an adeno-associated virus (AAV). In some embodiments, QC assay or the method of any preceding aspect detects viral titer and genomic packaging of an adeno-associated virus (AAV) using fluorescent microscopy including, but not limited to total internal reflection fluorescence microscopy (TIRFM), confocal microscopy, fluorescence resonance energy transfer (FRET), immunofluorescence, super-resolution microscopy, flow cytometry, x-ray fluorescence microscopy in the SEM, Fluorescence-integrated transmission electron microscopy (TEM), TEM- Energy Dispersed X-ray and Electron Energy-Loss Spectroscopy (TEM-EDX-EELS), Scanning transmission electron microscopy-Energy Dispersed X-ray (STEM-EDX), Scanning electron microscopy-Energy Dispersed X-ray (SEM-EDX), and fluorescent-mass photometry. In some embodiments, the method comprises a biochip for detecting viral titer and genomic packaging of the AAV of any preceding aspect. In some embodiments, the method introduces the transgene into a subject’s genome for treatment or prevention of a disease or disorder of any disclosed aspect. Docket No.103361-584WO1 In some aspects, disclosed herein is a biochip for detecting viral titer and genomic packaging of an adeno-associated virus (AAV), wherein said biochip comprises the quality control (QC) assay of any preceding aspect. As used herein, a “biochip” refers to a manufactured surface engineered to comprises large numbers of simultaneous biochemical reactions. It should be noted that the QC assay and / or methods of any preceding aspect can be applied to the treatment and / or prevention of numerous diseases or disorders including, but not limited to genetic diseases, Duchenne muscular dystrophy (DMD), acoustic neuroma, adenocarcinoma, adrenal gland cancer, anal cancer, angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma), appendix cancer, benign monoclonal gammopathy, biliary cancer (e.g., cholangiocarcinoma), bladder cancer, breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, mammary cancer, medullary carcinoma of the breast), brain cancer (e.g., meningioma; glioma, e.g., astrocytoma, oligodendroglioma; medulloblastoma), bronchus cancer, carcinoid tumor, cervical cancer (e.g., cervical adenocarcinoma), choriocarcinoma, chordoma, craniopharyngioma, colorectal cancer (e.g., colon cancer, rectal cancer, colorectal adenocarcinoma), epithelial carcinoma, ependymoma, endotheliosarcoma (e.g., Kaposi's sarcoma, multiple idiopathic hemorrhagic sarcoma), endometrial cancer (e.g., uterine cancer, uterine sarcoma), esophageal cancer (e.g., adenocarcinoma of the esophagus, Barrett's adenocarinoma), Ewing's sarcoma, eye cancer (e.g., intraocular melanoma, retinoblastoma), familiar hypereosinophilia, gall bladder cancer, gastric cancer (e.g., stomach adenocarcinoma), gastrointestinal stromal tumor (GIST), head and neck cancer (e.g., head and neck squamous cell carcinoma, oral cancer (e.g., oral squamous cell carcinoma (OSCC), throat cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer)), hematopoietic cancers (e.g., leukemia such as acute lymphocytic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acute myelocytic leukemia (AML) (e.g., B-cell AML, T-cell AML), chronic myelocytic leukemia (CML) (e.g., B-cell CML, T-cell CML), and chronic lymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL); lymphoma such as Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) and non-Hodgkin lymphoma (NHL) (e.g., B-cell NHL such as diffuse large cell lymphoma (DLCL) (e.g., diffuse large B-cell lymphoma (DLBCL)), follicular lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphomas (e.g., mucosa- associated lymphoid tissue (MALT) lymphomas, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma), primary mediastinal B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma (i.e., “Waldenstrom's macroglobulinemia”), hairy cell leukemia (HCL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma and primary Docket No.103361-584WO1 central nervous system (CNS) lymphoma; and T-cell NHL such as precursor T-lymphoblastic lymphoma / leukemia, peripheral T-cell lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g., mycosis fungiodes, Sezary syndrome), angioimmunoblastic T-cell lymphoma, extranodal natural killer T-cell lymphoma, enteropathy type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, anaplastic large cell lymphoma); a mixture of one or more leukemia / lymphoma as described above; and multiple myeloma (MM)), heavy chain disease (e.g., alpha chain disease, gamma chain disease, mu chain disease), hemangioblastoma, inflammatory myofibroblastic tumors, immunocytic amyloidosis, kidney cancer (e.g., nephroblastoma a.k.a. Wilms' tumor, renal cell carcinoma), liver cancer (e.g., hepatocellular cancer (HCC), malignant hepatoma), lung cancer (e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung), leiomyosarcoma (LMS), mastocytosis (e.g., systemic mastocytosis), myelodysplastic syndrome (MDS), mesothelioma, myeloproliferative disorder (MPD) (e.g., polycythemia Vera (PV), essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a. myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myelocytic leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES)), neuroblastoma, neurofibroma (e.g., neurofibromatosis (NF) type 1 or type 2, schwannomatosis), neuroendocrine cancer (e.g., gastroenteropancreatic neuroendoctrine tumor (GEP-NET), carcinoid tumor), osteosarcoma, osteoporosis, bone fracture, RPE65-mutation-associated retinal dystrophy, Hemophilia A and Hemophilia B, ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma), papillary adenocarcinoma, pancreatic cancer (e.g., pancreatic adenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), Islet cell tumors), penile cancer (e.g., Paget's disease of the penis and scrotum), pinealoma, primitive neuroectodermal tumor (PNT), prostate cancer (e.g., prostate adenocarcinoma), rectal cancer, rhabdomyosarcoma, salivary gland cancer, skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC)), small bowel cancer (e.g., appendix cancer), soft tissue sarcoma (e.g., malignant fibrous histiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma), sebaceous gland carcinoma, sweat gland carcinoma, synovioma, testicular cancer (e.g., seminoma, testicular embryonal carcinoma), thyroid cancer (e.g., papillary carcinoma of the thyroid, papillary thyroid carcinoma (PTC), medullary thyroid cancer), urethral cancer, vaginal cancer, vulvar cancer (e.g., Paget's disease of the vulva), Alzheimer’s disease, ataxia, Huntington’s disease, Parkinson’s disease, amyotrophic lateral sclerosis (ALS), Friedreich ataxia, Lewy body disease, spinal muscular atrophy, Alpers’ disease, Batten disease, Cerebro-oculo-facio-skeletal syndrome, Leigh syndrome, Prion diseases, Docket No.103361-584WO1 monomelic amyotrophy, multiple system atrophy, striatonigral degeneration, motor neuron disease, multiple sclerosis (MS), Creutzfeldt-Jakob disease, Parkinsonism, spinocerebellar ataxia, dementia, common cold, influenza (including, but not limited to human, bovine, avian, porcine, and simian strains of influenza), measles, acquired immune deficiency syndrome / human immunodeficiency virus (AIDS / HIV), anthrax, botulism, cholera, campylobacter infections, chickenpox, chlamydia infections, cryptosporidosis, dengue fever, diphtheria, hemorrhagic fevers, Escherichia coli (E. coli) infections, ehrlichiosis, gonorrhea, hand-foot-mouth disease, hepatitis A, hepatitis B, hepatitis C, legionellosis, leprosy, leptospirosis, listeriosis, malaria, meningitis, meningococcal disease, mumps, pertussis, polio, pneumococcal disease, paralytic shellfish poisoning, rabies, rocky mountain spotted fever, rubella, salmonella, shigellosis, small pox, syphilis, tetanus, trichinosis (trichinellosis), tuberculosis (TB), typhoid fever, typhus, west nile virus, yellow fever, yersiniosis, zika, coronary artery disease, high / low blood pressure, cardiac arrest / heart failure, congestive heart failure, congenital heart defects / diseases (including, but not limited to atrial septal defects, atrioventricular septal defects, coarctation of the aorta, double- outlet right ventricle, d-transposition of the great arteries, Ebstein anomaly, hypoplastic left heart syndrome, and interrupted aortic arch), arrhythmia, peripheral artery disease, stroke, cerebrovascular disease, renal artery stenosis, aortic aneurysm, cardiomyopathies, hypertensive heart disease, pulmonary heart disease, cardiac dysrhythmias, endocarditis, inflammatory cardiomegaly, myocarditis, eosinophilic myocarditis, valvular heart diseases, rheumatic heart diseases, asthma, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, pneumonia, bronchitis (chronic or acute bronchitis), emphysema, cystic fibrosis / bronchiectasis, pleural effusion, acute chest syndrome, acute respiratory distress syndrome, asbestosis, aspergilosis, severe acute respiratory syndrome (including, but not limited to SARS-CoV-1 and SARS-CoV- 2), respiratory syncytial virus (RSV), middle eastern respiratory syndrome (MERS), mesothelioma, pneumothorax, pulmonary arterial hypertension, pulmonary hypertension, pulmonary embolism, sarcoidosis, sleep apnea, albinism, amniotic band syndrome, anencephaly, Angelman syndrome, Barth syndrome, chromosomal abnormalities (including, but not limited to abnormalities to chromosome 9, 10, 16, 18, 20, 21, 22, X chromosome, and Y chromosome), cleft lip / palate, club foot, congenital adrenal hyperplasia, congenital hyperinsulinism, congenital sucrase-isomaltase deficiency (CSID), cystic fibrosis, De Lange syndrome, fetal alcohol syndrome, first arch syndrome, gestational diabetes, Haemophilia, heterochromia, Jacobsen syndrome, Katz syndrome, Klinefelter syndrome, Kabuki syndrome, Kyphosis, Larsen syndrome, Laurence-Moon syndrome, macrocephaly, Marfan syndrome, microcephaly, Nager’s syndrome, neonatal jaundice, neurofibromatosis, Noonan syndrome, Pallister-Killian syndrome, Pierre Docket No.103361-584WO1 Robin syndrome, Poland syndrome, Prader-Willi syndrome, Rett syndrome, sickle cell disease, Smith-Lemli-Optiz syndrome, spina bifida, congenital syphilis, teratoma, Treacher Collins syndrome, Turner syndrome, Umbilical hernia, Usher syndrome, Waardenburg syndrome, Werner syndrome, Wolf-Hirschhorn syndrome, Wolff-Parkinson-White syndrome, heartburn, irritable bowel syndrome, lactose intolerance, gallstones, cholecystitis, cholangitis, anal fissure, hemorrhoids, proctitis, colon polyps, infective colitis, ulcerative colitis, ischemic colitis, Crohn’s disease, radiation colitis, celiac disease, diarrhea (chronic or acute), constipation (chronic or acute), diverticulosis, diverticulitis, acid reflux (gastroesophageal reflux (GER) or gastroesophageal reflux disease (GERD)), Hirschsprung disease, abdominal adhesions, achalasia, acute hepatic porphyria (AHP), anal fistulas, bowel incontinence, centrally mediated abdominal pain syndrome (CAPS), clostridioides difficile infection, cyclic vomiting syndrome (CVS), dyspepsia, eosinophilic gastroenteritis, globus, inflammatory bowel disease, malabsorption, scleroderma, volvulus, diabetes mellitus Type I, diabetes mellitus Type II, familial hypercholesterolemia, Gaucher disease, Hunter syndrome, Krabbe syndrome, metachromatic leukodystrophy, Niemann-Pick syndrome, phenylketonuria (PKU), Tay-Sachs disease, hemochromatosis, Fabconis anemia, beta-thalassemia, lipoprotein lipase deficiency,Wilson’s disease, hemachromatosis, mitochondrial disorders or diseases (including, but not limited to Alpers Disease; Barth syndrome; beta.-oxidation defects:carnitine-acyl-carnitine deficiency; carnitine deficiency; coenzyme Q10 deficiency; Complex I deficiency; Complex II deficiency; Complex III deficiency; Complex IV deficiency: Complex V deficiency; cytochrome c oxidase (COX) deficiency, LHON Leber Hereditary Optic Neuropathy; MM Mitochondrial Myopathy: LIMM Lethal Infantile Mitochondrial Myopathy; MMC Maternal Myopathy and Cardiomyopathy; NARP Neurogenic muscle weakness, Ataxia, and Retinitis Pigmentosa; Leigh Disease: FICP—Fatal Infantile Cardiomyopathy Plus, a MELAS-associated cardiomyopathy: MELAS Mitochondrial Encephalomyopathy with Lactic Acidosis and Strokelike episodes; LDYT Leber's hereditary optic neuropathy and Dystonia; MERRF Myoclonic Epilepsy and Ragged Red Muscle Fibers; MHCM Maternally inherited Hypertrophic CardioMyopathy; CPEO Chronic Progressive External Opthalmoplegia; KSS Kearns Sayre Syndrome; DM Diabetes Mellitus; DMDF Diabetes Mellitus+DeaFness; CIPO Chronic Intestinal Pseudoobstruction with myopathy and Opthalmoplegia; DEAF Maternally inherited DEAFness or aminoglycoside-induced DEAFness; PEM Progressive encephalopathy; SNHL SensoriNeural Hearing Loss; Encephalomyopathy; Mitochondrial cytopathy: Dilated Cardiomyopathy: GER Gastrointestinal Reflux: DEMCHO Dementia and Chorea; AMDF Ataxia, Myoclonus; Exercise Intolerance: ESOC Epilepsy, Strokes, Optic atrophy, & Cognitive decline; FBSN Familial Bilateral Striatal Docket No.103361-584WO1 Necrosis: FSGS Focal Segmental Glomerulosclerosis: LIMM Lethal Infantile Mitochondrial Myopathy; MDM Myopathy and Diabetes Mellitus: MEPR Myoclonic Epilepsy and Psychomotor Regression; MERME MERRF / MELAS overlap disease; MHCM Maternally Inherited Hypertrophic CardioMyopathy; MICM Maternally Inherited Cardiomyopathy; MILS Maternally Inherited Leigh Syndrome; Mitochondrial Encephalocardiomyopathy; Multisystem Mitochondrial Disorder (myopathy, encephalopathy, blindness, hearing loss, peripheral neuropathy); NAION Nonarteritic Anterior Ischemic Optic Neuropathy; NIDDM Non-Insulin Dependent Diabetes Mellitus; PEM Progressive Encephalopathy; PME Progressive Myoclonus Epilepsy; RTT Rett Syndrome: SIDS Sudden Infant Death Syndrome: MIDD Maternally Inherited Diabetes and Deafness; and MODY Maturity-Onset Diabetes of the Young, and MNGIE), alcoholic cardiomyopathy, systemic carnitine deficiency, malonyl carboxylase deficiency, malonic aciduria, carnitine-acylcarnitine translocase deficiency, carnitine palmitoyltransferase II deficiency, deficiencies to mitochondrial beta-oxidation (including, but not limited to medium- chain acyl-coenzyme A (coA) dehydrogenase (MCAD) deficiency, short-chain acyl-coA dehydrogenase (SCAD) deficiency, very-long-chain acyl-coA dehydrogenase (VLCAD) deficiency, and long-chain 3-hydroxyacyl-coA dehydrogenase (LCHAD) deficiency), deficiencies to the mitochondrial electron respiratory chain (including, but not limited to Kearns- Sayre syndrome, MELAS syndrome, MERRF syndrome, Barth syndrome, Leigh’s syndrome, Pearson syndrome, respiratory chain complex I deficiency, and Complex III deficiency), Glycogen storage disease type II (Pompe disease), Glycogen storage disease type III, Niemann- Pick disease, Gaucher disease, I-cell disease, mucopolysaccharidosis type I (Hurler syndrome), mucopolysaccharidosis type II (Hunter syndrome), mucopolysaccharidosis type III (Harris- Sanfilippo syndrome), mucopolysaccharidosis type IV (Morquio syndrome), mucopolysaccharidosis type VI (Maroteaux-Lamy syndrome), GM1 gangliosidosis, galactosialidosis, carbohydrate deficient glycoprotein syndromes, Sandhoff’s disease, congenital heart defects, and other related diseases. A number of embodiments of the disclosure have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims. By way of non-limiting illustration, examples of certain embodiments of the present disclosure are given below. Docket No.103361-584WO1 EXAMPLES The following examples are set forth below to illustrate the compositions, devices, methods, and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention which are apparent to one skilled in the art. Example 1: AAV Characterization Specific Aims. Adeno-Associated Virus (AAV) vectors have emerged as powerful tools for genetherapy, thanks to their non-pathogenic nature and efficient human infectivity. Therefore, AAV-based gene therapy can “cure” genetic diseases following a single administration. The success of this therapy hinges upon the critical requirement for precise and reliable quantification of AAV titers, which is essential to ensure the optimal dosage and efficacy of gene therapy treatments, maximizing their therapeutic potential. Standardized methods and quality control (QC) measurements used to quantify AAV titers and infectivity helped to improve the accuracy andreproducibility of AAV production. However, current methods do not simultaneously determineAAV titers with intact or damaged genomes – due to packaging of incomplete genomes or to cross- packaging – and quantify empty AAV capsids. This leads to misestimation of the titer, which could incur serious consequences leading to administering low AAV titers with sub-optimal therapeutic efficacy or high AAV titers with the potential of causing severe adverse events or death to thetreated patient. At present, standardized methods used for AAV titer quantification includequantitative polymerase chain reaction (qPCR), droplet digital PCR (ddPCR), and enzyme-linked immunosorbent assay (ELISA). qPCR and ddPCR are designed to measure the single- stranded or self-complementary DNA genome of AAV (genome titer). Still, they cannot simultaneously measure the titer of intact and cross-packaged genomes and AAV empty capsids, which can be found in any AAV preparation, and their proportion depends on the size and complexity of theAAV genome and the purification method. This critical limitation could lead to adverse events(i.e., toxicity) and reduced therapeutic efficacy. Moreover, methods to measure the capsid titer (e.g., ELISA) cannot distinguish between empty and full AAV capsids (AAV packaging a genome), making them unreliable in determining an accurate titer of an efficacious and safe to administer AAV. Therefore, there is an unmet need for developing analytical methods that provide an efficient, cost-effective, and easy GMP implementation for multiparametric quantification of produced AAVs (e.g., capsid and genomic titer and AAV integrity). Herein, methods have been developed that are capable of capturing bioparticles, including Docket No.103361-584WO1 extracellular vesicles (EVs) and viruses from biofluids and then simultaneously quantifying their surface proteins and intraluminal molecular cargo in situ at the single particle level. Given the resemblance in size and molecular characteristics between EVs and viruses, the technology can be pivoted for quantitative analysis of AAVs. The applicability has been demonstrated to detect capsid (VP1-3, conformational epitope) proteins and genomic DNA in intact single AAV particles. In this application, a high-throughput platform for multiparametric detection and characterization of genomic DNA packed in single AAVs is developed, enabling the simultaneous quantification of genomic titer integrity and loading efficiency per capsid. Specifically, the present disclosure focuses on characterizing titers of AAV gene replacement (AAVrh74.microdystrophin) and exon- skipping (AAV9.U7.snRNA) therapy for Duchenne Muscular Dystrophy (DMD), which is the most common form of muscular dystrophy affecting 1 in every 3,500 boys and caused by mutations in the DMD gene. Development of a micropattern array and multiwell platform for high-throughput in situ single AAV analysis, theAAVBiochip. An integrated multiwell platform is designed to characterize capsid proteins and genomic DNA in single AAVs in situ. Micropattern arrays are cleaved from a polymer-coated glass surface by a UV photopatterning device. Each micropattern is positively charged to direct the capture of AAVs by electrostatic interactions. The micropatterns are arranged in separate multiwells for high-throughput and multiparametric testing conditions. DNA-targeting capsid penetrating nucleic acid probe (DNACPNPs) are hybridized to the genomic DNA from single AAVs and generate fluorescent signals detected by total internal reflection fluorescence microscopy (TIRFM). Fluorescence-labeled antibodies are also used to quantify capsid (VP1, VP2, and / or VP3) proteins. DNA / Protein and DNA / DNA colocalization fluorescent signals within single AAVs are quantified for genomic titer integrity and loading efficiency. Theinitial feasibility demonstration forAAVBiochip is conducted using well-characterized AAV9titers for which the capsid proteins, conformational epitope (3D structure) of assembled capsids, and the inverted terminal repeats (ITRs) sequences of the genomic DNA are targeted for the AAVs captured on the platform. Standard methods to quantify AAV titers are used for comparison. TheAAVBiochip is a QC method for AAV titers for DMD therapy. Different AAV serotypes (i.e., AAVrh74 and AAV9) are tested for gene replacement and exon-skipping therapy for DMD. ITRs, promoters, genome of interest (GOIs), and polyA sequences of the genomic DNA are targeted usingDNACPNP. Colocalization analysis simultaneously quantifies the integrity and cross-packaging of the DNAs in the capsids and loading efficiency at the single AAV particle level. This technology's in situ nature enables for comparison of critical metrics for each AAV titer, removing the limitations of the current standard methods. Docket No.103361-584WO1 The present disclosure ultimately enables an ultrasensitive single AAV characterization technology to be used as a QC tool for gene therapy in DMD, which is translatable and non- limiting to other AAV-based gene therapies. AAVs comprise a viral protein capsid that packages a therapeutic DNA payload. AAVDNA packaging is limited to 4.7 kb, and AAV DNA payload is used to encode gene editing andgene silencing machinery and to package genes replacing the defective ones. Upon reaching thecell, AAV is internalized and trafficked to the nucleus, where it sheds its capsid and delivers theDNA payload to express the encoded therapeutic product. The safety and efficacy of thetherapeutic outcome of any encoded gene is based on the titration accuracy of functional full AAV capsids and on the use of AAV preparations that have the very minimal presence of empty andpartial capsids, and capsids with host cell DNA fragments. Full AAV capsids contain the entiresequence of the AAV genome that encodes for a functional therapeutic product. The empty capsidhas no DNA payload, and the partial capsid may contain pieces of the complete AAV genome.These capsid forms do not contribute to the AAV therapeutic potency. They may increase AAV immunogenicity by increasing the titer of AAV capsid proteins and delivering immunogenic DNA fragments, reducing AAV therapeutic efficacy and potency and triggering adverse events likecomplement activation, thrombocytopenia, and liver toxicity. Cross-packaged ITRs with DNAfragments may also express toxic RNA and peptide sequences. Thus, although AAV remains theleading vector system for tissue transduction, safety, and immunogenicity are now primaryconcerns. Therefore, in an AAV-based gene therapy approach for any disease, ensuring theoptimal dosage and efficacy of a safe and fully functional gene therapy treatment is of utmost importance. Available methods for AAV characterization, including ELISA, UV-Vis, and ddPCR, donot simultaneously determine empty versus full AAV capsids, and AAV titers with intact, incomplete, or cross-packaged DNA that are important metrics to determine the optimal dosageand efficacy of gene therapy. Moreover, charged-based methods are relatively easy to use andimplement in a good manufacturing practice (GMP) environment but have poor analytical strength and ability to accurately quantify the concentration of functional (infectious) AAV particles. Mass size-based methods do not have high analytical strength, except SV-AUC, which separates full,partial, and empty capsids based on their Svedberg coefficient, making it a standard method formany AAV programs. However, these methods are not easy to use and implement in a GMPenvironment, like in imaging techniques such as cryo-TEM and mass photometry. The latter alsohas poor analytical strength (Table 1). All these limitations require combining the results of different techniques to get a more comprehensive evaluation of the quality of the AAV preparation. Docket No.103361-584WO1 However, the chances of compounding different sources of error increase, leading to high assay variability and inaccuracy. Therefore, there is an unmet need for developing analytical methods that provide an efficient, cost-effective, and easy GMP implementation for multiparametric quantification of produced AAVs (e.g., capsid and genomic titer and AAV integrity). Herein, the present disclosure develops and validates theAAVBiochip as a comprehensive, accurate, and reliable QC method to quantify capsid and genomic titers, providing a precise ratio of empty / full AAV capsids in one assay. TheAAVBiochip is an essential analytical tool for determining titers and empty vs. partial / full ratios of AAV preparations by direct measurements from single AAVs, thus enabling a better AAV production process for optimal dosage and efficacy. TheAAVBiochip consists of micropattern arrays in multiwells for high-throughput simultaneous capsid proteins and genomic DNA measurements at the single AAV level. The micropattern arrays are cleaved from a polyethylene glycol (PEG)-coated glass surface by a photoinitiator using a UV photopatterning PRIMO module. The non-cleaved PEG antifouling coatingprevents the binding of AAVs outside the micropatterns. The micropatterns are coated with apositively charged polyelectrolyte (e.g., poly- L-lysine) that allow capturing of the negativelycharged regions of the AAVs by electrostatic interactions. DNA-targeting capsid penetratingDNA probes (DNACPNPs) generates fluorescent signals from the genomic DNA on the immobilized single AAVs and be detected by total internal reflection fluorescence microscopy (TIRFM). Fluorescence-labeled antibodies are also tested to quantify capsid protein contents. Protein / DNA and DNA / DNA colocalization fluorescence signal analysis within single AAVs are optimized to determine empty vs. partial / full capsid and genomic titer ratios. The micropattern arrays are designed in a multiwell format compatible with a commercially available liquid- handling robot for high-throughput testing conditions. Herein, it is demonstrated the feasibility of the technology by using well-characterized AAV9 titers for which the capsid proteins, conformational epitope (3D structure) of assembled capsids, and ITR genomic regions are targeted. Statistical analysis of TIRFM images provides the concentration distribution of specific DNA and capsid proteins of individual AAVs captured in each micropattern of the array. The measurements are compared with conventional methods for quantifying capsid and genomic titers. Two AAV serotypes (i.e., AAV9 and AAVrh74) used for gene replacement and exon-skipping therapy forDMD are characterized with theAAVBiochip. ITRs, promoters, genome of interest (GOIs), andpolyA sequences of the genomic DNA are targeted usingDNACPNPs. Colocalization analysis simultaneously quantifies the integrity and cross-packaging of the DNAs in the capsids and loading efficiency at the single AAV particle level. Docket No.103361-584WO1 The significance of the present disclosure is two-fold: (1) A unique, innovative technology that enables a comprehensive analytical quantification of AAV titers overcoming the limitations of the current standard characterization methods. Thus, simultaneous quantification of capsid and genomic titers, with subsequent determination of empty / partial / full capsids at the single AAV level for DMD will be achieved. (2) The multi- disciplinary team with complementary expertise in nanotechnology, microfluidics and extracellular vesicles and virus detection technology development (Eduardo Reátegui, Ph.D., OSU); gene therapy and neuromuscular diseases (Nizar Y. Saad, Ph.D., NCH); and biostatistical analysis (Biostatistical Core, OSU) that will enable the development of an ultrasensitive AAV characterization technology to be used as a QC tool for gene therapy in DMD. In situ detection of proteins and RNAs in single Extracellular Vesicles (EVs). A single EV analysis technologies have been previously developed that is capable of multiplexing proteinand RNA biomarker detection. Figure 3A shows the concept of the approach. Antibodies tetheredon the device surface capture single EVs based on their membrane proteins. Then, specific fluorescently labeled antibodies and nucleic acid probes (e. g., molecular beacons (MBs)) are added to target and detect specific proteins and RNAs. One of the types of nucleic acid probes such as MBs have a hairpin-like structure, where the single-strand loop (~20 nts) is designed to recognize a specific sequence section on a nucleic acid target like a PCR primer. At the same time, the double-strand stem (6~8 base pairs) is tagged to a fluorescence dye molecule and a quencher molecule at the two ends of the stem, respectively. TIRFM provides an evanescent wavefront that exponentially decreases from the device surface, allowing the visualization of biomolecules in single EVs. Moreover, a micropattern-based design on the surface allows for a facile EVmultiplexed analysis. Figure 3B shows the detection of CD63 protein and microRNA hsa-miR-21-5p. Each green signal represents a single EV expressing CD63, while each red signal represents a single EV carrying hsa-miR-21-5p. Each yellow signal thus demonstrated the colocalization of both biomarkers in single EVs. TIRFM images could be quantified as distributions of fluorescence intensity of the single EVs to analyze the heterogenous expression of biomarkers (Figure 3C). Moreover, the technology was demonstrated to be ~100 fold more sensitive than thegold-standard qRT-PCR and ELISA methods detecting cancer biomarkers.Simultaneous detection of viral proteins and RNAs in single virus particles. Given theresemblance in size and molecular content between EVs and viruses, this technology is used toquantify viral proteins and nucleic acids from SARS-CoV-2. Viruses were captured on the surface of our platform using ACE2 proteins. Then, fluorescently labeled antibodies and nucleic acid probes (e. g., molecular beacons (MBs)) were delivered to target spike glycoproteins and viral Docket No.103361-584WO1 RNAs in single virions (Figure 4A). TIRFM was utilized to visualize the fluorescent signals from the immobilized single virions that can be quantified as distributions of fluorescent emissions (Figure 4B and 4C). Colocalized signals were observed and investigated multi- dimensionally. Figure 4D shows the co-targeting of the spike glycoprotein, nucleocapsid-protein- encoding RNA,and the S-L452R mutation (Delta variant).TheAAVBiochip enables an unparallel level of characterization of AAV vectorpreparations that will ensure optimal dosage and efficacy of a safe and fully functional gene therapy treatment with the following innovations: • Combining capsid protein and genomic DNA measurements allows for quantify capsid and genomic titers, determine empty vs. partial / full AAV capsids, measure GOIs, verify genome integrity, and evaluate DNA cross- packaging. Currently, no single method is available to provide such a comprehensive characterization of AAV preparations. Thus, different analytical tools have to be used (Table 1). • TheAAVBiochip provides high analytical strength - through the use of specific antibodies against the AAV protein capsid and specificDNACPNPs targeting different regions of the AAV genomic DNA and transfer plasmid backbone (Figure 13) - and be easy to use and implement in a GMP environment. • AAVs immobilized on micropattern arrays by electrostatic interactions instead of capture antibodies for rapid TIRFM imaging acquisition and analysis at the single particle level. • The data herein show the feasibility of the approach. AAV9 vectors were prepared using a standard method. The concentration of AAV9s was measured at 1.5*109(DNase-resistantparticles [DRP] / mL) and concentrated at 0.5*1010[DRP] / mL using an MWCO 3kDa spincolumn for subsequent immobilizing by electrostatic interactions on the surface of the platform. The present disclosure aimed to target capsid proteins and genomic DNA content for the AAV9s. Figure 5A shows the dual detection of the capsid (VP1-3) protein and DNA by TIRFM using fluorescently labeled antibodies and Ethidium homodimer-1 (EthD-1) for DNA. Images were quantified as distributions of fluorescence intensity to analyze different expression levels (Figure 5B). Colocalization analysis of fluorescent signals showed that ~84% of AAV capsids contain genomic DNA (Figure 5C). Moreover, it is remarkable that the approach can distinguish characteristic 2D projections of the icosahedral 3D structure of AAV9s. It was also possible to distinguish the presence of genomic DNA. Single AAV9s have different amounts of DNA (Figure 5D), showing the heterogeneity of DNA packaging. The TIRFM analysis was also validated using TEM Docket No.103361-584WO1 (Figure 5E). Developing a micropattern array and multiwell platform for high-throughput in situ single AAV analysis, theAAVBiochip. In situ analysis of AAVs enables a comprehensive, accurate, and reliable QC method to quantify capsid and genomic titers simultaneously. The platform us an essential analytical tool for determining empty vs. partial / full ratios of AAV preparations by direct measurements from single AAV particles, enabling a better production process for optimal AAV dosage and efficacy. Development and initial evaluation of the technology optimizes using AAV9 titers. ELISA, ddPCR, UV-Vis, SV-AUC, and cryo-TEM analysis are used for orthogonal comparisons. Production of AAVs from HEK293 cell lines. AAV9 and AAVrh74 vectors are produced in HEK293 cells using the calcium phosphate method. Briefly, on days 1 and 2 after co-transfection of pre-plated HEK293 cells with the adenovirus helper plasmid, rep / cap plasmid, and ITR-flanked AAV transgene expression cassette, the spent culture medium are replaced with fresh serum-free culture medium. HEK293 cells are lysed for AAVs, and AAVs are purified using the iodixanolisopycnic density gradient and the FPLC anion exchange purification method. Fixation andpermeabilization methods are used for the AAV particles. Briefly, different concentrations of a formaldehyde solution in PBS are tested for different incubation times at room temperature. Fixed AAVs are washed with PBS using a small cut-off centrifugal filter. A permeabilization step of the AAVs is conducted by Tris EDTA (TE) buffer. Previously, a similar fixation and permeabilization approach is used to deliver nucleic acid probes inside EVs and virus particles that hybridized insitu with the viral RNA and produced fluorescent signals (Figure 3 and 4).Design, assembly, and testing of theAAVBiochip. Micropattern arrays are assembled on a glass coverslip with a polymeric coating and photoetched with UV light. The platform has an antifouling capability. The coating consists of poly-L-lysine (PLL) physiosorbed on the glass surface and the covalent attachment of methoxy-poly(ethylene glycol)-succinimidyl valerate (mPEG-SVA). Different concentrations, sizes, and ratios of mPEG-SVA / PLL are tested to determine optimal antifouling parameters for preventing AAV binding in unpatterned regions. Photoetching of the micropatterns will be achieved using a PRIMO module via UV patterning projections translated by a digital-micromirror device in the presence of 4- benzoyl benzyl-trimethylammonium chloride (PLPP) as a photoactivator that cleaves the PEG monolayer (Figure 13). The UV light dose is computer controlled to determine the percentage of PEG removal that is verified with fluorescence microscopy. The light-controlled photoetching will expose the PLL on the micropatterns, enabling electrostatic interactions with the negatively charged regions of the AAVs surface.10x1030 µm Docket No.103361-584WO1 diameter micropatterns per well with 48 wells per device is to be achieved for high-throughput testing conditions (Figure 13). The micropattern size is chosen to maximize the field of view (FOV) of a single TIRFM image that covers 120 µm x 120 µm of the device surface. However, the micropattern size can be adjusted. Different antibodies quantify capsid (VP1-3) proteins andassembled capsids' conformational epitope (3D structure). Moreover,DNACPNPs in buffertargeting ITR genomic regions will be added to detect and measure the concentration of DNA (Figure 13). As a proof of concept, we have characterized capsid proteins and DNA content in AAV9s at the single particle level (Figure 5). A GMP protocol using a liquid handling robot will be implemented to minimize variability, reagent waste, and cross-contamination for the different assay steps, and a computer code are developed in Matlab to analyze TIRFM imaging data sets. Characterization of the integrity of captured AAVs with theAAVBiochip. Standardizedempty and full AAV9 capsids are used from amsbio as calibration materials. AAV9 preparationsat different concentrations (as described above) are used to test the effects of fixation and permeabilization in the capture efficiency of virus particles on the micropatterns of the device. Virus particles are tested for the presence of capsid (VP1-3) proteins and the integrity of conformational epitopes (3D) using fluorescent antibodies and measured by TIRFM (Table 2). The capsid titer is determined by quantifying fluorescent signals in situ from single AAVs captured on the surface of the device. Calibration curves are generated using the standardized AAV9 materials at known concentrations and for which their protein fluorescence signals are obtained by the method and compared to the measurements of theAAV9 preparations to determine concentrations. Moreover, capsid integrity is determined by quantifying the percentage of colocalized signals between (VP1-3) and conformational epitope (3D) proteins. Cryo-TEM us used to verify the integrity and size distribution of the treated virus particles. Nonfixed and nonpermeabilized empty and full AAV9 capsids will also be tested and used for comparison. Using theAAVBiochip to measure empty, partial, and full AAV9 capsids. Colocalization analysis of fluorescent signals determines the genomic titer integrity and AAV loading efficiency without lysing the virus particles.DNACPNPs targeting ITR and GOI regions are designed and added to the fixed and permeabilized AAV9 preparations to detect their genomic DNA signals by TIRFM. Different fluorescent reporters will be appropriately selected and conjugated to theDNACPNPs (Table 2). The genomic titer (e.g., ITR, GOI) is determined by quantifying DNA fluorescent signals from single AAVs captured on the surface of the device. Calibration curves are generated using the standardized full AAV9 material at known genomic titers, and for which their fluorescence signals are obtained by the method and compared to the measurements of our AAV9 preparations to determine their genomic titers. Different ITR or GOI Docket No.103361-584WO1 regions are targeted simultaneously to quantify the genomic titer integrity of the AAV9 preparations. Moreover, empty vs. partial / full capsid ratios are determined using appropriate combinations of antibodies andDNACPNPs probes that target capsid proteins and genomic DNA. Previously, the present disclosure has demonstrated the targeting and measuring of fluorescentsignals from surface proteins and nucleic acids in single EVs and virus particles (Figure 3 and 4).Thus, different concentrations of empty AAV9 capsids (amsbio) are spiked with AAV9 preparation (0.5:1, 1:1, and 1:2) to quantify capsid proteins and genomic DNA by fluorescence analysis and compared to the known spiked concentration values. Genome cross-packaging is a phenomenon where AAV capsids can package heterologous or non-native AAV genome sequences. In single AAV preparations, sequences originating from bacteria, host cells, and helper or transfer plasmids (bearing GOI flanked with two ITRs) can be cross-packaged into AAV capsids. Genome cross-packaging is titered using specificDNACPNPs (i.e.,BckbCPNPs probes to target ITR contiguous plasmid backbone sequences) with a similar experimental analysis (Table 2). Both averaged and distributed capsid protein and genomic DNA expression on each micropattern are analyzed and reported. The analysis is repeated by comparing single-stranded vs self-complementary AAV9 vectors. Comparison of the accuracy ofAAVBiochip metrics with other methods. Standard methods, including ELISA, ddPCR, UV-Vis, SV-AUC, and cryo-TEM, are used to obtain capsidand genomic titers of the AAV9 preparations and controls. TheAAVBiochip Analysis isevaluated on whether it can quantify the different titers more accurately than the conventional approaches. Criteria for success is a quantitative analysis of capsid and genomic titers in a singleassay. TheAAVBiochip provides >95% reproducibility. Achieving >95% correlation betweenresults obtained with theAAVBiochip and other methods.TheAAVBiochip is a QC method for AAV titers for Duchenne muscular dystrophy (DMD) therapy. X-linked recessive DMD is the most prevalent type of muscular dystrophy,affecting approximately 1 in every 3,500 boys. The underlying cause of DMD lies in loss-of-function mutations in the DMD gene encoding dystrophin protein. These mutations hinder theproduction of a fully functional dystrophin protein that plays a crucial role in stabilizing the membranes of myofibers during muscle contractions. Becker muscular dystrophy (BMD) is a milder form of DMD with specific in-frame exon deletions in the DMD gene, allowing the expression of partially functional dystrophin. This has inspired various treatment approaches, including therapies that involve exon-deleted DMD genes - such as microdystrophin - or methods Docket No.103361-584WO1 that skip internal exons, resulting in the expression of functional dystrophin protein isoforms. For best muscle transduction and therapeutic efficacy, these AAV-based gene therapies are administered at very high doses, which could raise the possibility of developing adverse events for every patient receiving these therapies, despite the stringent patient selection process. The latter disqualifies patients with severe symptoms, certain medical conditions or contraindications, and patients with pre-existing immunity against AAV. However, current AAV QC methods fail to provide all the characterization parameters needed to ensure the optimal dosage and efficacy of the AAV gene therapy, increasing concerns about the possibility of developing unpredicted adverse events associated with AAV quality. The platform disclosed herein enables for profiling capsid and genomic titers with higher sensitivity than current standard methods, allowing for high clinical utility and adoptability. TheAAVBiochip is tested using AAVs designed to deliver gene replacement therapy or surrogate gene therapies for DMD. AAV preparations for DMD. AAV preparations are sued for (i) gene replacement therapy (AAV.microdystrophin) and (ii) exon-skipping gene therapy (U7.snRNA). i) AAVrh74.MHCK7.microdystrophin gene replacement therapy was recently approved by the FDA for treating ambulatory pediatric patients aged 4 through 5 years with DMD. Due to the large size of the DMD open reading frame (11.5 kb), it is impossible to package it into an AAV vector, with a size limit of 4.7 kb for vector genome packaging. As a result, the current microdystrophin therapyis based on previous research that identified a partially functional dystrophin protein. ii)scAAV9.U7snRNA-based exon skipping demonstrated preclinical efficacy in the Dup2 mousemodel. This approach targets exon skipping using a small hairpin RNA (U7snRNA) carried by aself-complementary AAV9 vector. It has now entered clinical trials. Both AAVs are preparedusing the same method described above. Measurement of capsid and genomic titers for scAAV9.U7snRNA and AAVrh74.MHCK7.microdystrophin using theAAVBiochip. A systematic AAV titer analysis study is conducted with the disclosed platform.DNACPNPs targeting different ITR and GOI regions are combined with fluorescent antibodies for capsid (VP1-3) proteins and detected by TIRFM imaging. Different ITR or GOI regions will be targeted to confirm the genomic titer integrity of the AAV preparations. Colocalization analysis of capsid proteins with DNA from ITR or GOI regions determine empty / partial / full capsid ratios for the different AAV preparations. Additionally, this unique in situ approach allows for target other genome regions of interest, including the promoter and polyA enabling an unparallel level of characterization metrics of AAV preparations in a single assay. Test the sensitivity ofAAVBiochip to check for cross- packaging. TheAAVBiochip Docket No.103361-584WO1 sensitivity is tested to detect cross-packaging of AAV preparations for DMD. Cross-packaging of AAV preparations is measured and obtained from the co-transfection into HEK293 cells of equimolar amounts of scAAV9.U7snRNA or AAVrh74.MHCK7. Microdystrophin and an ITR- flanked GFP-luciferase plasmid at 10-fold dilutions (2.6 µg, 260 ng, 26 ng, 2.6 ng, and 260 pg) alongside equal quantities of adenovirus helper and rep / cap plasmids. The exact amount of DNA is maintained using an ITR-free plasmid. To measure background, mock AAV preparations are obtained by co-transfecting ITR-free plasmids with the same dilutions. The titers are then measured with theAAVBiochip using CPNPs targeting the U7snRNA, microdystrophin, and GFP and compare them to those obtained by ddPCR. Criteria for success is a quantitative analysis of capsid and genomic titers in a single assay with >95% reproducibility for the DMD AAV preparations. Achieving >95% correlation betweenresults obtained with theAAVBiochip and other methods. Cross-packaging of GFP decreasesupon diluting transfected plasmids. TheAAVBiochip is more sensitive than PCR in detectingcross- packaging at high dilutions. The design includes: (i) validated human cell lines, (ii) validated molecular probes and antibodies, (iii) high-quality reagents; (iv) negative controls in all experimental groups, (v) AAV vectors, (vi) steps to ensure AAV quality, integrity, identity, and purity. The reproducibility ofAAVBiochip is measured in triplicates with three technical replicates within a single experiment. One-way analysis of variance (ANOVA) is used to estimate the intraclass correlation coefficient (ICC) as a measure of test-retest reliability. Bootstrapping are used to get a 95% confidence interval of estimated ICC. The method herein is compared with each standard approach according to the mean square error (MSE). For each experiment, the one with a smaller MSE wins. Holm’s step- down procedure is used for multiple groups comparison to control the family-wise error rate at level 0.05. Measurements from different approaches are visualized using the Bland–Altman plot. The data herein is modeled using a linear mixed model and estimate ICC as the correlation between measurements from two methods. Data is analyzed using SAS and JMP (SAS, Inc; Cary, NC). It will be apparent to those skilled in the art that various modifications and variations can be made in the present disclosure without departing from the scope or spirit of the invention. Other embodiments of the disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the methods disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims. Docket No.103361-584WO1 TABLES Table 1. Comparison of AAV characterization and titration methods.adigital droplet PCR,bEnzyme-Linked Immunosorbent Assay,cUV-Vis spectroscopy,dSize Exclusion Chromatography-Multi Angle Light Scattering,eAnion Exchange Chromatography,fCapillary Isoelectric Focusing,gSedimentation velocity- Analytical Ultracentrifugation,hCharge Detection Mass Spectrometry,iAtomic Force Microscopy, cryo-TEM, Mass photometry,AAVBiochip.+Not well adapted,++partially adapted,+++very well adapted. Docket No.103361-584WO1 Table 2. List of antibodies and CNPs that will be used with theAAVBiochip. *Detects AAV conformational 3D structure (ADK9). † is specific for AAVrh74 (ADK8). ITR: Inverted Terminal Repeats; Prom: Promoter; PolyA: polyA signal sequence; Bckb: backbone. Table 3. Comparison ofAAVBiochip specifications with leading market products. Docket No.103361-584WO1 Table 4. Examples of CPNP and detection antibodies for AAVBiochip tests with FDA-approved AAV-based therapies.

Claims

Docket No.103361-584WO1 CLAIMS What is claimed is:

1. A quality control (QC) assay for detecting viral titer and genomic packaging of an adeno- associated virus (AAV), wherein the QC assay comprises at least one AAV capsid probe, an AAV gene probe, and a transgene probe, wherein the QC assay simultaneously detects a viral titer of the AAV and packaging of a target nucleic acid within the AAV.

2. The (QC) assay of claim 1, wherein the at least one AAV capsid probe binds to a membrane protein of the AAV.

3. The QC assay of claim 1 or 2, wherein the at least one AAV capsid probe comprises an antibody, or a fragment thereof.

4. The QC assay of any one of claims 1-3, wherein the at least one AAV capsid probe binds to a conformational epitope.

5. The QC assay of any one of claim 1-4, wherein the at least one AAV capsid probe binds to a VP1, VP2, VP3 membrane protein, or a combination thereof.

6. The QC assay of any one of claims 1-5, wherein the at least one AAV capsid probe comprises a first fluorescent molecule.

7. The QC assay of any one of claims 1-6, wherein the AAV gene probe binds an AAV nucleic acid.

8. The QC assay of claim 7, wherein the AAV nucleic acid comprises a DNA or RNA.

9. The QC assay of any one of claims 1-8, wherein the AAV gene probe comprises an antibody, a capsid penetrating nucleic acid probe (CPNP), or fragments thereof.

10. The QC assay of any one of claims 1-9, wherein the AAV gene probe comprises a second fluorescent molecule.Docket No.103361-584WO1 11. The QC assay of any one of claims 1-10, wherein the transgene probe binds to a target nucleic acid within the AAV.

12. The QC assay of claim 11, wherein the target nucleic acid comprises a DNA or RNA.

13. The QC assay of claim 11 or 12, wherein the target nucleic acid comprises a non-native nucleic acid not derived from the AAV.

14. The QC assay of any one of claims 1-13, wherein the transgene probe comprises an antibody, a capsid penetrating nucleic acid probe (CPNP), or fragments thereof.

15. The QC assay of any one of claims 1-14, wherein the transgene probe comprises a third fluorescent molecule.

16. The QC assay of any one of claims 1-15, wherein the AAV comprises a single-stranded AAV (ssAAV), a self-complementary AAV (scAAV), or a recombinant AAV (rAAV).

17. The QC assay of any one of claims 1-16, wherein the AAV comprises AAV1, AAV2, AAV3, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh74, or other serotypes thereof.

18. The QC assay of any one of claims 1-17, wherein the QC assay is performed on a micropattern array, a microchip platform, a multi-well platform, or a combination thereof.

19. The QC assay of any one of claims 1-18, wherein a signal from the first fluorescent molecule indicates the viral titer.

20. The QC assay of claim 19, wherein a signal from the second fluorescent molecule indicates a viral genome.

21. The QC assay of any one of claims 1-20, wherein a signal from the third fluorescent molecule indicates packaging of the target nucleic acid.Docket No.103361-584WO1 22. A biochip for detecting viral titer and genomic packaging of an adeno-associated virus (AAV), wherein said biochip comprises the quality control (QC) assay of any one of claims 1-21.

23. A method of detecting viral titer and genomic packaging of an adeno-associated virus (AAV), the method comprising: a. binding at least one AAV capsid probe to an AAV, wherein the at least one AAV capsid binds to a membrane protein of the AAV and comprises a first fluorescent molecule, b. binding an AAV gene probe to an AAV nucleic acid within the AAV, wherein the AAV gene probe comprises a second fluorescent molecule, c. binding a transgene probe to a target nucleic acid within the AAV, wherein the transgene probe comprises a third fluorescent probe, and d. detecting one, two, three, or more fluorescent signals from the fluorescent molecules of steps a) - c), wherein detecting the first fluorescent molecule indicates a viral titer, detecting the second fluorescent molecule indicates a viral genome, and detecting the third fluorescent molecule indicates packaging of the target nucleic acid.

24. A method of treating a subject with Duchenne muscular dystrophy (DMD), the method comprising: a. performing a QC assay to assess viral titer and genomic packaging of an adeno- associated virus (AAV), wherein the QC assay comprises: i.binding at least one AAV capsid probe to an AAV, wherein the at least one AAV capsid binds to a membrane protein of the AAV and comprises a first fluorescent molecule, ii.binding an AAV gene probe to an AAV nucleic acid within the AAV, wherein the AAV gene probe comprises a second fluorescent molecule, iii.binding a transgene probe to a target nucleic acid within the AAV, wherein the transgene probe comprises a third fluorescent probe, and iv.detecting one, two, three, or more fluorescent signals from the fluorescent molecules of steps i) - iii), wherein detecting the first fluorescent molecule indicates a viral titer, detecting the second fluorescent molecule indicates a viral genome, and detecting the third fluorescent molecule indicates packaging of the target nucleic acid, and b. subsequently administering to the subject the AAV comprising the target nucleic, wherein target nucleic acid treats or prevents DMD.Docket No.103361-584WO1 25. The method of claim 23 or 24, wherein the at least one AAV capsid probe comprises an antibody, or a fragment thereof.

26. The method of any one of claims 23-25, wherein the AAV capsid probe binds to a conformational epitope.

27. The method of any one of claims 23-26, wherein the at least one AAV capsid probe binds to a VP1, VP2, VP3 membrane protein, or a combination thereof.

28. The method of any one of claims 23-27, wherein the AAV nucleic acid comprises a DNA or RNA.

29. The method of any one of claims 23-28, wherein the AAV gene probe comprises an antibody, a capsid penetrating nucleic acid probe (CPNP), or fragments thereof.

30. The method of any one of claims 23-29, wherein the target nucleic acid comprises a DNA or RNA.

31. The method of any one of claims 23-30, wherein the target nucleic acid comprises a non- native nucleic acid not derived from the AAV.

32. The method of any one of claims 23-31, wherein the transgene probe comprises an antibody, a capsid penetrating nucleic acid probe (CPNP), or fragments thereof.

33. The method of any one of claims 23-32, wherein the AAV comprises a single-stranded AAV (ssAAV), a self-complementary AAV (scAAV), or a recombinant AAV (rAAV).

34. The method of any one of claims 23-33, wherein the AAV comprises AAV1, AAV2, AAV3, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh74, or other serotypes thereof.

35. The method of any one of claims 23-34, wherein the method is performed on a micropattern array, a microchip platform, a multi-well platform, or a combination thereof.Docket No.103361-584WO1 36. The method of any one of claims 23-35, wherein the method comprises a biochip for detecting viral titer and genomic packaging of an adeno-associated virus (AAV).

37. The method of any one of claims 23-36, wherein the method introduces the transgene into a subject’s genome for treatment or prevention of DMD.

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