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EP-4739804-A1 - METHODS AND COMPOSITIONS FOR CHARACTERIZING VIRAL PARTICLES IN A BIOLOGICAL SAMPLE

EP4739804A1EP 4739804 A1EP4739804 A1EP 4739804A1EP-4739804-A1

Abstract

The present disclosure provides methods and compositions for determining (i) the presence, absence, and/or amount of one or more groups of viral particles and/or (ii) the composition of viral particles, in a biological sample using a functionalized nanostructure-based sensor.

Inventors

  • QUAN, QIMIN
  • GARG, Angad
  • RAZ, TAL

Assignees

  • NanoMosaic Inc.

Dates

Publication Date
20260513
Application Date
20240709

Claims (20)

  1. 1. A method of determining the presence, absence, and/or amount of one or more groups of viral particles in a biological sample, the method comprising: (a) contacting a sensor defining a plurality of functionalized nanostructures with a first test sample comprising one or more nucleic acid molecules under conditions that permit the functionalized nanostructures to bind the one or more nucleic acid molecules, wherein the one or more nucleic acid molecules are harvested from the viral particles in the biological sample or are amplicons derived therefrom; (b) after step (a), detecting a change in a property of the nanostructures thereby to determine the presence, absence, and/or amount of the one or more nucleic acid molecules; and (c) after step (b), using the presence, absence, and/or amount of the one or more nucleic acid molecules and, optionally a concentration of viral particles in the biological sample, to determine the presence, absence, and/or amount of the one or more groups of viral particles within the first test sample.
  2. 2. The method of claim 1, wherein the functionalized nanostructures each comprise a capture agent.
  3. 3. The method of claim 1 or 2, wherein the amplicons are produced by amplification of nucleic acid molecules harvested from the viral particles.
  4. 4. The method of claim 3, wherein the amplicons comprise a detection label capable of binding to a detection agent.
  5. 5. The method of claim 4, wherein the detection label and the detection agent are selected from the group consisting of: biotin and streptavidin, fluorescein and an antifluorescein antibody, digoxigenin and an anti-digoxigenin antibody, 3, 3', 5,5'- Tetramethylbenzidine (TMB) and an antibody conjugated to horseradish peroxidase (HRP), an oligonucleotide and a complementary oligonucleotide, an oligonucleotide and an aptamer, an oligonucleotide and its binding partner, a click chemistry pair (e.g., an azide and an alkyne, an azide and a dibenzocyclooctyne, or a tetrazine and a transcy cl ooctene) .
  6. 6. The method of any one of claims 2-5, wherein the capture agent is an antibody or an antigen-binding fragment thereof capable of binding the one or more nucleic acid molecules.
  7. 7. The method of any one of claims 1-6, wherein the amplicons comprise a capture label.
  8. 8. The method of claim 7, wherein the capture agent and the capture label are selected from the group consisting of: a target antigen and an antibody, an aptamer-binding partner and an aptamer, biotin and streptavidin, a fluorophore (e.g., fluorescein, Tamra, Cy5, or Cy3) and an antibody that binds the fluorophore, digoxigenin and an anti-digoxigenin antibody, a nucleic acid molecule and a complementary nucleic acid molecule, a nucleic acid molecule and an aptamer capable of binding the nucleic acid molecule, a poly-A nucleic acid sequence and a poly-T nucleic acid sequence, a poly- A nucleic acid sequence and an antibody that binds the poly-A nucleic acid sequence, a poly-T nucleic acid sequence and an antibody that binds the poly-T nucleic acid sequence, a poly-C nucleic acid sequence and a poly-G nucleic acid sequence, a poly- C nucleic acid sequence and an antibody that binds the poly-C nucleic acid sequence, a poly-G nucleic acid sequence and an antibody that binds the poly-G nucleic acid sequence, and a click chemistry pair (e.g., an azide and an alkyne, an azide and a dibenzocyclooctyne, or a tetrazine and a transcyclooctene).
  9. 9. The method of claims 7 or 8, wherein the functionalized nanostructures each comprise an antibody or antigen binding fragment thereof that binds the capture label.
  10. 10. The method of any one of claims 1-9, further comprising, prior to step (c): (i) contacting an additional substrate comprising a plurality of functionalized nanostructures with an additional test sample comprising a plurality of viral particles, wherein the functionalized nanostructures of the additional substrate are capable of binding the viral particles; and (ii) after step (i), detecting a change in a property of the nanostructures of the additional substrate thereby to determine the concentration of the viral particles in the biological sample.
  11. 11. The method of claim 10, wherein the first test sample and the additional test sample are derived from a single viral culture and/or cell culture grown in a bioreactor.
  12. 12. The method of any one of claims 10 or 11, further comprising, prior to step (ii), contacting the viral particles with a detection antibody capable of binding the viral particles.
  13. 13. The method of claim 12, wherein the detection antibody is selected from the group consisting of an antibody that binds a viral protein of a specific serotype of the viral particle (e.g., an anti- VP 1 antibody, an anti-VP2 antibody, an anti-VP3 antibody), a serotype non-specific antibody, an antibody that binds a specific serotype of the viral particle (e.g., an anti-AAV9 antibody, an anti-AAV5 antibody, an anti-AAV2 antibody, or an anti-AAV8 antibody), an antibody that binds a lentiviral particle, an antibody that binds a lentiviral capsid protein, an antibody that binds to a baculovirus particle, and an antibody that binds to baculovirus capsid protein.
  14. 14. The method of any one of claims 10-13, wherein the detection antibody is conjugated to a second detection agent selected from the group consisting of: streptavidin, HRP, HRP and TMB, HRP and 3,3 '-Diaminobenzidine (DAB)), a nucleic acid molecule, a bead (e.g., a polystyrene bead or a magnetic bead), and a nanoparticle (e.g., a gold nanoparticle).
  15. 15. A method of determining the presence, absence, and/or amount of two or more groups of viral particles in a biological sample, the method comprising: (a) contacting a first region, a second region, and a third region of a sensor defining a plurality of functionalized nanostructures with a test sample comprising one or more nucleic acid molecules and one or more viral particles or viral capsid proteins thereof under conditions that permit the functionalized nanostructures to bind the one or more nucleic acid molecules and/or the one or more of the viral capsid proteins, wherein the first region of the sensor is configured to bind a first nucleic acid molecule, the second region of the senor is configured to bind a second, different nucleic acid molecule, and the third region of the sensor is configured to bind the viral particles or viral capsid proteins, and wherein the one or more nucleic acid molecules are harvested from the viral particles in the biological sample or are amplicons derived therefrom; (b) after step (a), detecting a change in a property of the nanostructures thereby to determine the presence, absence and/or amount of the first nucleic acid molecule, the second nucleic acid molecule, and the one or more viral particles or viral capsid proteins; and (c) after step (b), using the presence, absence, and/or amount of the one or more nucleic acid molecules of the first test sample, the one or more nucleic acid molecules of the second test sample, and the one or more viral particles or viral capsid proteins in the biological sample to determine the presence, absence, and/or amount of the two or more groups of viral particles within the biological sample.
  16. 16. The method of claim 15, wherein the functionalized nanostructures comprise a capture agent.
  17. 17. The method of claim 15 or 16, wherein the amplicons are produced by amplification of nucleic acid molecules harvested from the viral particles.
  18. 18. The method of claim 17, wherein the amplicons comprise a detection label capable of binding to a detection agent.
  19. 19. The method of claim 18, wherein the detection label and the detection agent are selected from the group consisting of biotin and streptavidin, fluorescein and an antifluorescein antibody, digoxigenin and an anti-digoxigenin antibody, 3, 3', 5,5'- Tetramethylbenzidine (TMB) and an antibody conjugated to horseradish peroxidase (HRP), an oligonucleotide and a complementary oligonucleotide, an oligonucleotide and an aptamer, an oligonucleotide and its binding partner, a click chemistry pair (e.g., an azide and an alkyne, an azide and a dibenzocyclooctyne, or a tetrazine and a transcy cl ooctene) .
  20. 20. The method of any one of claims 16-19, wherein the capture agent is an antibody or an antigen-binding fragment thereof capable of binding the one or more nucleic acid molecules.

Description

METHODS AND COMPOSITIONS FOR CHARACTERIZING VIRAL PARTICLES IN A BIOLOGICAL SAMPLE CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63/525,716 filed on July 9, 2023, U.S. Provisional Patent Application No. 63/602,618 filed on November 26, 2023, and U.S. Provisional Patent Application No. 63/569,729 filed on March 26, 2024, the disclosures of each of which are hereby incorporated by reference in their entireties for all purposes. FIELD [0002] The disclosure relates to methods and compositions for characterizing viral particles and their composition in a biological sample using a nanostructure-based sensor. BACKGROUND [0003] The detection and quantification of viral particles and the composition of viral genomes included within the viral particles is required in a variety of applications. For example, during the manufacture of viral particles used in gene therapy it is critical to assess the presence and amount of viral particles, and the completeness of the viral genome at various stages during the upstream (cell line development and viral harvest) and downstream (viral purification and formulation) processes. The genetic characterization of the viral particles is critical for informing treatment options, such as appropriate dosages to be administered to a subject. [0004] Existing methods for characterizing viral particles suffer from slow speed (sample- to-answer time), low throughput, low correlation across analytical platforms (conventional approaches require consolidation of multiple technologies, instruments, consumable, and workflows), large sample amount and/or purity requirement (an inability to work with a crude sample), which can become a bottleneck during process development and manufacturing. A major challenge with such approaches is based on the requirement for calibration of the results across multiple measurements from different assay workflows and various testing instruments, including, for example, enzyme-linked immunosorbent assays (ELISA), UV/visible spectroscopy, mass spectroscopy (MS), transmission electron microscopy (TEM), quantitative PCR (qPCR), digital PCR (dPCR) and AUC/MS quantifications. Such methods typically require nucleic acid purification, dilution, amplification, and an additional variety of steps before detection of genetic alteration can be accomplished. These processes are cumbersome and result in significant loss of sample and in quantification bias. [0005] Despite the advances made to date there is still a need for new methods and compositions for characterizing viral particles present in biological samples, especially viral particles that will be administered to humans during a gene therapy protocol. SUMMARY [0006] The present disclosure generally relates to nanosensor-based methods and compositions that facilitate highly sensitive detection of viral particles and genomic DNA contained within the viral particles. Furthermore, the present disclosure details the utilization of nanosensors for detection and quantification of nucleic acids of a sequence of interest, directly in crude samples such as cell lysates and body fluids (e.g., plasma, serum, cerebrospinal fluid, urine, or tears) without requiring sample purification. For example, the methods and compositions described herein provide an approach for the routine analysis of viral particles within a biological sample to determine, for example, whether a viral particle contains a full transgene, an empty capsid, a partial transgene, or a transgene or partial transgene containing a nucleotide insertion, deletion or substitution. Furthermore, the methods and compositions described herein facilitate the routine analysis of biological samples to determine whether the viral capsids in the sample are empty, full, partially full and/or have the desired integrity. The methods and compositions described herein can characterize viral particle titers and genetic composition, using a single instrument, the same assay plate, and no sample purification. [0007] In a first aspect, the disclosure provides a method of determining the presence, absence, and/or amount of one or more groups of viral particles in a biological sample, the method comprising: (a) contacting a sensor defining a plurality of functionalized nanostructures with a first test sample comprising one or more nucleic acid molecules under conditions that permit the functionalized nanostructures to bind the one or more nucleic acid molecules, wherein the one or more nucleic acid molecules are harvested from the viral particles in the biological sample or are amplicons derived therefrom; (b) after step (a), detecting a change in a property of the nanostructures thereby to determine the presence, absence, and/or amount of the one or more nucleic acid molecules; and (c) after step (b), using the presence, absence, and/or amount of the one or more nucleic acid molecules and, option