Methods for characterizing lentiviruses

Abstract

A method for characterizing a lentivirus includes providing a sample containing a lentivirus population including fully loaded lentivirus, partially loaded lentivirus, and / or empty lentivirus; contacting a substrate with the sample to capture the lentivirus population; contacting the captured lentivirus population with a first fluorescent agent comprising a first fluorescent label and a first binding molecule that binds to an envelope protein of the lentivirus, a second fluorescent agent comprising a second fluorescent label and a second binding molecule that binds to a capsid protein of the lentivirus, and a third fluorescent agent comprising a third fluorescent label that binds to the payload; illuminating the captured lentivirus population with light to excite the fluorescent agents; detecting fluorescence emitted from the fluorescent agents at different wavelengths; and characterizing the lentivirus based on the detected fluorescence.

Description

[Technical Field]

[0001] The present disclosure provides methods for characterizing lentiviral vectors using multiple fluorescent agents that each bind to the envelope protein, capsid protein, and / or payload of the lentiviral vector. By detecting fluorescence at different wavelengths, fully loaded, partially loaded, and empty lentiviruses can be distinguished and quantified. These methods provide rapid, convenient, and simultaneous characterization of lentiviral vectors and aid in optimizing lentiviral vector yields. [Background technology]

[0002] Lentiviral vectors are biological vehicles that can be used to deliver therapeutic genes ("payloads") to cells. Current lentiviral manufacturing processes typically produce only approximately 20% fully loaded lentiviral vectors, which contain the three viral components and the payload. Large amounts of empty or partially loaded lentiviral vectors can reduce the efficacy of lentiviral gene delivery, increase the immunogenic burden of transduced cells in vitro or in vivo, and potentially cause serious side effects in patients. A method for identifying fully loaded lentiviral vectors, as well as partially loaded vectors, is needed. The present invention fulfills this need. Summary of the Invention

[0003] In some embodiments, the method includes providing a sample comprising a lentiviral vector population comprising fully loaded lentiviral vectors, partially loaded lentiviral vectors, and / or empty lentiviral vectors; contacting a substrate with the sample to capture the lentiviral vector population on the substrate, wherein the substrate comprises an anti-envelope protein antibody; and interrogating the captured lentiviral vector population with a first fluorescent agent comprising a first fluorescent label and a first binding molecule that binds to an envelope protein of the lentiviral vector, a second fluorescent agent comprising a second fluorescent label and a second binding molecule that binds to a capsid protein of the lentiviral vector, and a pre- Provided herein is a method for characterizing a lentiviral vector, comprising: contacting a population of captured lentiviral vectors with a third fluorescent agent comprising a third fluorescent label that binds to a payload of the lentiviral vector; illuminating the captured lentiviral vector population with light from an illumination source, thereby exciting the first fluorescent agent, the second fluorescent agent, and the third fluorescent agent; detecting a first fluorescence emitted from the first fluorescent agent at a first fluorescent wavelength, a second fluorescence emitted from the second fluorescent agent at a second fluorescent wavelength, and a third fluorescence emitted from the third fluorescent agent at a third fluorescent wavelength; and characterizing the lentiviral vector according to the detected first, second, and third fluorescence, wherein the first, second, and third fluorescent wavelengths are different. [Brief explanation of the drawings]

[0004] [Figure 1] 1 shows the structure of a lentiviral vector carrying a payload. [Figure 2-1] 1 illustrates a method for three-layer visualization of lentiviral vectors according to some embodiments of the present disclosure. [Figure 2-2] This is a continuation of Figure 2-1. [Figure 3] 1 shows packaging plasmids, envelope plasmids, and transfer plasmids for producing lentiviral vectors according to some embodiments of the present disclosure. [Figure 4]1 shows different types of capsid loading of recombinant adeno-associated virus (rAAV) in rAAV production. [Figure 5] 1 shows lentiviral vector profiles made with three different ratios of plasmids according to some embodiments of the present disclosure. [Figure 6-1] 1 shows lentiviral vector profiles made with five different ratios of plasmids according to some embodiments of the present disclosure, which express green fluorescent protein (GFP). [Figure 6-2] This is a continuation of Figure 6-1. DETAILED DESCRIPTION OF THE INVENTION

[0005] The words "a" or "an," when used in conjunction with the word "comprising" in the claims and / or specification, may mean "one," but may also be used consistently with the meanings of "one or more," "at least one," and "one or more than one."

[0006] Throughout this application, the term "about" is used to indicate that a value includes the variation of error inherent in the method / device being used to determine the value. Typically, this term is meant to include a variation of less than about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, depending on the context.

[0007] Although the use of the term "or" in the claims is used to mean "and / or" unless expressly stated to refer to alternatives only or the alternatives are not mutually exclusive, the present disclosure supports a definition that refers to alternatives only and "and / or."

[0008] As used in this specification and claims, the words "comprising" (and any form of "comprising," e.g., comprise and comprise), "having" (and any form of having, e.g., have and has), "including" (and any form of "including," e.g., includes and include), or "containing" (and any form of containing, e.g., contains and contain) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0009] As used herein, "nucleic acid," "nucleic acid molecule," or "oligonucleotide" refers to a polymeric compound comprising covalently linked nucleotides. The term "nucleic acid" includes polyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA), both of which may be single-stranded or double-stranded. DNA includes, but is not limited to, complementary DNA (cDNA), genomic DNA, plasmid or vector DNA, and synthetic DNA. RNA includes, but is not limited to, mRNA, tRNA, rRNA, snRNA, microRNA, miRNA, or miRNA.

[0010] As used herein, "gene" refers to an assembly of nucleotides that encodes a polypeptide, and includes cDNA and genomic DNA nucleic acid molecules. "Gene" also refers to a nucleic acid fragment that can function as a regulatory sequence before (5' non-coding sequences) and after (3' non-coding sequences) the coding sequence. In some embodiments, the gene is integrated in multiple copies. In some embodiments, the gene is integrated in a predetermined number of copies.

[0011] As shown in Figure 1, a functional lentiviral vector (also referred to herein as a functional lentivirus) has three essential components: an outer envelope composed of, for example, the vesicular stomatitis virus glycoprotein (VSV-G) protein; a capsid housing the genetic material composed of, for example, the p24 protein; and a payload and / or genomic RNA, often a therapeutic nucleic acid. Currently, no single assay provides a complete picture of a lentiviral vector after its creation. Current assays are time-consuming, low-throughput, and often labor-intensive. Furthermore, most assays only measure a single characteristic of the lentiviral vector, not the complete vector structure.

[0012] In certain aspects, the present disclosure provides methods for characterizing lentiviral vectors using three-layer visualization, which in some embodiments do not require purification of the lentiviral vector from culture media and allows for high-throughput analysis of multiple samples simultaneously.

[0013] In some embodiments, a method for characterizing lentiviral vectors includes providing a sample containing a lentiviral vector population including fully loaded lentiviral vectors, partially loaded lentiviral vectors, and / or empty lentiviral vectors, capturing the lentiviral vector population on a substrate by contacting the sample with a substrate, wherein the substrate comprises an anti-envelope protein antibody, and contacting the captured lentiviral vector population with a first fluorescent agent comprising a first binding molecule that binds to an envelope protein of the lentiviral vector and a first fluorescent label, a second fluorescent agent comprising a second binding molecule that binds to a capsid protein of the lentiviral vector and a second fluorescent label, and a third fluorescent agent comprising a third fluorescent label that binds to the payload. The method further includes illuminating the captured lentiviral population with light from an illumination source, thereby exciting the first fluorescent agent, the second fluorescent agent, and the third fluorescent agent. The method further includes detecting fluorescence of a first wavelength emitted from the first fluorescent agent, fluorescence of a second wavelength emitted from the second fluorescent agent, and third fluorescence of a third fluorescent wavelength emitted from the third fluorescent agent, and then characterizing the lentivirus according to the detected first, second, and third fluorescence. In some embodiments, the first, second, and third fluorescent wavelengths are different.

[0014] Figures 2-1 and 2-2 show a three-layer visualization method for lentiviral vectors according to some embodiments of the present disclosure, which provides three-layer visualization using a fluorescent anti-VSV-G antibody, a fluorescent anti-p24 antibody, and a fluorescent nucleic acid stain. In some embodiments, the present disclosure targets the payload using a permeable fluorescent nucleic acid stain that selectively stains the RNA payload.

[0015] A suitable lentiviral sample is contacted with a substrate, such as a microarray chip, containing an envelope capture antibody, such as an anti-VSV-G capture antibody. As shown in Figures 2-1 and 2-2, the capture antibody captures the lentiviral vector through interaction between the surface-bound antibody and an antibody on the exterior of the lentiviral vector. Representative envelope capture antibodies include VSV-G capture antibodies, F and HN, as described herein. The captured and bound sample is fixed, permeabilized, and stained with a cocktail containing fluorescent antibodies (e.g., fluorescent anti-VSV-G antibody and fluorescent anti-p24 antibody, which target the envelope and capsid) and a permeable fluorescent nucleic acid stain that targets the payload. The selected fluorescent nucleic acid stain is specific for nucleic acids with a high affinity for RNA and remains non-fluorescent when not bound to nucleic acids. The processed sample is then scanned on a fluorescent imaging platform to monitor the colocalization of three fluorescent signals. The colocalization of the three fluorescent signals indicates the proportion of intact lentivirus in the sample. In this manner, the present disclosure allows for visualization of lentiviral vector characteristics at three different levels for a comprehensive understanding of the particle. In some embodiments, the fluorescence imaging platform is the EXOVIEW® imaging platform.

[0016] [The method described herein utilizes fluorescent visualization of lentiviral characteristics at three different levels (envelope, capsid, and payload) and demonstrates the colocalization of these three attributes at the single viral particle level in a sample. As a result, the percentage of truly intact, potentially functional particles can be obtained in a small-volume, high-throughput manner. This disclosure is advantageous because there are currently no methods on the market that provide all this information in a single assay. Currently available assays measure single lentiviral attributes, and data must be combined to gain a comprehensive understanding of the particle.]

[0017] The terms "lentivirus," "lentiviral vector," and "lentiviral particle" are used interchangeably herein. As used herein, "lentiviral vector" refers to a vector into which a desired gene can be inserted for use in research, therapeutic, or gene therapy applications. Appropriately, lentiviral vectors are derived from the HIV family. Lentiviral vectors are a well-studied vector system based on the human immunodeficiency virus (HIV-1). Because lentiviral vectors integrate into the genome of host cells, the vectors enable sustained transgene expression. Other lentiviral systems have also been developed as gene transfer systems, including HIV-2, simian immunodeficiency virus, non-primate lentivirus, feline immunodeficiency virus, and bovine immunodeficiency virus. Driven by safety concerns due to the pathogenicity of HIV-1 in humans, the most widely used lentiviral systems for clinical and research and development purposes are based on a four-plasmid system (third-generation lentiviral vector) that expresses the lentivirus group-specific antigen (GAG) and lentiviral polymerase (POL) proteins, an envelope protein (usually vesicular stomatitis virus glycoprotein (VSV-G)), the HIV virion protein expression regulator (REV) protein, and a transfer vector (TV) containing a gene of interest (GOI). The GOI can then be introduced into cells of interest for therapies and disease treatments, such as immunodeficiency and neurodegenerative disorders. In some embodiments, other types of lentiviral vectors can also be used, such as the three-vector second-generation system.

[0018] In some embodiments, lentiviral vectors are generated by transfecting host cells with the above-described plasmids. As used herein, "transfection" refers to the introduction of an exogenous nucleic acid molecule, including a plasmid and / or a vector, into a cell. A "transfected" cell contains an exogenous nucleic acid molecule within the cell, and a "transformed" cell is one in which the exogenous nucleic acid molecule within the cell induces a phenotypic change within the cell. The transfected nucleic acid molecule may be integrated into the genomic DNA of the host cell and / or may be maintained by the cell transiently or extrachromosomally long-term. In some embodiments, "transduction" refers to the infection of a mammalian cell with a viral vector and is used interchangeably with "transfection" in this disclosure.

[0019] Host cells or organisms that express exogenous nucleic acid molecules or fragments are referred to as "recombinant," "transformed," or "transgenic" organisms. Suitable host cells for use in the various methods described herein are mammalian cells and cell lines or cell cultures. As used herein, the term "mammalian cells" includes cells derived from any member of the mammalian order, such as human cells, mouse cells, rat cells, monkey cells, hamster cells, etc. In some embodiments, the cells are mouse cells, human cells, Chinese hamster ovary (CHO) cells, CHOK1 cells, CHO-DXB11 cells, CHO-DG44 cells, CHOK1SV cells, including all variants (e.g., POTELLIGENT®, Lonza, Slough, UK), or CHOK1SV GS-KO (glutamine synthetase knockout) cells, including all variants (e.g., XCEED™, Lonza, Slough, UK). Exemplary human cells include human embryonic kidney (HEK) cells, such as HEK-293, HeLa, or HT1080 cells. In some embodiments of the present disclosure, the lentiviral vectors are produced from HEK-293 cells, human Caucasian colon adenocarcinoma HT-29 cells, or mesenchymal stem cells (MSCs).

[0020] Mammalian cells include mammalian cell cultures, which can be either adherent or suspension cultures. Adherent cultures refer to cells that grow on a substrate surface, such as a plastic plate, dish, or other suitable cell culture growth platform, and may be attachment-dependent. Suspension cultures refer to cells that can be maintained, for example, in culture flasks or large suspension vessels, which allow for a large surface area for gas and nutrient exchange. Suspension cell cultures often utilize stirring or agitation mechanisms to provide adequate mixing. Media and conditions for maintaining cells in suspension are generally known in the art. Exemplary suspension cell cultures include human embryonic kidney (HEK293) clonal cells.

[0021] In some embodiments, the cells and their product, lentiviral vectors, are produced in a bioreactor. The cells can be prepared in any suitable bioreactor (also referred to herein as a reactor), including, but not limited to, stirred tank, airlift, fiber, microfiber, hollow fiber, ceramic matrix, fluidized bed, fixed bed, and / or entrained bed bioreactors. As used herein, a "bioreactor" can include a fermenter or fermentation unit or any other reaction vessel, and the terms "bioreactor" and "reactor" are used interchangeably with "fermenter." The terms fermenter or fermentation refer to both microbial and mammalian cultures. For example, in some embodiments, an exemplary bioreactor unit can perform one or more or all of the following: feeding nutrients and / or carbon sources; injecting a suitable gas (e.g., oxygen); inlet and outlet flows of fermentation or cell culture media; separating gas and liquid phases; maintaining temperature; maintaining oxygen and CO2 levels; maintaining pH levels; agitation (e.g., stirring); and / or cleaning / sterilization. An exemplary reactor unit, such as a fermentation unit, can include multiple reactors within the unit; for example, a unit can have 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 or more bioreactors within each unit and / or facility, and / or a facility can include multiple units with single or multiple reactors within the facility. In various embodiments, the bioreactors can be suitable for batch, semi-fed-batch, fed-batch, perfusion, and / or continuous fermentation processes. Any suitable reactor diameter can be used. In embodiments, the bioreactors can have a volume of from about 100 mL to about 50,000 L.Non-limiting examples include 100 mL, 250 mL, 500 mL, 750 mL, 1 liter, 2 liters, 3 liters, 4 liters, 5 liters, 6 liters, 7 liters, 8 liters, 9 liters, 10 liters, 15 liters, 20 liters, 25 liters, 30 liters, 40 liters, 50 liters, 60 liters, 70 liters, 80 liters, 90 liters, 100 liters, 150 liters, 200 liters, 250 liters, 300 liters, 350 liters, 400 liters, 450 liters, 500 liters, and 550 liters. Suitable reactors include those with volumes of 1000 liters, 600 liters, 650 liters, 700 liters, 750 liters, 800 liters, 850 liters, 900 liters, 950 liters, 1000 liters, 1500 liters, 2000 liters, 2500 liters, 3000 liters, 3500 liters, 4000 liters, 4500 liters, 5000 liters, 6000 liters, 7000 liters, 8000 liters, 9000 liters, 10,000 liters, 15,000 liters, 20,000 liters, and / or 50,000 liters. Additionally, suitable reactors may be multi-use, single-use, disposable, or non-disposable, and may be formed from any suitable material, including metal alloys such as stainless steel (e.g., 316L or any other suitable stainless steel) and Inconel, plastic, and / or glass.

[0022] In some embodiments, the host cells generate a lentiviral vector population. In some embodiments, the host cells are transduced with two packaging plasmids, one envelope plasmid, and one transfer plasmid, as shown in Figure 3. Depending on the type and ratio of plasmids, the host cells, the host cell culture conditions, ion exchange purification to concentrate the lentiviral vectors, and size exclusion chromatography to remove damaged lentiviral particles, the generated lentiviral vector population may contain different proportions of fully loaded lentiviral vectors, partially loaded lentiviral vectors, and / or empty lentiviral vectors.

[0023] As used herein, a fully loaded lentiviral vector refers to a vector containing envelope proteins formed as a lentiviral envelope, capsid proteins formed as a lentiviral capsid, and one or more copies of the payload sufficient for payload delivery. As used herein, a partially loaded lentiviral vector refers to a vector containing envelope proteins (formed as an envelope or partial envelope) and capsid proteins (formed as a capsid or partial capsid), but only a payload or fragments of host cell DNA or RNA. Host cell DNA / RNA or partial payloads may not be suitable for research or therapeutic applications. As used herein, an empty lentiviral vector refers to a vector containing envelope proteins (formed as an envelope or partial envelope) and capsid proteins (formed as a capsid or partial capsid), but substantially lacking a payload. In some embodiments, lentiviral vector populations may further contain other components or impurities, such as small aggregates formed by envelope or capsid proteins. These aggregates do not contain a payload.

[0024] Figure 4 provides an overview of the main capsid types generated during recombinant adeno-associated virus (rAAV) production. While rAAV may not contain an envelope, fully loaded lentiviral vectors do (Gimpel et al., Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies, Molecular Therapy Methods & Clinical Development, 2021, 20:740-754). This illustrates various scenarios of viral genome packaging, a process well studied for AAV but less understood for lentiviruses. As shown in Figure 4, complete capsids have correct genome packaging, partially loaded capsids package only partial vector or host cell DNA, and empty capsids do not package a genome. The percentage of complete capsids at harvest is less than 30%, but can be increased to over 70% after purification. As described herein, partially loaded capsids, empty capsids, and aggregates, as shown in Figure 4, may not only fail to provide sufficient transduction of the desired payload, but injection into patients may increase the risk of immunotoxicity.

[0025] As described above, a sample, such as a cell culture containing a lentiviral vector, is contacted with a substrate, allowing the substrate to capture the lentiviral vector from the sample. The substrate (see Figures 2-1 and 2-2) is preferably an optical substrate designed to generate an enhanced fluorescent signal to increase the sensitivity of lentiviral vector detection. In some embodiments, the substrate has a planar reflective surface, and anti-envelope protein antibodies are immobilized on the surface. In some embodiments, the planar surface of the substrate can include one or more layers that enhance excitation and / or emission of fluorescence from the first, second, and third fluorescent labels. In some embodiments, the substrate is a silicon substrate, such as a glass slide. In some embodiments, the substrate includes an oxide layer on a silicon base. In some embodiments, the substrate is a microarray chip. In some embodiments, the substrate is a bead, such as a magnetic bead. In some embodiments, each chip is suitable for characterizing lentiviral vectors in a single sample. Multiple chips can be arranged in a multiwell plate, such as a 6-well, 24-well, 48-well, or 96-well plate, allowing multiple samples corresponding to multiple substrates or chips to be characterized simultaneously, preferably automatically. In some embodiments, each chip can have multiple spots for characterization of multiple samples.

[0026] In some embodiments, the substrate has a reflectance greater than a certain minimum value across one or more wavelengths and / or spectral ranges of interest. Exemplary spectral ranges include, but are not limited to, the ultraviolet spectral range of about 400 nm to about 450 nm, the blue spectral range of about 460 nm to about 500 nm, the green spectral range of about 520 nm to about 560 nm, the red spectral range of about 640 nm to about 680 nm, and the deep red spectral range of about 710 nm to about 750 nm. For example, the reflective substrate can have a "reflectance" or "reflective capacity" greater than or equal to 25% across one or more wavelengths and / or spectral bands of interest (e.g., greater than 30%, e.g., greater than 40%, e.g., greater than 50%, e.g., greater than 60%, greater than 70%, etc.). In certain embodiments, the reflective substrate has a reflectance greater than 80% across one or more wavelengths and / or spectral bands of interest. In some embodiments, the reflective substrate comprises an oxide layer on a silicon base. In certain embodiments, the reflective substrate comprises multiple layers.

[0027] As described above, the substrate surface is coated with anti-envelope protein antibodies, which are immobilized on the substrate surface. The envelope protein of the lentiviral vector can be at least one of VSV-G, fusion protein, hemagglutinin, and hemagglutinin-neuraminidase (HN). Accordingly, the substrate is immobilized with at least one of anti-VSV-G antibody, anti-fusion protein (anti-F) antibody, anti-hemagglutinin (anti-HA) antibody, and anti-HN antibody. The substrate bearing the anti-envelope antibody can capture the lentiviral vector bearing the corresponding envelope protein. In some embodiments, the lentiviral vector bears the VSV-G envelope protein, and the substrate surface is coated with anti-VSV-G antibody. In some embodiments, the anti-VSV-G antibody is attached to the substrate surface by physical adsorption, chemical bonding, or biological means.

[0028] In some embodiments, contacting the sample with the substrate surface is achieved by incubating the sample with the substrate for a predetermined time at a predetermined temperature. In some embodiments, the incubation is at 20°C, 25°C, 30°C, 35°C, or 37°C. In some embodiments, the predetermined time is about 5 minutes to about 90 minutes. In some embodiments, the predetermined time is about 15 minutes to 60 minutes. In some embodiments, the predetermined time is about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 70 minutes, about 80 minutes, or about 90 minutes. In some embodiments, the predetermined time is about 15 minutes. In some embodiments, the predetermined time is about 30 minutes. In some embodiments, the predetermined time is about 60 minutes. After incubation, the lentivirus population from the sample is immobilized on the substrate surface by binding of the VSV-G protein of the lentiviral vector to the anti-VSV-G antibody on the substrate surface. In some embodiments, the contacted sample after incubation can be removed (thus removing any unbound viral vectors or moieties / aggregates), for example, by simply lifting the substrate to decant the sample solution, and then optionally drying the substrate for a short period of about 5 to about 10 minutes.

[0029] After the sample is contacted with the substrate, the method further contacts the substrate with a first, second, and third fluorescent agent. Optionally, the method can contact the sample and the substrate with the three fluorescent agents simultaneously, or with two of the three reagents simultaneously. The fluorescent agent comprises a fluorescent label.

[0030] As used herein, the term "label" or "tag" refers to a compound capable of producing a detectable signal indicative of the presence of a target in an assay sample. Suitable labels include radioisotopes, nucleotide chromophores, enzymes, substrates, fluorescent molecules, chemiluminescent moieties, magnetic particles, bioluminescent moieties, and the like. Thus, a label is any compound detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. In some embodiments, the label is a fluorescent label, including a fluorescent molecule.

[0031] In some embodiments, the first fluorescent agent comprises a first fluorescent label and a first binding molecule. The first binding molecule is configured to target a lentiviral envelope protein of the lentiviral vector. Where appropriate, the first binding molecule may be an anti-VSV-G antibody, an anti-F antibody, an anti-HA antibody, or an anti-HN antibody. In some embodiments, the first binding molecule is an anti-VSV-G antibody. In some embodiments, the first binding molecule is the same as the capture molecule on the substrate, with the difference being that the capture molecule is immobilized on the substrate surface, while the first binding molecule is bound to a first fluorescent label.

[0032] In some embodiments, the second fluorescent agent comprises a second fluorescent label and a second binding molecule. The second binding molecule is configured to target a capsid protein of the lentiviral vector. Suitably, the second binding molecule can be an anti-p7 antibody, an anti-p24 antibody, or an anti-p27 antibody. In some embodiments, the second binding molecule is an anti-p24 antibody.

[0033] In some embodiments, the third fluorescent agent comprises a third fluorescent label. The third fluorescent label is configured to bind to the payload of the lentiviral vector. The payload can be a nucleic acid, such as DNA or RNA, or a protein. Accordingly, the third fluorescent label can be a DNA stain, an RNA stain, a nucleic acid stain, or an antibody targeting the payload protein. In some embodiments, the third fluorescent label can be an anti-integrase antibody, an anti-reverse transcriptase antibody, or an anti-protease antibody targeting these various functional proteins.

[0034] In some embodiments, the sample is diluted with an appropriate buffer prior to contact with the substrate. In some embodiments, the sample is diluted to at least about 1:10, 1:20, 1:50, 1:100, 1:200, 1:500, 1:1000, 1:1500, 1:2000. In some embodiments, the dilution is such that the concentration of lentiviral vectors (lentiviral particles) in the sample is about 10 per milliliter (ml). 5 ~10 8 In some embodiments, the concentration of the lentiviral vector is about 10 6 ~10 8 In certain embodiments, the concentration of the lentiviral vector in the sample is estimated using any suitable means, such as interference microscopy, flow cytometry, fluorescence spectroscopy, titration, polymerase chain reaction (PCR), or based on previous experiments or experience.

[0035] In some embodiments, after the substrate is contacted with the sample, the first, second, and third fluorescent agents are simultaneously contacted with the substrate. In some embodiments, after the substrate is contacted with the sample, the substrate is first contacted with the third fluorescent agent, and then with the second and third fluorescent agents. In some embodiments, the third fluorescent agent comprises a permeable fluorescent nucleic acid stain that allows the stain to penetrate into the lentiviral vector. In some embodiments, the third fluorescent agent comprises a fluorescent nucleic acid stain and a permeabilization reagent, and the lentiviral vector immobilized on the substrate is treated with the permeabilization reagent for a certain period of time and then contacted with the fluorescent nucleic acid stain. Alternatively, the permeabilization reagent and the fluorescent nucleic acid stain are simultaneously contacted with the lentiviral vector for a certain period of time. The permeabilization reagent can be, for example, an organic solvent such as methanol or acetone, a detergent such as Triton X, Tween, or NP-40, or a selective surfactant such as saponin, digitonin, or leucopem. In some embodiments, the period of permeabilization (permeabilization period) is less than 5 hours, less than 4 hours, less than 3 hours, less than 2 hours, or less than 1 hour. In some embodiments, the permeabilization period is about 0.5 to 2 hours. In some embodiments, the permeabilization period is less than 30 minutes, less than 15 minutes, or less than 10 minutes. Limiting the permeabilization period can prevent or substantially reduce damage to the lentiviral vector.

[0036] In some embodiments, the step of contacting the captured lentiviral vector population is carried out by incubating the substrate bearing the captured lentiviral vector population with the first, second, and third fluorescent agents for a period of time (incubation period). In some embodiments, the incubation period is about 15 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, or about 5 hours. In some embodiments, the incubation period is about 15 to 60 minutes. In some embodiments, the incubation period is at least 15 minutes. In some embodiments, the incubation period is about 30 minutes. In some embodiments, the incubation period is carried out at a temperature of about 15°C to 40°C. For example, the substrate can be contacted with the fluorescent agents for about 30 minutes to about 2 hours, about 30 minutes to about 1.5 hours, about 45 minutes to about 1.5 hours, about 1 hour to about 1.5 hours, or about 1.5 hours, at a temperature of about 15°C to 40°C, e.g., about 20°C, about 25°C, or about 37°C.

[0037] Following labeling with the three fluorescent agents, the substrate with the captured lentivirus population can be washed one or more times to remove unbound fluorescent agents and other debris. In some embodiments, no washing steps are necessary.

[0038] After contacting the sample with the substrate with the first, second, and third fluorescent agents, the lentiviral population captured on the substrate surface binds to the fluorescent label and can be detected using fluorescence.

[0039] In some embodiments, the fluorescent properties of the lentiviral vector are determined using a fluorescence detection device, which suitably includes an illumination source, a fluorescence detector, a computing device, and optionally an interference microscope.

[0040] The illumination light source generates excitation light, which is directed toward the substrate surface, and the fluorescent labels can emit fluorescent signals after being activated by the excitation light. In some embodiments, the illumination light source includes one or more light-emitting diodes (LEDs) or one or more lasers. In some embodiments, the light source is a coherent light source that generates light at specific wavelengths. In some embodiments, the illumination light source generates light at three specific wavelengths corresponding to the three fluorescent labels. In some embodiments, each specific wavelength is a narrow range of wavelengths. In some embodiments, the wavelength of the excitation light can be, for example, about 400 nm to about 450 nm (ultraviolet), about 460 nm to about 500 nm (blue), about 520 nm to about 560 nm (green), about 640 nm to about 680 nm (red), or about 710 nm to about 750 nm (deep red). In some embodiments, the wavelength of the illumination light source is tunable. In some embodiments, the illumination light source has three to five channels, and each channel is configured to irradiate a coherent light source at a predetermined wavelength.

[0041] After absorbing excitation light, the first, second, and third fluorescent labels in the lentiviral vector are configured to emit light at first, second, and third fluorescent wavelengths, respectively. In some embodiments, the first, second, and third fluorescent wavelengths are in the range of about 250 nm to about 700 nm. In some embodiments, one of the first, second, and third fluorescent wavelengths is in the range of about 460 nm to about 510 nm, another of the first, second, and third fluorescent wavelengths is in the range of about 520 nm to about 570 nm, and another of the first, second, and third fluorescent wavelengths is in the range of about 640 nm to about 680 nm.

[0042] In some embodiments, the emission wavelengths of the first, second, and third fluorescent labels are in the ultraviolet range of about 330 nm to about 380 nm, the blue range of about 420 nm to about 495 nm, the yellow range of about 520 nm to about 580 nm, or the red range of about 620 nm to about 750 nm. In some embodiments, the difference in emission wavelengths between two of the first, second, and third fluorescent labels (i.e., the difference in emission wavelengths) is at least about 20 nm to about 80 nm. In some embodiments, the difference is at least about 40 nm to 60 nm. In some embodiments, the difference is about 50 nm. In some embodiments, the emission wavelengths of the first, second, and third fluorescent labels are about 460 nm to about 510 nm, about 520 nm to about 570 nm, or about 640 nm to about 680 nm.

[0043] In some embodiments, the excitation light for the first, second, and third fluorescent labels has a wavelength of about 488 nm, and the corresponding emission light has a wavelength of about 510 nm. In some embodiments, the first fluorescent label is THERMOPHISHER's NovaFluor Blue 510 dye. In some embodiments, the excitation light for the second fluorescent label has a wavelength of about 561 nm, and the corresponding emission light has a wavelength of about 568 nm. In some embodiments, the second fluorescent label is THERMOPHISHER's NovaFluor Yellow 570 dye. In some embodiments, the excitation light for the third fluorescent label has a wavelength of about 640 nm, and the corresponding emission light has a wavelength of about 685 nm. In some embodiments, other types of fluorescent labels or fluorescent label / excitation light combinations can be used, so long as the three fluorescent labels have easily distinguishable fluorescent emissions. In embodiments, suitable dyes are CF® 555 (VSV-G) and CF® 647 (p24).

[0044] In some embodiments, the first fluorescent label is chemically or biologically conjugated to a first binding molecule and the second fluorescent label is chemically or biologically conjugated to a second binding molecule.

[0045] In some embodiments, the third fluorescent label is a nucleic acid stain. The nucleic acid stain can be, for example, Quant-iT™ PicoGreen dsDNA stain, Quant-iT™ OliGreen ssDNA stain, Quant-iT™ RiboGreen RNA stain, TOTO®-1 green fluorescent nucleic acid stain, YOYO®-1 green fluorescent nucleic acid stain, Hoechst 33258 blue fluorescent nucleic acid stain, SYBR™ Green I nucleic acid gel stain, or SYTO™ RNASelect™ green fluorescent stain. In some embodiments, the third fluorescent label is a permeable fluorescent nucleic acid stain that penetrates the envelope and capsid of the lentiviral vector and selectively stains the RNA payload within the capsid. In some embodiments, the third fluorescent label is carboxyfluorescein cinnamidyl ester (6-carboxyfluorescein cinnamidyl ester, 5(6)-CFDA-SE) (CFSE), a dye that binds via its cinnamidyl group to intracellular lentiviral vector molecules, specifically to intracellular lysine residues and other amine sources.

[0046] In some embodiments, interference microscopy is used to characterize the location and size of lentiviral vectors on a substrate surface. For example, an IM microscope scans the substrate surface to acquire IM images, and a computing device uses the IM images to identify lentiviral vectors within the IM images. In some embodiments, particles less than 10 nm in diameter are discarded, particles between about 10 nm and about 50 nm in size represent labeled fragments, and particles between about 50 nm and about 200 nm in size represent labeled lentiviral vectors. In some embodiments, only lentiviral particles greater than 50 nm in size are subjected to subsequent fluorescence-based analysis.

[0047] The fluorescence emitted from the first, second, and third fluorescent labels can be detected by a fluorescence detector or spectrophotometer. In some embodiments, the detector is a charge-coupled device (CCD) camera. The CCD collects images of each sample at each wavelength, and for each sample, three corresponding images are collected at approximately the same time at the same light-receiving position. Thus, the fluorescence signals at corresponding positions cover the same sample material.

[0048] As defined herein, a "spectrophotometer" is a light intensity measuring device (a device that measures the intensity of light) that can measure the intensity relative to the color of light, more specifically, the wavelength of light. In some embodiments, the spectrophotometer is configured to measure the light intensity at the first, second, and third emission wavelengths of the first, second, and third fluorescent labels. In some embodiments, the spectrophotometer is configured to measure the light intensity ratio between the first, second, and third emission wavelengths. Measurement of the three fluorescence intensities or the ratio between the three fluorescence intensities can be performed using one, two, or three spectrophotometers. In some embodiments, the present disclosure is performed on a LENTIVIEW™ imaging platform from NanoView Biosciences. In some embodiments, the present disclosure is performed on a VIRUS COUNTER® imaging platform from SARTORIUS, Inc., where the spectrophotometer is, for example, a Virus Counter 3100. In certain embodiments, the present disclosure can also be performed on an EXOVIEW™ imaging platform adapted for lentiviral vector analysis.

[0049] In some embodiments, the spectrophotometer is configured to scan the same region of the substrate surface with fluorescence intensity images at first, second, and third emission wavelengths (fluorescence wavelengths), thereby obtaining a first fluorescence image reflecting the fluorescence signal at the first emission wavelength, a second fluorescence image reflecting the fluorescence signal at the second emission wavelength, and a third fluorescence image reflecting the fluorescence signal at the third emission wavelength. Each fluorescence intensity image contains multiple fluorescence signals, and each fluorescence signal in the fluorescence image can correspond to a lentiviral vector.

[0050] In some embodiments, the fluorescence device does not include an interference microscope, and the lentiviral vector is recognized directly from the first fluorescence image. In some embodiments, the lentiviral vector is recognized by an image object detection algorithm. For example, each pixel in the first fluorescence image is evaluated as a fluorescent pixel if its fluorescence signal exceeds a threshold. Adjacent fluorescent pixels are combined to form a fluorescent spot, and if the longest diameter of this spot is greater than 50 nm, it represents a lentiviral vector. In some embodiments, the lentiviral vector can also be detected from the first, second, and third fluorescence images by the image object detection algorithm.

[0051] If IM images are available, in some embodiments, three fluorescent images of each substrate surface are overlaid with the corresponding IM images to characterize the lentiviral vectors. In some embodiments, for locations where lentiviral vectors are present in the IM images, the computing device determines whether there are fluorescent signals in the first, second, and third fluorescent images. For each lentiviral vector or particle recognized in the IM images, the computing device calculates first, second, and third fluorescent signals from the first, second, and third fluorescent images corresponding to the recognized lentiviral vector and compares the first, second, and third fluorescent signals with first, second, and third fluorescent thresholds. If the first fluorescent signal (intensity) exceeds the first fluorescent threshold, the recognized lentiviral vector is deemed to have envelope fluorescence. If the second fluorescent signal (intensity) exceeds the second fluorescent threshold, the recognized lentiviral vector is deemed to have capsid fluorescence. If the third fluorescent signal (intensity) exceeds a third fluorescent threshold, the recognized lentiviral vector is considered to have payload fluorescence, and if the third fluorescent signal is below the third fluorescent threshold but above a fourth fluorescent threshold, the lentiviral vector is considered to have partial payload fluorescence. In some embodiments, the first, second, and third fluorescent thresholds are predefined. In some embodiments, the first, second, and third fluorescent thresholds are calibrated. In some embodiments, the amount of payload required for each lentiviral vector may differ based on the amount of payload to be delivered and the disease to be treated. Therefore, the third fluorescent threshold and, optionally, the fourth fluorescent threshold for different RNA payloads will also be different.

[0052] For each lentiviral vector recognized or discovered in the IM images, if all three fluorescent images have fluorescent signals at the same location (colocalization), the fluorescent signal is determined to correspond to a fully loaded lentiviral vector because the images contain fluorescent signals for envelope proteins, capsid proteins, and the payload. If the first and second fluorescent images have fluorescent signals at the same location but the third fluorescent image does not, the fluorescent signal is determined to correspond to an empty lentiviral vector because the image contains fluorescent signals for envelope proteins and capsid proteins but not for the payload. If the first and second fluorescent images have fluorescent signals at the same location but the third fluorescent image has a fluorescent signal below the third threshold but above the fourth threshold, the fluorescent signal is determined to correspond to a partially loaded lentiviral vector because the image contains fluorescent signals for envelope proteins and capsid proteins but only a weak signal for the payload. In some embodiments, other types of lentiviral vectors can be defined using their IM signal and the first, second and third fluorescent signals.

[0053] In some embodiments, characterizing the lentiviral vectors includes determining the first, second, and third fluorescent signals and calculating the ratio of the number of lentiviral vectors having the first, second, and third fluorescent signals (fully loaded lentiviral vectors) to the number of lentiviral vectors having the first fluorescent signal (or the number of lentiviral vectors recognized from the IM image - i.e., all lentiviral vectors). This ratio indicates the proportion of fully loaded lentiviral particles to the total number of lentiviral particles.

[0054] In certain aspects, the present disclosure provides methods for optimizing lentiviral vector yields, achieved through a combination of process improvement steps, using novel high-throughput fluorescence detection analytical methods that interrogate each layer of lentiviral morphology.

[0055] As previously described, the present disclosure targets lentiviral vectors at three different levels (envelope, capsid, and payload) to identify the components of vector particles. Lentiviral samples are captured on a microarray chip by an envelope capture antibody, appropriately via an anti-VSV-G capture antibody. The bound sample is fixed, permeabilized, and stained with a cocktail of fluorescent antibodies (e.g., those targeting the VSV-G envelope or p24 capsid) and a permeable fluorescent nucleic acid stain that selectively targets the payload, such as the RNA payload. The stained sample is scanned with a fluorescent imaging platform, such as the EXOVIEW® imaging platform, to monitor the colocalization of all three fluorescent signals. The ratios of complete lentiviral vector, empty lentiviral vector, and uncompleted particles are obtained through the fluorescent images. Based on this, the disclosure further develops formulas for optimizing process parameters, including specific component combinations in specific ratios to improve productivity, plasmid design, and identifying parameters critical to the process. Furthermore, the disclosure obtains predictive estimates of lentiviral function, helping the optimization process maximize yield. This data-driven approach accelerates the speed of process development.

[0056] In some embodiments, the optimization method includes predefining the ratios of packaging plasmid, envelope plasmid, transfer plasmid, and payload; transducing host cells with the ratio of the plasmids for each ratio; culturing the transduced cells; obtaining a population of lentiviral vectors from the cell culture; determining the ratio of fully loaded lentiviral vectors or the total amount of fully loaded lentiviral vectors in the lentiviral vector population; selecting the ratio with the highest ratio or highest amount of fully loaded lentiviral vectors as the selected ratio; and producing lentiviral vectors using the selected ratio. In some embodiments, the optimization procedure can be repeated, for example, by defining a new set of ratios based on the selected ratios and obtaining further selected ratios. When a predetermined number of iterations is reached or there is no obvious difference in the selected ratios between the last two iterations, the final selected ratio can be determined as the optimized ratio and used for high-efficiency lentiviral vector production.

[0057] In some embodiments, in addition to optimizing plasmid ratios, the optimization process described above can also be used to optimize different plasmid designs, optimize transduction conditions, optimize culture conditions, optimize ion exchange chromatography for lentiviral vector purification, or evaluate a size exclusion step to remove damaged lentiviral vectors.

[0058] In some embodiments, the optimization process described above is adapted for high throughput, using multi-well plates to hold multiple microarrays, each microarray being used for one experimental test. In some embodiments, the microarrays have multiple test spots, each configured to correspond to one experimental test. In some embodiments, all or some of the optimization steps are automated, improving the efficiency of the process.

[0059] In some embodiments, the present disclosure provides methods of producing lentiviral vectors, wherein the production is carried out using the optimized conditions described above.

[0060] [Embodiment] In a first embodiment, a method for detecting lentiviral vectors includes providing a sample containing a lentiviral vector population comprising fully loaded lentiviral vectors, partially loaded lentiviral vectors, and / or empty lentiviral vectors; capturing the lentiviral vector population on a substrate by contacting the sample with the substrate, wherein the substrate comprises an anti-envelope protein antibody; and interrogating the captured lentiviral vector population with a first fluorescent agent comprising a first fluorescent label and a first binding molecule that binds to an envelope protein of the lentiviral vector, a second fluorescent agent comprising a second fluorescent label and a second binding molecule that binds to a capsid protein of the lentiviral vector, and and a third fluorescent agent comprising a third fluorescent label that binds to the payload of the lentiviral vector; illuminating the captured lentiviral vector population with light from an illumination source to excite the first fluorescent agent, the second fluorescent agent, and the third fluorescent agent; detecting a first fluorescence having a first fluorescent wavelength emitted from the first fluorescent agent, a second fluorescence having a second fluorescent wavelength emitted from the second fluorescent agent, and a third fluorescence having a third fluorescent wavelength emitted from the third fluorescent agent; and characterizing the lentiviral vector according to the detected first, second, and third fluorescence, wherein the first, second, and third fluorescent wavelengths are different.

[0061] Embodiment 2 includes the method of embodiment 1, wherein the substrate comprises an anti-vesicular stomatitis virus-G protein (anti-VSV-G) antibody.

[0062] Embodiment 3 includes the method of embodiment 1 or 2, wherein the first binding molecule comprises an anti-VSV-G antibody, an anti-fusion protein (anti-F) antibody, an anti-hemagglutinin (anti-HA) antibody, or an anti-hemagglutinin-neuraminidase (anti-HN) antibody.

[0063] Embodiment 4 includes the method of any of embodiments 1-3, wherein the second binding molecule comprises an anti-p7 antibody, an anti-p24 antibody, or an anti-p27 antibody.

[0064] Embodiment 5 includes the method of any of embodiments 1 to 4, wherein the third fluorescent label specifically binds to a nucleic acid or a protein.

[0065] Embodiment 6 includes the method of embodiment 5, wherein the third fluorescent label comprises an anti-integrase antibody, an anti-reverse transcriptase antibody, or an anti-protease antibody.

[0066] Embodiment 7 includes the method of any one of embodiments 1 to 6, wherein detection of the first and second fluorescence characterizes the empty lentiviral vector, and detection of the first, second, and third fluorescence characterizes the fully loaded lentiviral vector.

[0067] Embodiment 8 includes the method of any one of embodiments 1 to 7, wherein the substrate is a microarray chip.

[0068] Embodiment 9 includes the method of any one of embodiments 1 to 8, wherein the fully loaded lentiviral vector comprises ribonucleic acid (RNA) or deoxyribonucleic acid (DNA).

[0069] Embodiment 10 includes the method of any one of embodiments 1 to 9, wherein each of the first, second, and third fluorescent wavelengths is within the range of about 250 nm to about 700 nm.

[0070] Embodiment 11 includes the method of embodiment 10, wherein one of the first, second, and third fluorescent wavelengths is within the range of about 460 nm to about 510 nm.

[0071] Embodiment 12 includes the method of embodiment 10 or 11, wherein one of the first, second, and third fluorescent wavelengths is within the range of about 520 nm to about 570 nm.

[0072] Embodiment 13 includes the method of any of embodiments 10-12, wherein one of the first, second, and third fluorescent wavelengths is within the range of about 640 nm to about 680 nm.

[0073] Embodiment 14 includes the method of any one of embodiments 1 to 13, wherein characterizing the lentiviral vector includes determining first, second, and third fluorescent signals according to the intensities of the first, second, and third fluorescent signals, and calculating a ratio between the area having the first, second, and third signals and the area having the first signal, wherein the ratio indicates the proportion of fully loaded lentiviral vector in the sample relative to the total amount of lentiviral vector in the sample.

[0074] Embodiment 15 includes the method of any of embodiments 1 to 14, wherein the captured population of lentiviral vectors is contacted with the first fluorescent agent, the second fluorescent agent, and the third fluorescent agent simultaneously.

[0075] Embodiment 16 includes the method of any one of embodiments 1 to 15, wherein contacting the captured lentiviral vector population with the first fluorescent agent, the second fluorescent agent, and the third fluorescent agent comprises contacting the captured lentiviral vector population with the third fluorescent agent, and then contacting the captured lentiviral vector population with the first fluorescent agent and the second fluorescent agent.

[0076] Embodiment 17 includes the method of any one of embodiments 1 to 16, wherein contacting the captured lentiviral vector population includes incubating the sample with the first, second, and third fluorescent agents for at least 15 minutes.

[0077] Example 18 includes the method of any of Examples 1-17, wherein the third fluorescent agent comprises a permeable fluorescent nucleic acid stain.

[0078] Embodiment 19 includes the method of any of embodiments 1-18, wherein the method further includes permeabilizing the lentiviral vector population prior to contacting with the first fluorescent agent, the second fluorescent agent, and the third fluorescent agent. [Example]

[0079] Example 1: Characterization of lentiviral vectors by three-layer fluorescence detection A fluorescence device is provided that includes three laser coherent light sources configured to generate light at approximately 552 nm, approximately 637 nm, and approximately 500 nm; one or more fluorescence detectors configured to detect fluorescence, e.g., at approximately 590 nm, approximately 685 nm, and approximately 525 nm, respectively; an interference microscope for collecting IM images; and a computing device configured to process images collected by the fluorescence detector and interference microscope. A microarray is provided, having 24 (4 x 6) wells. Each well has a surface immobilized with anti-VSV-G antibodies. The wells are used to hold replicate samples or lentivirus populations generated using different parameters.

[0080] An RNA payload and first, second, and third fluorescent agents are provided. The first fluorescent agent comprises an anti-VSV-G antibody conjugated to a first fluorescent label. The first fluorescent label is NovaFluor Yellow 590 dye, which has an excitation maximum of about 552 nm and an emission maximum of about 590 nm. The second fluorescent agent comprises an anti-p24 antibody conjugated to a second fluorescent label. The second fluorescent label is NovaFluor Red 685 dye, which has an excitation maximum of about 637 nm and an emission maximum of about 685 nm. The third fluorescent label is Quant-iT™ RiboGreen, which has an excitation maximum of about 500 nm and an emission maximum of about 525 nm.

[0081] Figure 3 shows the plasmid design for balanced expression of packaging, envelope, and transgenes for improved lentiviral vector packaging. As shown in Figure 3, the constructed plasmids include two packaging plasmids, one envelope plasmid, and one transfer plasmid. The packaging plasmid contains the GAG-POL and REV genes, the envelope plasmid contains the VSV-G gene, and the transfer plasmid contains the green fluorescent protein (GFP) gene as an exemplary GOI.

[0082] HEK-293 cells are provided. They are cultured in Dulbecco's Modified Aigle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37°C, 5% CO2, and 60% humidity until they reach the desired confluent density. The cultured HEK-293 cells are transiently transfected with a packaging plasmid, an envelope plasmid, a transfer plasmid, and an RNA payload. The molar ratio of the three plasmids to the payload plasmid is pVSV-G:pREV:pGAG-POL:pGOI / GFP (1:1:2:3). The transfected HEK-293 cells are further cultured, and a sample containing a lentivirus population is obtained from the transfected HEK-293 cells. The number of particles in the sample is estimated by interference microscopy or other appropriate means. The sample is diluted as needed until the concentration of the diluted sample is approximately 10. 6 ~about 10 8 The lentiviral particles are then added.

[0083] The microarray is placed in a well of the plate, and 25 μl of diluted sample is applied to the microarray. The microarray with the diluted sample is incubated at 20°C for 1 hour. The diluted sample contains a lentiviral vector population. The lentiviral vector population can include fully loaded lentiviral vectors, partially loaded lentiviral vectors, empty lentiviral vectors, and small aggregates. Fully loaded lentiviral vectors have an envelope formed by VSV-G protein, a capsid located inside the envelope and formed by capsid proteins, and an RNA payload packaged within the capsid. Partially loaded lentiviral vectors have an envelope and capsid, but the capsid contains only a fragment of the RNA payload, an insufficient amount of RNA payload, and / or partial vector or host cell DNA / RNA. Empty lentiviral vectors contain an envelope and capsid but are substantially lacking in RNA payload. Small aggregates can be formed by only envelope protein, or optionally, by only capsid protein. The microarray surface is fixed with anti-VSV-G antibodies, and lentiviral particles carrying VSV-G proteins are fixed to the microarray surface by binding to the anti-VSV-G antibodies on the surface.

[0084] The sample solution is removed and the microchips are allowed to dry for approximately 5 minutes. 80 μl of each of the three fluorescent dyes is added and used to immerse the microarrays in the wells. The plate is incubated at 37°C for 30 minutes with shaking at 50 revolutions per minute, protected from light.

[0085] The microarray is scanned with an interference microscope, and the scanned interference microscope (IM) image shows the number of particles within the scanned area. Particles with a diameter of less than 10 nm are discarded, particles with a size of approximately 10 nm to approximately 50 nm are labeled aggregates, and particles with a size of approximately 50 nm to approximately 200 nm are labeled lentiviral vectors.

[0086] The excitation fluorescence light is irradiated onto the surface of the microarray at wavelengths of 552 nm, 637 nm, and 500 nm, respectively, and the fluorescence emitted from the microchip surface is collected by a detector(s) of the fluorescence device at wavelengths of about 590 nm, about 685 nm, and about 525 nm, respectively, to obtain first, second, and third fluorescence images.

[0087] In some embodiments, the fluorescence device does not include an interference microscope, and the lentiviral vector is recognized directly from the first fluorescence image. In some embodiments, the lentiviral vector is recognized by an image object detection algorithm. For example, each pixel in the first fluorescence image is evaluated as a fluorescent pixel if its fluorescence intensity exceeds a threshold. Adjacent fluorescent pixels are combined to form a fluorescent spot, and if the longest diameter of this spot is greater than 50 nm, it represents a lentiviral vector. In some embodiments, the lentiviral vector can also be detected from the first, second, and third fluorescence images by the image object detection algorithm.

[0088] In some embodiments, lentiviral vectors are identified from the IM image and also recognized by image object detection from all three fluorescent images. There may be overlap in the identified or detected lentiviral vectors, and some lentiviral vectors may be recognized only from the IM image or only from one of the three fluorescent images.

[0089] After acquiring the IM image, the fluorescence image, and the recognized lentiviral vector, the computing device overlays the IM image on the first, second, and third fluorescence images. For each lentiviral vector recognized in the IM image, the computing device calculates first, second, and third fluorescence signals from the first, second, and third fluorescence images corresponding to the recognized lentiviral vector and compares the first, second, and third fluorescence signals with first, second, and third fluorescence thresholds. If the first fluorescence signal (intensity) exceeds the first fluorescence threshold, the recognized lentiviral vector is deemed to have envelope fluorescence. If the second fluorescence signal (intensity) exceeds the second fluorescence threshold, the recognized lentiviral vector is deemed to have capsid fluorescence. If the third fluorescence signal (intensity) exceeds the third fluorescence threshold, the recognized lentiviral vector is deemed to have payload fluorescence. If the third fluorescence signal (intensity) is below the third fluorescence threshold but above the fourth fluorescence threshold, the lentiviral vector is deemed to have partial payload fluorescence. In some embodiments, the first, second, and third fluorescence thresholds are predefined. In some embodiments, the first, second, and third fluorescence thresholds are calibrated. In some embodiments, the amount of payload required for each lentiviral vector may differ based on the amount of payload required to be delivered and the disease being treated. Thus, the third fluorescence threshold for different RNA payloads will also differ.

[0090] The computing device determines whether a lentiviral vector has envelope, capsid, and payload fluorescence, whether it is a fully loaded lentiviral vector, whether it has envelope and capsid fluorescence but no payload fluorescence, whether it is an empty lentiviral vector, or whether it has other IM / fluorescence patterns, which may include partially loaded lentiviral vectors with envelope, capsid, and partial payload fluorescence, and aggregated lentiviral vectors with only envelope fluorescence.

[0091] In some embodiments, the computing device further counts the total number of lentiviral vectors and optionally aggregates from the IM images, counts the total number of fully loaded lentiviral vectors, and calculates the ratio of the total number of fully loaded lentiviral vectors to the total number of lentiviral vectors and / or aggregates, which is a strong indicator of whether host cells transiently transfected with the plasmid are suitable for lentiviral vector production.

[0092] In some embodiments, the ratio of the fluorescence intensities of corresponding spots in the first, second, and third images is used to characterize the lentiviral vector.

[0093] Example 2: Yield optimization of lentiviral vectors based on three-layer fluorescence detection The fluorescent device and microarray are provided as described in Example 1. The RNA payload and the first, second, and third fluorescent agents are prepared as described in Example 1. The packaging plasmid, envelope plasmid, and transfer plasmid are constructed as described in Example 1. HEK-293 cells are provided as described in Example 1. The difference is that the first fluorescent label (binding to anti-VSV-G antibody), the second fluorescent label (binding to anti-p24 antibody), and the third fluorescent label (targeting the RNA payload) are green, red, and blue fluorescent, respectively. Furthermore, the parameters in Example 1 may be varied, such as upstream optimization including plasmid design and plasmid ratios, and downstream optimization including an ion exchange (IEX) purification step for enriching intact lentiviral vectors and a size exclusion step for damaged lentiviral vector particles, to determine the optimal parameters for producing fully loaded lentiviral vectors.

[0094] In this Example 2, three sets of molar ratios between the packaging plasmid, envelope plasmid, transfer plasmid, and RNA payload are predefined: in Set 1, the molar ratio between the envelope plasmid (VSV-G), packaging plasmid (REV), packaging plasmid (GAG-POL), and transfer plasmid expressing the RNA payload is 1:1:2:3; in Set 2, the molar ratio is 1:1:2:1; and in Set 3, the molar ratio is 1:1:2:0.1.

[0095] As in Example 1, for each ratio, HEK-293 cells were cultured and transfected with the plasmid and RNA payload at the aforementioned ratio. The transfected cells were further cultured, and a lentiviral vector population was collected from the cultured cells. The lentiviral vector population was then incubated with a microchip. The incubated microchip was further incubated with three fluorescent agents. The lentiviral vectors were scanned using an interference microscope. The further incubated microchip was excited with a laser light source with three excitation wavelengths. Fluorescence at three different emission wavelengths was collected and processed to determine whether the lentiviral vectors were fully loaded, partially loaded, or empty. The ratio of fully loaded lentiviral vectors to the total lentiviral vectors, or the total number of fully loaded lentiviral vectors, was obtained.

[0096] Microarrays can be designed with multiple wells to simultaneously characterize three different sets of conditions. For example, a 4 x 6-well plate is provided, with four rows and six columns. Each well contains a microarray. Each well / microarray in the first row receives a 25 μl sample of the plasmid corresponding to the first ratio. This sample has two different dilutions, resulting in an expected lentiviral vector concentration of approximately 10 per milliliter. 6 ~about 10 8Each dilution is provided in triplicate. Similarly, six microarrays in the second column of the plate contain a plasmid sample corresponding to the second ratio, and six microarrays in the third column contain a plasmid sample corresponding to the third ratio. Specific microarrays in the fourth column are designed as positive and negative controls. The positive control uses a confirmed number of lentiviral vectors as samples, and the negative control uses medium (without cultured cells) as samples.

[0097] In some embodiments, microchips with multiple wells can be configured for handling by robotic equipment, and in this way, optimization can be performed in a high-throughput manner.

[0098] Figure 5 shows the results of Example 2, in which the complete lentiviral vector has fluorescent signals for all of the envelope, capsid, payload, and IM signals, while the empty lentiviral vector does not contain a fluorescent signal for the payload RNA. As shown in Figure 5, a molar ratio of 1:1:2:3 provided 26.9% complete lentiviral vector, which is advantageous compared to the 20.8% complete lentiviral vector produced by the 1:1:2:1 molar ratio and the 6.2% complete lentiviral vector produced by the 1:1:2:0.1 molar ratio. Thus, complete characterization of lentiviral vectors indicates, among other things, that reducing nucleic acid content during the manufacturing process reduces the percentage of lentiviral particles produced.

[0099] Example 3: Yield optimization of lentiviral vectors based on three-layer fluorescence detection Example 3 is similar to Example 2. Instead of testing three ratios, five plasmid ratios were tested in Example 3, with the GOI set as GFP. As shown in Figures 6-1 and 6-2, the molar ratio of 1:1:2:3 was the most promising, producing 15.8% intact lentiviral vector.

[0100] In summary, the present disclosure provides fluorescent detection of all critical components of the lentiviral structure, including the payload critical for fully functional lentiviral particles, and also provides formulas for process optimization, including combinations of specific components in specific ratios to optimize productivity, and also provides analytical models for optimizing the process based on variations in process inputs to maximize yield. Furthermore, the disclosed optimization process can be performed at high throughput and does not require sample pretreatment.

[0101] While certain embodiments have been illustrated and described herein, it is to be understood that the claims are not limited to the specific forms or arrangements of elements described and shown. Although exemplary embodiments are disclosed herein and specific terms are employed, these terms are used in a generic and descriptive sense only and are not intended to be limiting. Modifications and variations of the embodiments are possible in light of the above teachings. It is therefore to be understood that the embodiments may be practiced otherwise than as specifically described.

[0102] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. providing a sample comprising a population of lentiviral vectors comprising fully loaded lentiviral vectors, partially loaded lentiviral vectors, and / or empty lentiviral vectors; capturing the lentiviral vector population on a substrate by contacting the substrate with the sample, the substrate comprising an anti-envelope protein antibody; contacting the captured lentiviral vector population with a first fluorescent agent comprising a first fluorescent label and a first binding molecule that binds to an envelope protein of the lentiviral vector, a second fluorescent agent comprising a second fluorescent label and a second binding molecule that binds to a capsid protein of the lentiviral vector, and a third fluorescent agent comprising a third fluorescent label that binds to a payload of the lentiviral vector; illuminating the captured population of lentiviral vectors with light from an illumination source to excite the first fluorescent agent, the second fluorescent agent, and the third fluorescent agent; detecting a first fluorescence having a first fluorescence wavelength emitted from the first fluorescent agent, a second fluorescence having a second fluorescence wavelength emitted from the second fluorescent agent, and a third fluorescence having a third fluorescence wavelength emitted from the third fluorescent agent; characterizing the lentiviral vector according to the detected first, second, and third fluorescence; A method for characterizing a lentiviral vector, wherein the first, second, and third fluorescent wavelengths are different.

2. 2. The method of claim 1, wherein the substrate comprises an anti-vesicular stomatitis virus-G protein (anti-VSV-G) antibody.

3. 3. The method of claim 1 or claim 2, wherein the first binding molecule comprises an anti-VSV-G antibody, an anti-fusion protein (anti-F) antibody, an anti-hemagglutinin (anti-HA) antibody, or an anti-hemagglutinin-neuraminidase (anti-HN) antibody.

4. The method of any one of claims 1 to 3, wherein the second binding molecule comprises an anti-p7 antibody, an anti-p24 antibody, or an anti-p27 antibody.

5. The method of any one of claims 1 to 4, wherein the third fluorescent label specifically binds to a nucleic acid or a protein.

6. The method of claim 5 , wherein the third fluorescent label comprises an anti-integrase antibody, an anti-reverse transcriptase antibody, or an anti-protease antibody.

7. 7. The method of any one of claims 1 to 6, wherein detection of the first and second fluorescence characterizes the empty lentiviral vector, and detection of the first, second and third fluorescence characterizes the fully loaded lentiviral vector.

8. The method of any one of claims 1 to 7, wherein the substrate is a microarray chip.

9. The method of any one of claims 1 to 8, wherein the fully loaded lentiviral vector comprises ribonucleic acid (RNA) or deoxyribonucleic acid (DNA).

10. 10. The method of claim 1, wherein each of the first, second, and third fluorescent wavelengths is in the range of about 250 nm to about 700 nm.

11. 11. The method of claim 10, wherein one of the first, second, and third fluorescent wavelengths is in the range of about 460 nm to about 510 nm.

12. 12. The method of claim 10 or claim 11, wherein one of the first, second, and third fluorescent wavelengths is in the range of about 520 nm to about 570 nm.

13. 13. The method of any one of claims 10 to 12, wherein one of the first, second, and third fluorescent wavelengths is in the range of about 640 nm to about 680 nm.

14. Characterization of the lentiviral vector includes: determining first, second, and third fluorescent signals according to the intensities of the first, second, and third fluorescent lights; 14. The method of any one of claims 1 to 13, comprising calculating a ratio between an area having the first, second, and third signals and an area having the first signal, wherein the ratio indicates a proportion of the fully loaded lentiviral vector in the sample relative to the total amount of lentiviral vector in the sample.

15. 15. The method of any one of claims 1 to 14, wherein the captured lentiviral vector population is contacted with the first fluorescent agent, the second fluorescent agent, and the third fluorescent agent simultaneously.

16. 16. The method of any one of claims 1 to 15, wherein contacting the captured lentiviral vector population with the first fluorescent agent, the second fluorescent agent, and the third fluorescent agent comprises contacting the captured lentiviral vector population with the third fluorescent agent, and then contacting the captured lentiviral vector population with the first fluorescent agent and the second fluorescent agent.

17. 17. The method of any one of claims 1 to 16, wherein contacting the captured population of lentiviral vectors comprises incubating the sample with the first, second, and third fluorescent agents for at least 15 minutes.

18. 18. The method of any one of claims 1 to 17, wherein the third fluorescent agent comprises a permeable fluorescent nucleic acid stain.

19. 19. The method of any one of claims 1 to 18, further comprising permeabilizing the population of lentiviral vectors prior to contacting with the first fluorescent agent, the second fluorescent agent, and the third fluorescent agent.

Images