A method for detecting in vitro the enzymatic activity of CYP4V2

Abstract

The present application relates to a method for detecting CYP4V2 enzyme activity in vitro. Specifically, the method comprises the following steps: a) incubating a luciferin precursor with a cell expressing CYP4V2, the incubation system comprising a culture medium; b) after the incubation, collecting the cell supernatant and detecting the content of luciferin in the cell supernatant; wherein the cell expressing CYP4V2 is a cell transfected with an adeno-associated virus (AAV) vector comprising a nucleic acid encoding CYP4V2.

Description

Technical Field

[0001] This application relates to the field of biomedicine, specifically to a method for detecting the enzyme activity of cytochrome oxidase CYP4V2 in vitro. Background Technology

[0002] Bietti crystalline dystrophy (BCD) is an autosomal recessive retinitis pigmentosa disorder characterized by the deposition of yellowish-white crystalline fatty metabolites in the retina and cornea, and progressive degeneration and atrophy of the retinal pigment epithelium (RPE) and choroid. The main pathogenic gene for BCD is the CYP4V2 gene located at 4q35, which belongs to the cytochrome P450 family of cytochrome oxidases and plays an important role in fatty acid metabolism.

[0003] CYP4V2 protein is a typical microsomal fatty acid ω-hydrogenase and mitochondrial peroxisome β-oxidase, playing a role in intracellular lipid metabolism. Its catalytic efficiency exhibits regioselectivity and fatty acid chain length selectivity, enabling it to ω-hydroxylate medium-chain saturated fatty acids (such as lauric acid and myristic acid) and polyunsaturated fatty acids (such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), with the catalytic reaction dependent on β-NADPH. CYP4V2 protein is highly expressed in the endoplasmic reticulum of human retinal cells and is the most important cytochrome oxidase in the eye responsible for fatty acid metabolism. Mutations in the CYP4V2 gene lead to insufficient catalytic activity of its encoded hydroxylase, affecting lipid metabolism and causing abnormal lipid metabolism in the retinopathy of prematurity (RPE), thereby damaging photoreceptor cells and leading to benign prostatic hyperplasia (BCD). However, the specific pathogenic mechanism remains unclear.

[0004] Gene therapy is a novel treatment approach that introduces exogenous genes into cells to correct or compensate for diseases caused by gene defects or abnormal gene expression. To evaluate the in vitro activity of gene replacement therapy products and ensure product quality, it is necessary to establish a stable, simple, rapid, and highly specific in vitro CYP4V2 enzyme activity detection method.

[0005] Because CYP4V2 can exert ω-hydroxylation on medium-chain saturated fatty acids, CN115851873A reports the use of arachidonic acid as a substrate to detect CYP4V2 activity. In addition, other literature reports the use of lauric acid and myristic acid as reaction substrates to detect CYP4V2 activity. Articles published by Nakano et al. in *Drug Metabolism and Disposition* in 2009 and by Jarrar et al. in *Annals of Human Genetics* in 2020 indicate that dodecanoic acid (lauric acid) and tetradecanoic acid (myristic acid) are relatively sensitive reaction substrates for CYP4V2, generating hydroxylated products. Therefore, based on this principle, a method for detecting CYP4V2 activity can be established. For example, cells containing CYP4V2 can be lysed, and then incubated with the necessary coenzyme and medium-chain saturated fatty acid substrate. Subsequently, the generated hydroxylated products can be detected using chromatography-mass spectrometry. The following is a chemical reaction formula using lauric acid as a substrate, CYP4V2 as a hydroxylase, and with the participation of NADPH and O2:

[0006]

[0007] Although the above-mentioned LC-MS / MS detection method based on medium-chain saturated fatty acid substrates and cell lysates can specifically reflect CYP4V2 enzyme activity, it has the following inherent limitations: (1) High consumption of viral vectors: The method requires a large number of transfected cells for enzyme activity assessment, which leads to a significant increase in the demand for viral vectors. (2) Cumbersome sample pretreatment process and damage to the microenvironment: Cells must be lysed to obtain lysates as enzyme sources, involving complex and time-consuming pretreatment steps such as cell digestion, lysis, and centrifugation. This process damages cell integrity, causing the enzyme to be removed from its natural subcellular microenvironment. In addition, in order to reconstruct the active reaction system in vitro, exogenous coenzyme factors (such as cytochrome b5, cytochrome P450 reductase, and NADPH) need to be added, which not only increases the complexity of operation but also potentially introduces methodological variability and uncertainty. (3) Detection mode limitation: This method is essentially an endpoint assay, which can only obtain enzyme activity data at a single time point, making it difficult to achieve continuous and real-time tracking of enzyme reaction kinetics. (4) Equipment dependence and low throughput bottleneck: Activity detection is highly dependent on the liquid chromatography-tandem mass spectrometry (LC-MS / MS) platform, involving complex chromatographic separation and mass spectrometry analysis steps, resulting in cumbersome operation procedures and low throughput (single sample analysis takes about 10-30 minutes), significantly prolonging the research cycle. Therefore, developing a simpler method that can detect CYP4V2 enzyme activity in situ, in real time or near real time in intact cells can meet unmet technical needs and has important technical value. Summary of the Invention

[0008] This application provides a novel and effective method for detecting CYP4V2 enzyme activity: the luciferin substrate method. The luciferin precursor is a series of substrates, all of which can be converted into beetle luciferin (D-luciferin) by CYP family proteins. These substrates are then oxidized by luciferase and a series of cofactors to produce light. The activity of the CYP family enzyme is determined based on the signal intensity, which is directly proportional to the CYP enzyme activity. The chemical reaction formula is as follows:

[0009]

[0010] Currently known luciferin precursor substrates all exhibit statistically significant activity against CYP proteins, but there is no clear report on the most suitable substrate and specific detection conditions for detecting CYP4V2 protein activity. This invention aims to develop a luciferin substrate method suitable for detecting CYP4V2 activity, for the activity assay of related gene therapy products.

[0011] The method for detecting CYP4V2 enzyme activity based on a fluorescent substrate, protected in this application, is briefly described below. First, a vector delivering CYP4V2 is added to cells stably transfected with AAVR to transfect the cells and induce CYP4V2 protein expression. Then, a luciferin precursor is added and incubated with the virus-transfected cells for an appropriate time. Both the luciferin precursor and the luciferin produced by the CYP4V2 enzyme reaction are cell-permeable. Due to this characteristic, the luciferin precursor, as a substrate, can enter the cell, be converted into the luciferin product by CYP4V2 within the cell, and the product can also cross the cell membrane to the extracellular space. Subsequently, a luciferin detection reagent reacts with the luciferin in the cell supernatant, and the enzyme activity of CYP4V2 is assessed based on the intensity of the generated light signal.

[0012] Firstly, this application protects a method for detecting the enzyme activity of CYP4V2 in vitro. The core of this method lies in using a luciferin precursor as a substrate, and through the hydroxylase activity of CYP4V2, hydroxylating the luciferin precursor to form luciferin (e.g., D-luciferin). Specifically, this method can detect the enzyme activity of CYP4V2 in cells.

[0013] In some embodiments, the method comprises co-incubating the luciferin precursor with a vector expressing CYP4V2 or with cells containing the vector. The incubation system preferably comprises a culture medium or buffer. The buffer may be a commonly used buffer existing in the art and likely to be used in the future, such as PBS, Tris-HCl, TAE, TBE, TE, RIPA, SDS-PAGE buffer, TBST, Ponceau S, or HEPES. The culture medium is preferably a cell culture medium, which may be a commercially available cell culture medium, such as BME, MEM or Opti-MEM, DMEM, GMEM, IMDM, RPMI-1640, F-10, F-12, F-12K, GlutaMAX-I, and McCoy's 5A, or a mixture of two or more of the above. More specifically, the culture medium may also contain essential cell culture supplies, such as serum, antibiotics, etc., the selection and concentration of which can be determined based on the experience of those skilled in the art.

[0014] In some embodiments, the method includes obtaining the product of a luciferin precursor catalyzed by the hydroxylase activity of CYP4V2. The product is specifically luciferin, more specifically D-luciferin. The product may be present in a medium, such as a medium in which the luciferin precursor and a CYP4V2-expressing vector are co-incubated, preferably in cell supernatant, and the acquisition may be a direct harvest of the co-incubated cell supernatant. Alternatively, the product may be present in a CYP4V2-expressing vector, preferably in a CYP4V2-expressing cell vector, and the collection may be achieved by lysing cells to obtain a cell lysate containing luciferin.

[0015] In a specific implementation, the method includes the following steps:

[0016] a) Incubating the luciferin precursor with cells expressing CYP4V2, wherein the incubation system contains a culture medium;

[0017] b) Collect the cell supernatant after incubation and detect the fluorescein content in the cell supernatant.

[0018] In some embodiments, the cells are cells containing a nucleic acid vector encoding CYP4V2. Specifically, the cells may be formed by transfection with a nucleic acid vector encoding CYP4V2. More specifically, the nucleic acid vector may be a plasmid, a virus, a vesicle vector, or a nanoparticle vector (such as a lipid nanoparticle vector).

[0019] In specific embodiments, the virus may be selected from one or more of the following: adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, herpes simplex virus, vaccinia virus, baculovirus, rhabdovirus, and paramyxovirus. Preferably, the viral vector is an adeno-associated virus (AAV) vector.

[0020] In some embodiments, the fluorescein precursor is a compound as shown in Formula I.

[0021]

[0022] Formula I

[0023] In some embodiments, R1 in Formula I is an organic substituent attached to an aromatic ring, which is connected to the fluorescein core via an ether bond formed by an oxygen atom; R2 is an alkyl or substituent attached to a carboxylic acid ester group. The fluorescein core refers to the benzothiazole moiety and the dihydrothiazole moiety between the ether bond and the carboxylic acid ester group in Formula I, more specifically 2-(4,5-dihydrothiazole-2-yl)benzo[d]thiazole.

[0024] In some embodiments, R1 is a substituent group that can be recognized and hydroxylated by CYP4V2, which, while maintaining substrate membrane permeability and metabolic stability, facilitates the efficient execution of CYP4V2-mediated enzymatic reactions. Specifically, in some embodiments, R1 is a straight-chain or branched alkyl group, such as methyl, ethyl, n-propyl, isopropyl, or tert-butyl; in some embodiments, R1 is an alkyl group containing an aromatic ring, such as benzyl or p-methoxybenzyl; in some embodiments, R1 is a heteroarylmethyl group, such as 3-furanmethyl, thiophenemethyl, or pyrrolemethyl; and in some embodiments, R1 is a functionalized substituent, such as fluorobenzyl, trifluoromethylbenzyl, hydroxybenzyl, or cyanomethyl.

[0025] In a preferred embodiment, R1 is methyl or 3-furanmethyl.

[0026] R2 is an alkyl group or substituent attached to a carboxylic acid ester group, used to create good cell permeability. In some preferred embodiments, R2 can be a low-carbon chain alkyl group, such as methyl, ethyl, or n-propyl; it can also be a branched alkyl group, such as isopropyl or tert-butyl; it can also be an aryl or aralkyl group, such as benzyl or p-tolyl; it can also be a heterocyclic derivative, such as furan ethyl or pyrrole methyl; or it can be other lipophilic groups with biocompatibility.

[0027] In a preferred embodiment, R2 is preferably a methyl group. This structure not only improves the cell membrane permeability of the substrate, but also, under the condition of esterase, can rapidly release free carboxyl groups to form a substrate that can be detected by fluorescein detection reagent.

[0028] In a preferred embodiment, the fluorescein precursor is fluorescein-MultiCYP (structural formula: ) or fluorescein-3FEME (structural formula: (More preferably, fluorescein-MultiCYP).

[0029] In some embodiments, the cells expressing CYP4V2 also express adeno-associated virus receptor (AAVR). In a preferred embodiment, the cells expressing CYP4V2 also exogenously express adeno-associated virus receptor (AAVR).

[0030] In some implementations, the AAVR may include any AAVR of any vertebrate origin, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats). Preferably, the AAVR is human-derived. Specifically, the AAVR may be a human protein numbered Q8IZA0 on the Uniprot website.

[0031] In some implementations, the AAVR is a wild-type AAVR.

[0032] In some embodiments, the AAVR is a mutated AAVR, where the mutation can be a deletion, substitution, and / or insertion of one or more amino acids. In some embodiments, in the mutated AAVR, the mutation does not occur in the CP1 region of the AAVR, i.e., not in the region of amino acids 29 to 264. In some embodiments, in the mutated AAVR, the mutation does not occur in the CP1 region, the single transmembrane region, and / or the cytoplasmic tail region of the AAVR, i.e., not in the region of amino acids 29 to 264, amino acids 740 to 760, and / or amino acids 761 to 835. In specific embodiments, the number of mutated amino acids may or may not be an integer multiple of 3.

[0033] In some embodiments, the AAVR is an AAVR with a complete deletion or insertion of amino acid fragments. In some embodiments, in the AAVR with a complete deletion or insertion of amino acid fragments, the deletion or insertion does not occur in the CP1 region of the AAVR, i.e., not in the region of amino acids 29 to 264. In some embodiments, in the AAVR with a complete deletion or insertion of amino acid fragments, the deletion or insertion does not occur in the CP1 region, the single transmembrane region, and / or the cytoplasmic tail region of the AAVR, i.e., not in the region of amino acids 29 to 264, amino acids 740 to 760, and / or amino acids 761 to 835.

[0034] In some embodiments, the AAVR is a domain-deleted AAVR. In some embodiments, in the domain-deleted AAVR, the domain deletion does not occur in the CP1 region of the AAVR, i.e., it does not occur in the region of amino acids 29 to 264 of the AAVR. In some embodiments, in the domain-deleted AAVR, the domain deletion does not occur in the CP1 region, the single transmembrane region, and / or the cytoplasmic tail region of the AAVR, i.e., it does not occur in the region of amino acids 29 to 264, amino acids 740 to 760, and / or amino acids 761 to 835 of the AAVR.

[0035] In some implementations, the AAVR is an AAVR with a structural domain insertion.

[0036] The amino acid site counts are based on the site counts of wild-type AAVR protein. Regardless of any mutations or deletions that occur in AAVR compared to the wild-type protein, the amino acid site counts remain unchanged.

[0037] In a more specific embodiment, the amino acid sequence of the AAVR is shown in SEQ ID NO: 1.

[0038] In some embodiments, the cells may be primary cells or cell lines.

[0039] In a preferred embodiment, the cells may be easily transformed cells. For example, the cells are selected from one or more of the following: HEK293, 293T, 293F, HeLa, COS-7, CHO-K1, CHO-S, NIH / 3T3, Jurkat, K562, HCT116, A549, MEF, HBEC, HMEC, and RK3E.

[0040] In a preferred embodiment, the cells are retinal epithelial cells. The retinal epithelial cells can be primary cells or cell lines. In a more preferred embodiment, the retinal epithelial cells are cell lines selected from one or more of the following: ARPE-19, hTERT-RPE1, D407, Lonza H-RPE, OpRegen® RPE, and porcine RPE.

[0041] In some embodiments, in step a), the culture medium is a serum-containing culture medium or a serum-free culture medium. The serum can be serum from any source within the field.

[0042] In some embodiments, the cells expressing CYP4V2 are prepared by infecting cells with a virus, wherein the virus contains a nucleotide sequence encoding CYP4V2, preferably an AAV viral particle containing a nucleotide sequence encoding CYP4V2. In some embodiments, the virus infects the cells with a multiple of infection (MOI) of about 1E3 to about 1E6, preferably about 5E3 to about 5E5, more preferably about 1E4 to about 2E5, more preferably about 2.4E5, and even more preferably about 1E4, about 2E4, about 3E4, about 4E4, about 5E4, about 6E4, about 7E4, about 8E4, about 9E4, about 1E5, about 1.1E5, about 1.2E5, about 1.3E5, about 1.4E5, about 1.5E5, about 1.6E5, about 1.7E5, about 1.8E5, about 1.9E5, or about 2E5. More specifically, the step of preparing the cells expressing CYP4V2 includes: adding a virus diluent to a cell culture medium and incubating, wherein the virus in the virus diluent contains a nucleotide sequence encoding CYP4V2. The cell culture medium contains cells cultured thereon, more specifically, the cells are adherent cells, semi-adherent cells, or suspension cells. Preferably, the incubation lasts for about 60 to about 84 hours, more preferably about 64 to about 80 hours, even more preferably about 68 to about 76 hours, even more preferably about 70 to about 74 hours, and most preferably about 72 hours. The virus diluent is diluted using a culture medium, preferably the culture medium used in the cell culture medium, specifically referring to the state of the culture medium before actual use (e.g., culturing cells).

[0043] In some embodiments, the step of preparing the cells expressing CYP4V2 further includes a culture process following incubation of the aforementioned virus dilution with the cell culture medium. The culture medium used for this culture is preferably the same as the one used in the cell culture medium, specifically referring to the state of the culture medium before actual use (e.g., culturing cells). Preferably, the culture lasts for about 36 to about 60 hours, more preferably about 40 to about 56 hours, even more preferably about 44 to about 52 hours, even more preferably about 46 to about 50 hours, and most preferably about 48 hours.

[0044] In a specific implementation, the cells are prepared through the following steps:

[0045] i. Add the virus dilution to the cell culture medium and incubate, wherein the virus in the virus dilution contains a nucleotide sequence encoding CYP4V2;

[0046] ii. After incubating the virus dilution with the cell culture medium for about 60 to about 84 hours, change the medium and continue culturing for about 36 to about 60 hours.

[0047] Preferably, the virus diluent is incubated with the cell culture medium for about 72 hours, then the medium is changed and the culture continues for about 48 hours.

[0048] In some embodiments, in step i, the virus infects the cells with a multiple of infection (MOI) of about 1E5 to about 1E6. Preferably, the MOI is about 2E5 to about 5E5.

[0049] In a more preferred embodiment, in step i, the virus infects the cell with a multiplicity of infection (MOI) of about 2.4E5 or about 4.8E5.

[0050] In some embodiments, in step a), the final concentration of the luciferin precursor in the culture medium is about 5 to about 200 μM, preferably about 10 to about 100 μM, more preferably about 10 μM, about 20 μM, about 30 μM, about 40 μM, about 50 μM, about 60 μM, about 70 μM, about 80 μM, about 90 μM or about 100 μM, and even more preferably about 10 μM, about 50 μM or about 100 μM.

[0051] In some implementations, the detected CYP4V2 is located inside the cell.

[0052] In some embodiments, in step a), the luciferin precursor is incubated with the CYP4V2-expressing cells for about 2 to about 4 hours, preferably about 3 hours or about 130 to about 140 minutes, more preferably about 135 minutes.

[0053] In some embodiments, the method for detecting the luciferin content in the system can be selected from high-performance liquid chromatography, fluorescence spectrophotometry, luciferase-ATP luminescence assay, classical extraction-gravimetric method, or it can be performed using commercially available detection reagents. In a preferred embodiment, the method for detecting the luciferin content in the system is performed using commercially available detection reagents. Specifically, after harvesting the medium from which the luciferin precursor is co-incubated with the CYP4V2-expressing vector, it is incubated with a luciferin detection reagent, preferably for about 10 to about 60 minutes, more preferably for about 20 to about 40 minutes, and even more preferably for about 30 minutes.

[0054] In some embodiments, the CYP4V2 is used to treat crystalline retinal degeneration (BCD). The method can predict the expected efficacy of the product against BCD by detecting the hydroxylase activity produced after CYP4V2 transfection of cells in a specific product.

[0055] Secondly, this application also provides the use of the method of the first aspect in predicting the expected therapeutic effect of a nucleic acid vector containing CYP4V2 against crystalline retinal degeneration (BCD). Preferably, the nucleic acid vector is a viral vector, a nanoparticle vector (such as a lipid nanoparticle vector), or a plasmid. More preferably, the viral vector is an adeno-associated virus (AAV) vector, and even more preferably a recombinant adeno-associated virus (rAAV) vector.

[0056] Thirdly, this application also provides the use of the compound of Formula I and cells expressing adeno-associated virus receptor (AAVR) in the preparation of a reagent for predicting the expected efficacy of a nucleic acid vector encoding CYP4V2 against crystal-like retinal degeneration (BCD). Preferably, the nucleic acid vector is a viral vector, a nanoparticle vector (such as a lipid nanoparticle vector), or a plasmid.

[0057] Fourthly, this application also provides a kit. The kit contains the luciferin precursor used in the method of the first aspect. The kit is used to detect the enzyme activity of CYP4V2, specifically hydroxylase activity.

[0058] In some embodiments, the kit further comprises cells expressing adeno-associated virus receptor (AAVR). In some embodiments, the kit further comprises instructions describing a method for detecting the enzyme activity of CYP4V2 according to any one of the first aspects.

[0059] In some embodiments, the kit also includes an instruction manual that describes the complete procedure for detecting the enzyme activity of CYP4V2 in the first aspect.

[0060] In a preferred embodiment, the kit is used to detect the enzyme activity of CYP4V2 in cells.

[0061] In some embodiments, the kit is used to detect the efficiency of cell transformation containing a nucleic acid vector encoding CYP4V2.

[0062] In some embodiments, the kit is used to predict the expected therapeutic effect of a nucleic acid vector containing CYP4V2 against crystal-like retinal degeneration. Currently, the commonly used method in the art for detecting CYP4V2 enzyme activity is mass spectrometry based on medium-chain saturated fatty acid substrates, briefly described as follows: After co-incubating the CYP4V2 delivery vector with susceptible cells, cell lysates are collected, and a series of relevant coenzyme factors (cytochrome b5, cytochrome P450 reductase, reduced coenzyme II NADPH) are added. Subsequently, substrates lauric acid or myristic acid are added, and the reaction is carried out for 0-120 minutes. Finally, the concentration of hydroxylated products (12-hydroxylauric acid or 14-hydroxymyristic acid) is detected by LC-MS / MS. In this enzymatic reaction system, cytochrome P450 reductase is an essential component of the cytochrome P450 protein family in catalyzing metabolic reactions, helping to transfer electrons from NADPH to P450 enzymes; cytochrome b5 also participates in electron transfer in the reaction; reduced coenzyme II, i.e., β-NADPH, provides the electrons required for the reaction; all of the above coenzyme factors are in excess, so that CYP4V2 can fully bind to the substrate and exert its catalytic function.

[0063] Compared with the medium-chain saturated fatty acid substrate method described above, the fluorescein substrate method protected in this application has the following advantages: (1) It has a high detection limit and a lower multiplicity of infection (MOI) when transfecting cells with a vector containing mRNA encoding CYP4V2; (2) It can directly use the cell's endogenous coenzyme system and achieve in situ enzyme activity reaction in the cell without the use of exogenous cofactors; (3) The fluorescein precursor used and the fluorescein produced in the reaction are permeable to membranes, and the product can be detected in the cell supernatant without lysing the cells. (4) Smaller error, the risk of error caused by lysis operation and introduction of exogenous coenzyme system can be eliminated; (5) The operation process is significantly simplified, and it can support continuous monitoring of enzyme activity in the time dimension, and more realistically reflect the intracellular enzyme dynamics state; (6) Based on the plate detection mode of the microplate reader, it can ensure higher detection throughput of the detection method and realize high-throughput detection; (7) The cost is significantly reduced, no exogenous coenzyme system is required, the reduction of infection multiple, the use of microplate reader, etc. can all promote the reduction of detection cost.

[0064] Other aspects and advantages of this application will readily be apparent to those skilled in the art from the detailed description below. Only exemplary embodiments of this application are shown and described in the following detailed description. As will be appreciated by those skilled in the art, the content of this application enables them to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the descriptions in the accompanying drawings and specification of this application are merely exemplary and not restrictive. Attached Figure Description

[0065] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention can be better understood by referring to the exemplary embodiments and drawings described in detail below. A brief description of the drawings is as follows:

[0066] Figure 1 This diagram shows the operation procedure for the fluorescent substrate method.

[0067] Figure 2 The image shows protein expression in HEK293 cells after AAV-CYP4V2 was transduced in vitro.

[0068] Figure 3 The results shown are fluorescein detection results for cell supernatant and cell lysate after different substrate reactions.

[0069] Figure 4 The results show a comparison of the enzyme activity of CYP4V2 detected by the luciferin precursors luciferin-MultiCYP and luciferin-3FEME in different cell cultures and at different MOIs.

[0070] Figure 5 The results show a comparison of AAVR receptor expression rates between wild-type HEK293 cells and HEK293-AAVR cells.

[0071] Figure 6 The results shown are fluorescence detection results of CYP4V2 expression levels after HEK293 and HEK293-AAVR transfection of AAV-CYP4V2.

[0072] Figure 7 The results show the incubation detection of HEK293-AAVR cells with fluorescent substrates after three viruses expressing different enzyme proteins were transfected with them.

[0073] Figure 8 The graph shows the time-signal relationship of CYP4V2 protein activity detected by fluorescence substrate method in different substrate incubation and culture systems.

[0074] Figure 9 The results show the effects of incubating HEK293-AAVR cells with different MOIs and fluorescent substrates for different durations.

[0075] Figure 10 The results show the effects of incubating HEK293-AAVR cells with different MOIs and fluorescent substrates for different durations.

[0076] Figure 11 The diagram shows the MOI-signal relationship between AAV-CYP4V2 virus in the detection of CYP4V2 protein activity using a fluorescent substrate method. Detailed Implementation

[0077] The following specific embodiments illustrate the implementation of the invention. Those skilled in the art can easily understand other advantages and effects of the invention from the content disclosed in this specification.

[0078] Terminology Definition

[0079] The term "CYP4V2" refers to cytochrome P450 family 4 subfamily V polypeptide 2, a fatty acid ω-hydroxylase that primarily catalyzes the terminal hydroxylation of medium- to very long-chain fatty acids. This enzyme is highly expressed in retinal pigment epithelial cells (RPE) and participates in maintaining retinal lipid homeostasis; its functional deficiency leads to Bietti crystalline retinal dystrophy (BCD), characterized by lipid crystal deposition in the retina and progressive vision loss. Furthermore, CYP4V2 is also expressed in tissues such as the liver, kidneys, small intestine, and adrenal glands, and is associated with non-alcoholic fatty liver disease (NAFLD / MASLD), cholesterol metabolism, and cardiovascular disease. In this application, "mouse CYP4V2" can refer to the CYP4V2 protein or the nucleic acid (including DNA and RNA) encoding the protein; the specific meaning can be determined from the context. The CYP4V2 can include any CYP4V2 from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats). Preferably, the CYP4V2 is human-derived. Specifically, the CYP4V2 can be the human protein designated Q6ZWL3 on the Uniprot website. The CYP4V2 protein can be a wild-type protein, or a mutant formed by substitution, deletion, or substitution of one or more amino acids based on the wild-type protein, or a mutant formed by insertion or deletion of one or more amino acid fragments based on the wild-type protein. It should be noted that, for the detection to proceed smoothly, the mutant of the CYP4V2 protein should have at least partial hydroxylase activity.

[0080] The term "crystalline retinal degeneration" or "BCD" is a rare, inherited degenerative retinal disease caused by mutations in the CYP4V2 gene, characterized by intraretinal lipid crystal deposition, choroidal atrophy, and progressive vision loss. In this application, "BCD" may refer to the disease itself or to complications or sequelae arising from the disease.

[0081] The term "adeno-associated virus receptor" or "AAVR" is the universal major receptor for most adeno-associated virus (AAV) serotypes (such as AAV1, AAV2, AAV5, AAV8, AAV9, etc.) to enter cells. The function of AAVR is to mediate the binding of AAV to host cells and promote viral internalization through clathrin or caveolin-mediated endocytosis, thereby determining the tissue tropism and transduction efficiency of the virus. Studies have found that removing AAVR significantly reduces the infectivity of AAV, indicating its crucial role in AAV gene therapy. AAVR, also known as KIAA0319L, is a multi-transmembrane protein, currently classified in the literature into four major structural / functional domains: signal peptide region (SP, amino acids 1-28), extracellular region (amino acids 29-739), single transmembrane region (TM, amino acids 740-760), and cytoplasmic tail region (C-tail, amino acids 761-835). The extracellular region consists of three tandem carboxypeptidase-like domains (CP1, CP2, and CP3), containing amino acids 29-264, 265-492, and 493-739, respectively. CP1 is the core for AAV binding. The three domains together form a catalytic pocket, but enzyme activity is not essential for the receptor function of AAVR. The single transmembrane region anchors the protein to the cell membrane; deletion or mutation in this region leads to decreased efficiency of AAV entry into cells expressing AAVR. The cytoplasmic tail region participates in downstream signal transduction and protein stability regulation; deletion or mutation in this region also leads to decreased efficiency of AAV entry into cells expressing AAVR. It is important to note that the amino acid site counts are based on the wild-type AAVR protein site count; regardless of any mutations or deletions in AAVR compared to the wild-type, the amino acid site count remains unchanged.

[0082] The expression level of AAVR in wild-type cells is related to cell type, and the expression level varies significantly among different cell types. Therefore, it should be noted that the phrase "cells expressing AAVR" and similar descriptions in this application should be understood as referring to cells with low AAVR expression levels, specifically "cells overexpressing AAVR" or "cells expressing exogenous AAVR." For cells with high AAVR expression levels, this can refer to wild-type cells themselves, or to "cells overexpressing AAVR" or "cells expressing exogenous AAVR." In short, "cells expressing AAVR" should be understood as cells with high AAVR expression levels, regardless of whether they are wild-type cells or transformed cells. The expression level can be obtained by flow cytometry, and "high expression level" refers to an AAVR expression rate greater than 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5%. The expression rate can specifically be the proportion of reporter gene expression in transformed cells, such as the expression proportion of fluorescent protein or drug resistance. More specifically, the expression rate refers to the proportion of cells expressing the reporter gene after transformation out of all transformed cells. A high expression level can also be characterized by a fold change in the mRNA or protein encoding AAVR (e.g., a fold change greater than 2, 3, 4, 5, 10, 20, 30, 40, 50, or 100 times).

[0083] The terms “activity,” “enzyme activity,” “catalytic activity,” “enzyme catalytic activity,” or “hydroxylase activity” in the context of this application refer to the hydroxylase ability of the CYP4V2 protein, specifically its ability to terminally hydroxylate fatty acids or other similar compounds.

[0084] The term "incubation" or "co-incubation" refers to the process of placing two or more systems together in the same container for a period of time. Typically, both systems are liquid, although there are exceptions.

[0085] In this application, the term "culture medium," unless otherwise specified, should be understood as any culture that has not been cultured, or a culture that has just begun, or simply refers to a product contained within a system. It should contain little or no substances not present at the time of manufacture, and its properties should be similar to those at the time of manufacture. The term "cell supernatant," unless otherwise specified, should be understood as the cell culture medium remaining within the system after the cell culture process. It may contain cell debris, cell secretions, and other substances that may be present in the cell culture system. Its properties have changed significantly compared to the culture medium at the time of manufacture, for example, a significant change in pH. It is worth noting that cell supernatant can be directly collected cell supernatant, cell supernatant present directly in the cell culture system without being collected, or cell supernatant collected and then centrifuged to remove cell debris and other solid substances. The term "cell culture medium" refers to the liquid substance present in a cell culture system at any time, and it may contain cell debris and other solid substances present in the system.

[0086] The term "system" refers to the mixture primarily studied at a particular stage of the detection process, and its specific meaning can be determined from the context. For example, in the cell culture stage, the system refers to the cell culture medium; in the medium change stage, the system can refer to the cell culture medium that has been replaced (also called supernatant or cell supernatant), or it can refer to the cell culture medium after the replacement; in the stage where the luciferin precursor reacts to form the luciferin product, the system can be a mixture containing adherent cells, cell culture medium, luciferin precursor, and the luciferin formed by the reaction; in the luciferin detection stage, the system can be cell supernatant or cell lysis buffer. The system can be before incubation, during incubation (the incubation period should be considered as a whole), or after incubation.

[0087] In this application, the term "comprising" generally means "including," "summarizing," "containing," or "encompassing," and these terms are used interchangeably. In some cases, it also means "to be" or "composed of."

[0088] In this application, the term "about" generally refers to a variation within a range of 0.5% to 10% above or below a specified value, such as a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

[0089] In this application, the term “selected from” generally refers to the selection of objects and all combinations thereof. For example, “selected from A, B and C” means all combinations of A, B and C, such as A, B, C, A+B, A+C, B+C, or A+B+C.

[0090] In addition to detecting the hydroxylase activity of CYP4V2 within cells, the method protected in this application can detect the hydroxylase activity of CYP4V2 in any state, such as free CYP4V2, CYP4V2 in viral particles, or CYP4V2 in a medium containing a nucleic acid vector. In specific embodiments, the free CYP4V2 can be present in any medium, such as cell culture medium, body fluids (e.g., blood, urine), or buffer solutions (e.g., PBS).

[0091] As is known to those skilled in the art, fluorescein produced by the hydroxylation of CYP4V2, especially intracellular CYP4V2, from a precursor of fluorescein exists in the cell supernatant. The method for detecting the fluorescein content, fluorescein signal value, or fluorescein luminescence signal value in the cell supernatant can be performed after the cell supernatant is collected; or it can be performed directly without collecting the cell supernatant, for example, by directly adding the fluorescein detection reagent to the cell culture system.

[0092] As is known to those skilled in the art, the hydroxylase activity of CYP4V2 can be characterized within the system. The characterized CYP4V2 can be a major component of the system or an impurity within the system. The impurity can be either an impurity that is allowed to exist in the system or an impurity that needs to be removed from the system.

[0093] In this application, for cells expressing CYP4V2 and AAVR, the order of expression of CYP4V2 and AAVR can be random. For example, CYP4V2 can be expressed first, followed by AAVR, or AAVR can be expressed first, followed by CYP4V2, or they can be expressed simultaneously. The expression can be accomplished by transfecting the nucleic acid encoding the corresponding protein into the cell. The transfection method can be any method selected within the art, such as microinjection, electroporation, DEAE-glucan treatment, lipid transfection, nanoparticle-mediated transfection, virus-mediated transfection, transposon-mediated transfection, protein transduction domain-mediated transfection, and PEG-mediated transfection.

[0094] In addition to predicting the expected therapeutic effect of nucleic acid vectors containing CYP4V2 against BCD, the in vitro detection method for CYP4V2 enzyme activity protected in this application can also be used for other diseases related to CYP4V2 deficiency, such as coronary heart disease, metabolic-associated fatty liver disease, and ischemic stroke. It can be used to predict the expected therapeutic effect of nucleic acid vectors containing CYP4V2 against these diseases, and may also be used for other possible purposes.

[0095] This application also relates to the following:

[0096] 1. A method for detecting the enzyme activity of CYP4V2 in vitro, comprising the following steps:

[0097] a) Incubate the luciferin precursor as shown in Formula I with cells expressing CYP4V2, wherein the incubation system contains a culture medium, wherein R1 in Formula I is an organic substituent connected to an aromatic ring via an ether bond formed by an oxygen atom, and R2 is an alkyl group connected to a carboxylic acid ester group; the alkyl group is preferably a C1-C3 alkyl group, more preferably a methyl group;

[0098] b) Collect or not collect the cell supernatant after incubation, and detect the fluorescein content in the cell supernatant.

[0099] Formula I

[0100] 2. The method according to item 1, wherein the cell is a cell transfected with a viral vector containing nucleic acid encoding CYP4V2.

[0101] 3. According to the method described in item 2, the viral vector is an adeno-associated virus (AAV) vector.

[0102] 4. The method according to any one of items 1-3, wherein the fluorescein precursor is or .

[0103] 5. The method according to any one of items 1-4, wherein the fluorescein precursor is .

[0104] 6. The method according to any one of items 1-5, wherein the cells expressing CYP4V2 also express adeno-associated virus receptor (AAVR), preferably by artificial modification to overexpress adeno-associated virus receptor (AAVR).

[0105] 7. The method according to any one of items 1-6, wherein in step a), the culture medium is a culture medium containing serum or a culture medium not containing serum.

[0106] 8. The method according to any one of items 1-7, wherein the cells expressing CYP4V2 are prepared by infecting cells with a virus, wherein the virus contains a nucleotide sequence encoding CYP4V2, preferably an AAV virus particle containing a nucleotide sequence encoding CYP4V2.

[0107] 9. According to the method described in item 8, the virus infects the cells at a multiplicity of infection (MOI) of about 5E3 to about 5E5.

[0108] 10. The method according to item 8 or 9, wherein the virus infects the cells at a multiple of infection (MOI) of about 1E4 to about 2E5, about 2.4E5 or about 4.8E5.

[0109] 11. The method according to any one of items 1-10, wherein the cells expressing CYP4V2 are prepared by the following steps:

[0110] i. Add the virus dilution to the cell culture medium and incubate, wherein the virus in the virus dilution contains a nucleotide sequence encoding CYP4V2;

[0111] ii. After incubating the virus diluent with the cell culture medium for 60-84 hours, change the medium and continue culturing for another 36-60 hours.

[0112] 12. According to any one of items 1-11, in step a), the final concentration of the fluorescein precursor in the culture medium is 5-200 μM.

[0113] 13. The method according to any one of items 1-12, wherein the detected CYP4V2 is located intracellularly.

[0114] 14. According to any one of items 1-13, in step a), the luciferin precursor is incubated with the cells expressing CYP4V2 for 2-4 hours.

[0115] 15. According to any one of items 1-14, in step b), the method for detecting the fluorescein content in the culture medium comprises the following steps:

[0116] (1) The cell supernatant was co-incubated with the fluorescein detection reagent;

[0117] (2) Detect the fluorescence signal value of the co-incubated solution.

[0118] 16. According to the method of item 15, in step (1), the incubation is carried out under light-protected conditions.

[0119] 17. According to the method of item 15 or 16, in step (1), the incubation is carried out at 37°C.

[0120] 18. According to any one of items 15-17, in step (1), the incubation lasts for 20-40 minutes.

[0121] 19. According to any one of items 15-18, in step (2), the detection of the fluorescence signal value can be performed by one or more of the following methods: fluorescence spectrophotometer, time-resolved fluorescence, fluorescence polarization, wide-field fluorescence microscopy, confocal microscopy, two-photon microscopy, TIRF total internal reflection, conventional flow cytometry, mass cytometry, imaging flow cytometry, fluorescence microplate reading, fluorescence polarization high-throughput screening, AlphaScreen / AlphaLISA, IVIS in vivo imaging system, fiber optic recording, and near-infrared fluorescence imaging.

[0122] 20. The method according to any one of items 1-19, wherein the CYP4V2 is used to treat crystalline retinal degeneration.

[0123] 21. The use of the method of any one of items 1-20 in predicting the expected therapeutic effect of a nucleic acid vector containing CYP4V2 against crystal-like retinal degeneration.

[0124] 22. Use of the compound of Formula I and cells expressing adeno-associated virus receptor (AAVR) in the preparation of a reagent for predicting the expected efficacy of a nucleic acid vector containing CYP4V2 against crystal-like retinal degeneration (BCD).

[0125] 23. A kit comprising a compound of formula I and cells expressing adeno-associated virus receptor (AAVR).

[0126] 24. The kit according to item 23 further comprises instructions for use, which describe the method of any one of items 1-20.

[0127] 25. The kit according to item 23 or 24, used for detecting the enzyme activity of CYP4V2, preferably the intracellular enzyme activity of CYP4V2.

[0128] 26. The kit according to item 23 or 24, for detecting the efficiency of cell transformation containing a nucleic acid vector encoding CYP4V2.

[0129] 27. The kit according to any one of items 23-26, used to predict the expected efficacy of a nucleic acid vector containing CYP4V2 against crystal-like retinal degeneration.

[0130] The embodiments described below are not intended to be limited by any theory, but are merely for illustrating the preparation method and uses of this application, and are not intended to limit the scope of the invention.

[0131] Example

[0132] Example 1. CYP4V2 protein expression after AAV-CYP4V2 transduction into HEK293 cells in vitro.

[0133] To explore the optimal multiplicity of infection (MOI) for inducing intracellular production of CYP4V2 protein, we co-cultured human embryonic kidney epithelial cells (HEK 293) with AAV-CYP4V2 viruses (containing recombinant AAV vectors or viral particles encoding the nucleotide sequence of CYP4V2) at different MOIs (0, 1E5, 5E5, 2E6, 5E6). Cells were collected after incubation for 48 to 72 hours. After sonication lysis, the expression level of CYP4V2 protein was detected using Western blotting (WB).

[0134] Cells and culture conditions: HEK293 cells, 37℃, 5% CO2 incubator.

[0135] Preparation of hydroxyurea (HU): In a clean bench, take hydroxyurea (Sigma) and add it to sterile water for injection to reconstitute. Filter it through a 0.22 μm filter membrane into a new centrifuge tube to obtain a concentration of 660 mM. Aliquot and store at -20℃.

[0136] Preparation of complete culture medium containing 2 mM HU: Take 40 mL of complete culture medium and add 121 μL of HU.

[0137] Operating steps:

[0138] 1) Cell pretreatment

[0139] Remove the cell culture dish, remove the culture medium, add 2 mL of 1×PBS and wash twice, then aspirate the PBS. Add 2 mL of TrypLE™ solution, incubate at 37°C for about 5 minutes, then add 2 mL of 1×PBS to stop the reaction. Transfer all solutions to centrifuge tubes and centrifuge at 1500 rpm for 5 minutes. Discard the supernatant, take an appropriate amount of complete culture medium to resuspend the cell pellet, take an appropriate amount of the resuspended cells and add them to complete culture medium containing HU, mix well, transfer to a culture dish, and incubate at 37±2°C for 24 hours±15 minutes.

[0140] 2) Cell inoculation

[0141] Take the pretreated cells, remove the culture medium, wash twice with 1 mL of 1×PBS, and aspirate the PBS. Add 2 mL of TrypLE™ solution, incubate at 37°C for about 5 minutes, then add 2 mL of 1×PBS to stop the reaction. Transfer all solutions to centrifuge tubes and centrifuge at 1500 rpm for 5 minutes. Discard the supernatant, take an appropriate amount of complete culture medium / complete culture medium-F12K to resuspend the cell pellet and adjust the density to 1E5 cells / mL. Seed 1 mL per well in a 24-well plate.

[0142] 3) Viral transduction

[0143] Add 100 µL / well of AAV-CYP4V2 dilution to the corresponding wells of a 24-well plate. Add 100 µL / well of DMEM basal medium (1×) to the blank control wells. Shake manually for 1 minute and incubate at 37℃±2℃ for about 48-72 hours.

[0144] 4) Cell lysis

[0145] Remove the 24-well plate, remove the culture medium, and add 500 µL of pre-chilled 1×PBS to each well for two washes. Remove the 1×PBS, add 200 µL of pre-chilled cell lysis buffer per well, and place the plate on ice for lysis for 5 minutes. Use a pipette to thoroughly mix the cells and lysis buffer, centrifuge at 12,000 rpm for 10 minutes at 4°C, and collect the supernatant. Mix thoroughly with an appropriate amount of LDS sample buffer (5×), incubate at 95°C for 10 minutes, and store at -80°C for later testing after the sample has returned to room temperature.

[0146] 5) WB detection

[0147] Table 1. WB detection

[0148]

[0149] The results of Western blotting (WB) of protein detection and their gray quantitative analysis (normalized to GAPDH protein expression levels) are shown below. Figure 2 The results showed that when HEK 293 cells were not co-cultured with AAV-CYP4V2 (i.e., MOI=0), no obvious CYP4V2 protein band was detected, indicating that the endogenous CYP4V2 protein expression level in this cell line was extremely low or undetectable. After incubation with AAV-CYP4V2, the expression level of CYP4V2 protein in HEK 293 cells was significantly upregulated compared with the untreated group. Within the MOI range of 5E5~5E6, the CYP4V2 protein expression level increased with increasing MOI, showing a clear concentration-response relationship, and all cells achieved significant CYP4V2 protein expression.

[0150] Example 2. Screening of derivatized fluorescent substrates suitable for the detection of CYP4V2 enzyme activity using fluorescent substrate methods.

[0151] To address the limitations of existing methods for detecting medium-chain saturated fatty acids—high viral vector consumption, low throughput, and variability and uncertainty introduced by multi-step sample pretreatment processes—there is an urgent need to develop a new method that meets the following core requirements: it can reflect enzyme activity in situ within intact cells, more closely mimicking the physiological environment; it can improve detection stability and repeatability; and it can significantly reduce the amount of viral vector used.

[0152] The inventors discovered that specific luciferin precursor substrates can be metabolized into beetle luciferin (D-luciferin) by cytochrome P450 (CYP) family enzymes. This product reacts with luciferase and cofactors (ATP, Mg) naturally present in living cells. 2+ In the presence of O2, an oxidation reaction occurs, producing a chemiluminescent signal that is proportional to the activity of CYP enzymes.

[0153] Based on the above principles, in the examples below, we designed a series of experiments to screen ideal luciferin precursor substrates. Such substrates can be specifically metabolized by CYP4V2 and facilitate highly sensitive in-situ enzyme activity detection. To verify feasibility, we systematically screened a variety of potential substrates, all of which are luciferin derivatives containing CYP enzyme-sensitive groups (such as ether bonds and alkyl chains), which release free D-luciferin after CYP4V2-catalyzed dealkylation / hydroxylation. A brief description of the detection method is as follows: Figure 1 As shown, the cells, treatment time, seeding density, multiplicity of infection (MOI), fluorescence detection method, and the type, concentration, and incubation time of the fluorescent substrate are all exemplary and should be based on the specific application.

[0154] The chemical structural formulas of the potential substrates are shown in the table below.

[0155] Table 2. Chemical structural formulas of potential substrates

[0156]

[0157] The specific experimental design is as follows. The HeLa-AAVR cells used in this example are a cell line stably overexpressing adeno-associated virus receptor (AAVR). The AAVR cDNA sequence is shown in NCBI number NM_024874.5. This cell line was established through lentivirus-mediated transduction. HeLa-AAVR cells were co-cultured with AAV-CYP4V2 virus with a multiplicity of infection (MOI) of 4.8E5. After 72 hours of culture, the medium was changed, and the cells were cultured for another 48 hours. Then, 50 μL of Opti-MEM serum-free medium containing different fluorescent substrates (luciferin-MultiCYP, luciferin-BE, luciferin-PFBE, luciferin-ME-EGE, luciferin-3FEME, and luciferin-CEE, final concentration: 10 µM) was added, and the cells were incubated at 37°C for 3 hours. D-luciferin detection reagent was added to the cell supernatant and cell lysis buffer, and the cells were incubated at 37°C in the dark for 30 minutes. Fluorescence signals were detected, and CYP4V2 enzyme activity was calculated based on the fluorescence signal intensity. The nucleotide sequence of the lentiviral plasmid containing the AAVR gene coding sequence is shown in SEQ ID NO: 2.

[0158] Cells and culture conditions: Hela-AAVR cells, 37℃, 5% CO2 incubator.

[0159] Preparation of complete culture medium 1: In a biosafety cabinet, add 50 mL of FBS to 450 mL of DMEM culture medium, and add 2.5 mL of double antibiotics (penicillin / streptomycin mixture), mix by inversion, store at 5℃±3℃, and use within 1 month.

[0160] Preparation of complete culture medium 2: In a biosafety cabinet, add 50 mL of FBS and 1.5 mg of bleasidium S to 450 mL of DMEM (1X) + GlutaMAX-I medium (Gibco, 10569-010), mix by inversion, store at 5℃±3℃ and use within 1 month.

[0161] Preparation of hydroxyurea (HU): In a clean bench, take hydroxyurea (Sigma) and add it to sterile water for injection to reconstitute. Filter it through a 0.22 μm filter membrane into a new centrifuge tube to obtain a concentration of 660 mM. Aliquot and store at -20℃.

[0162] Preparation of 0.1 mM HU: Take 10 mL of complete culture medium 1 or 2 (the same as the culture medium used for culturing cells), add 6 μL of HU, and prepare fresh before use.

[0163] Preparation of substrate stock solutions: The following solutions can be prepared in proportions according to specific usage conditions. After all stock solutions are prepared, they should be dispensed and stored at ≤-65 ℃.

[0164] Table 3 Preparation of substrate stock solutions

[0165]

[0166] *:ME-EGE is a solution and does not require preparation.

[0167] The specific operation steps are as follows, and the exemplary simplified steps are as follows: Figure 1 As shown.

[0168] 1) Cell pretreatment, exemplary cell pretreatment corresponding to Figure 1 The first step in the process involved treating HEK293-AAVR cells with HU (0.1 mM) for 24 h, followed by 5 × 10⁻⁶ cells. 4 1 cell / well, 96-well cell plate, incubated overnight at 37±2°C.

[0169] Remove the cell culture dish, remove the culture medium, and wash twice with 1×PBS. Add TrypLE™ fast-acting enzyme solution, incubate at 37 ℃ for about 5 minutes, then add 1×PBS to stop the reaction. Transfer all solutions to centrifuge tubes and centrifuge at 1500 rpm for 5 minutes. Discard the supernatant, take an appropriate amount of resuspended cells, add complete culture medium 1 containing 0.1 mM HU, mix well, and transfer to a culture dish. Incubate at 37±2 ℃ for 24 hours±15 minutes. Take the pretreated cells, remove the culture medium, and wash twice with 1×PBS. Add TrypLE™ fast-acting enzyme solution, incubate at 37 ℃ for about 5 minutes, then add 1×PBS to stop the reaction. Transfer all solutions to centrifuge tubes and centrifuge at 1500 rpm for 5 minutes. Discard the supernatant, take an appropriate amount of complete culture medium 1 to resuspend the cell pellet and adjust the cell density to 2.5E5 cells / mL. Take 200 μL / well and add it to a 96-well cell culture plate, i.e., 5×10⁶ cells / well. 4 Cells / well

[0170] 2) Viral transduction, exemplary viral transduction corresponding to Figure 1 The second step is to "remove the cell culture plate, add VGR-R01 to the cell culture plate at an MOI of 1.2E5, incubate for 72 hours (D3), change the medium, and continue culturing for 48 hours (D5)".

[0171] After diluting the AAV-CYP4V2 virus buffer to the appropriate MOI=4.8E5, 20 μL of the diluted virus solution was added dropwise to each well of a 96-well plate. The plate was manually shaken for 1 minute and then incubated at 37℃±2℃ in a CO2 incubator. An equal volume of complete culture medium and one cell as a blank control were added simultaneously. The cells were cultured as follows: After co-incubating the virus and cells for 72 hours, the 96-well plate was removed, the medium was changed, the supernatant was removed, and 200 μL of fresh culture medium was added. The plate was then cultured for another 48 hours (D5).

[0172] 3) Fluorescent substrate incubation, exemplary fluorescent substrate incubation corresponding to Figure 1 The third step in the process is: "After the culture is completed, remove the cell culture plate and remove the supernatant. Add 50 μL of Multi-CYP substrate prepared in Opti-MEM medium to a final concentration of 100 μM, and incubate at 37℃ and 5% CO2 for 0 minutes, 30 minutes, 60 minutes, 90 minutes, and 120 minutes respectively."

[0173] After culture, remove the cell culture plate and discard the supernatant. Add various substrates (final concentration: 10 µM) prepared with 50 μL of Opti-MEM serum-free medium (brand: Gibco, catalog number: 31985-070) to each well. Incubate the culture plate in a 37℃±2℃ CO2 incubator for 3 hours.

[0174] 4) Sample testing, exemplary sample testing corresponding to Figure 1 The fourth step in the process is: "After the reaction is complete, take 25 μL of the cell supernatant and mix it with 25 μL of D-luciferin detection reagent at a 1:1 ratio. Incubate at 37°C in the dark for 30 minutes and then perform chemiluminescence detection."

[0175] Cell supernatant detection: After the reaction, 25 µL of the cell supernatant was mixed with 25 µL of D-fluorescein detection reagent at a 1:1 ratio, and then added to a Pierce™ 96-well polystyrene plate (white and opaque). The plate was placed in a 37°C incubator and incubated in the dark for 30 minutes before detecting the fluorescence signal value.

[0176] Cell lysis buffer was used for detection: After the reaction, 80 μL of cell lysis buffer containing luciferase was added into the cells, and the cells were incubated at 37°C in the dark for 30 minutes. The supernatant of the lysis buffer was then used for detection.

[0177] After incubation with six different probe substrates at the same concentration, the cell supernatant and cell lysate were analyzed. The results are as follows: Figure 3 As shown, among the six substrates tested, fluorescein-MultiCYP and fluorescein-3FEME produced significantly higher detection results than other substrates; in different treatment groups of these two substrates, the detection results of cell supernatant were significantly higher than those of cell lysate.

[0178] Example 3. Screening of fluorescent substrates and substrate incubation conditions suitable for the detection of CYP4V2 protein activity using fluorescent substrate methods.

[0179] To further evaluate the two substrates, luciferin-MultiCYP and luciferin-3FEME, and to screen and determine suitable substrate incubation conditions, we employed the following experimental design: HEK 293-AAVR cells were co-cultured with AAV-CYP4V2 virus at MOIs of 4.8E5 and 1E5, respectively. After transduction for 72 hours, the medium was changed, and the cells were cultured for another 48 hours. Then, luciferin-MultiCYP and luciferin-3FEME were added to serum-free Opti-MEM medium and complete medium 2 at final concentrations of 10 µM, 50 µM, and 100 µM, respectively. The cells were incubated at 37°C for 3 hours. The resulting cell supernatant was mixed 1:1 with D-luciferin detection reagent and incubated at 37°C in the dark for 30 minutes. Fluorescence signals were detected, and CYP4V2 enzyme activity was calculated based on the fluorescence signal intensity.

[0180] Cells and culture conditions: HEK293-AAVR cells, 37℃, 5% CO2 incubator

[0181] The operation steps in this embodiment are as follows, and the exemplary simplified steps are as follows: Figure 1 As shown.

[0182] 1) Cell pretreatment is the same as in Example 2, except that complete culture medium 1 is replaced with complete culture medium 2 due to the different cell lines;

[0183] 2) The virus transfection time is the same as that in Example 2. operate;

[0184] 3) Fluorescent substrate incubation

[0185] After the culture is completed, remove the cell culture plate, remove the supernatant, add 50 μL of Opti-MEM serum-free medium containing fluorescein-MultiCYP, 3FEME (final concentration: 10 µM, 50 µM, 100 µM) to each well, and incubate in a 37℃±2℃ CO2 incubator for 3 hours.

[0186] 4) Sample testing is the same as the sample testing in Example 2. operate.

[0187] To assess the effect of substrate concentration, fluorescein-MultiCYP and 3FEME were incubated in Opti-MEM serum-free and complete media under two different multiplicity of infection (MOI = 4.8E5 or 1E5). The results showed that... Figure 4 (Table 4)

[0188] 1) MOI effect: For both substrates (fluorescein-MultiCYP and fluorescein-3FEME), under the condition of MOI=4.8E5, regardless of whether Opti-MEM serum-free medium or complete medium was used, and regardless of the substrate concentration (10 µM, 50 µM, 100 µM), the detection signal value was significantly higher than the detection result under the corresponding MOI=1E5 condition.

[0189] 2) Substrate concentration and culture medium optimization (MOI=4.8E5):

[0190] Fluorescein-MultiCYP: In complete medium, the differences between AAV-CYP4V2(+) and AAV-CYP4V2(-) at substrate concentrations of 10 µM, 50 µM, and 100 µM were 3200, 1994, and 2018, respectively. The largest detection window was obtained at a concentration of 10 µM. In Opti-MEM serum-free medium, the differences between AAV-CYP4V2(+) and AAV-CYP4V2(-) at substrate concentrations of 10 µM, 50 µM, and 100 µM were 2737, 5051, and 6051, respectively. The largest detection window was obtained at a concentration of 100 µM.

[0191] Fluorescein-3FEME: In complete medium, the differences between AAV-CYP4V2(+) and AAV-CYP4V2(-) at substrate concentrations of 10 µM, 50 µM, and 100 µM were 598, 5560, and 18168, respectively. In Opti-MEM serum-free medium, the differences between AAV-CYP4V2(+) and AAV-CYP4V2(-) at substrate concentrations of 10 µM, 50 µM, and 100 µM were 123, 2092, and 5748, respectively. The largest detection window was obtained at a concentration of 100 µM.

[0192] In summary, under the same detection conditions, the fluorescein-3FEME substrate exhibited a higher background signal value compared to the fluorescein-MultiCYP substrate. Given that a high background signal may interfere with detection specificity, fluorescein-MultiCYP was selected as the preferred substrate for further investigation in subsequent experiments.

[0193] For the luciferin-MultiCYP substrate, experimental data showed that a concentration of 10 µM yielded the largest detection window (difference: 3200) when incubated with complete medium. When incubated with Opti-MEM serum-free medium, a concentration of 100 µM yielded the largest detection window (difference: 6051).

[0194] Table 4. Summary of results for different substrates

[0195]

[0196] Example 4. Study on the AAVR expression efficiency of wild-type HEK293 cells and HEK293-AAVR cells.

[0197] AAVR possesses the dual function of binding and transporting AAV, eliminating the need for other primary receptors or co-receptors and significantly improving AAV transfection efficiency. The inventors used HEK293-AAVR cells, which were obtained by delivering AAVR expression cassettes to HEK293 wild-type cells via lentivirus and undergoing drug resistance selection to obtain a stable AAVR-expressing cell line. The construction process of this cell line is briefly described below.

[0198] First, the lentiviral expression plasmid was cloned. After the coding fragment of the human AAVR gene was synthesized, it was inserted into the lentiviral vector plasmid pBMLY007, forming the pBMLY007-Lenti-AAVR expression plasmid. The main elements of this plasmid include long terminal repeats (LTRs), packaging signals (Ψ), CMV promoter, AAVR CDS region, WPRE element, PGK promoter, and the resistance selection gene budasidin (Blasticidin).

[0199] The next step was lentivirus preparation. Transfection-grade plasmids were prepared and lentiviruses were packaged by Genewiz Biotechnology Co., Ltd., and the titer was determined to be 1.07E+09TU / mL.

[0200] Then, the cell line was constructed. Wild-type HEK293 cells were infected with the aforementioned lentivirus, and two days post-infection, acetaminophen S was added for AAVR-positive cell selection. Two weeks after drug screening, the cell pool was continuously expanded and cryopreserved. Finally, one cell line was thawed and AAV infection titer was assessed, with wild-type HEK293 cells serving as a control. The AAV infection titer of HEK293-AAVR cells was significantly higher than that of wild-type cells, thus confirming that the cell line expresses AAVR.

[0201] To evaluate the impact of AAVR receptor-carrying cells versus receptor-free cells on AAVR expression efficiency, we used a self-developed cell line carrying the AAVR receptor: HEK293-AAVR cells and wild-type HEK293 cells. Flow cytometry was used to detect AAVR expression efficiency. The specific experimental design is as follows: HEK293-AAVR cells and wild-type HEK293 cells were incubated with the AAVR-specific detection antibody KIAA0319L polyclonal antibody (Brand: Proteintech, Catalog No.: 21016-1-AP), respectively. After incubation, a fluorescent staining antibody, Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody (Alexa Fluor™ 488, Brand: Thermo, Catalog No.: A-11008), was added for further incubation. AAVR cell expression rate was then detected using flow cytometry.

[0202] The operation steps in this embodiment are as follows:

[0203] 1) Preparation of cell suspension

[0204] HEK293 and HEK293-AAVR cells were cultured in a 37°C, 5% CO2 incubator. After removing the cell culture dishes and the culture medium, the cells were washed twice with 1×PBS. TrypLE™ fast-acting enzyme solution was added, and the cells were incubated at 37°C for approximately 5 minutes, then 1×PBS was added to terminate the reaction. All solutions were transferred to centrifuge tubes and centrifuged at 1500 rpm for 5 minutes. The supernatant was discarded, and an appropriate amount of culture medium was used to resuspend the cell pellet, which was then counted. After counting, 100,000 cells were placed in 1.5 mL EP tubes, 1 mL of PBS was added, and the cells were centrifuged at 400 g for 5 minutes at room temperature. The supernatant was discarded, and the cells were resuspended in 50 μL of PBS to obtain a cell suspension.

[0205] 2) Cell staining

[0206] Add 1 µL of KIAA0319L polyclonal antibody to each tube and incubate at room temperature in the dark for 30 min. Then add 1 mL of PBS, centrifuge at 400g for 5 min at room temperature, and discard the supernatant. Resuspend the cells in 50 μL of PBS, add 1 µL of goat-derived anti-rabbit IgG heavy and light chain cross-adsorbed secondary antibody (Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488) to each tube, centrifuge at 400g for 5 min at room temperature, and discard the supernatant.

[0207] 3) After resuspending the cells in 50 μL PBS, they were detected by flow cytometry.

[0208] The expression rate of AAVR receptor in wild-type HEK293 cells and HEK293-AAVR cells was detected using an AAVR-specific detection antibody. With an isotype control (the group detected using an isotype control antibody) as a control, the AAVR receptor expression rates in HEK293 and HEK293-AAVR cells were 11.26% and 97.39%, respectively, with mean fluorescence intensity (MFI) values ​​of 4363.2 and 21115.0, respectively. The results indicate that the AAVR receptor expression rate in HEK293-AAVR cells was significantly higher than that in wild-type HEK293 cells (see...). Figure 5 ).

[0209] Example 5. Effect of AAVR receptor on in vitro transduction of AAV-CYP4V2 to express CYP4V2 protein.

[0210] To evaluate the impact of AAVR receptor-carrying cells versus receptor-free cells on AAV-CYP4V2 virus transfection efficiency and on the fluorescence substrate method for detecting CYP4V2 protein activity, we used a self-developed cell line carrying the AAVR receptor (HEK293-AAVR cells) to study the fluorescence substrate method for detecting CYP4V2 protein activity. The specific experimental design was as follows: AAV-CYP4V2 virus with a multiplicity of infection (MOI) of 4.8E5 was co-incubated with wild-type HEK293 or HEK293-AAVR cells. After 72 hours of transduction, the medium was changed, and the cells were cultured for another 48 hours. Then, fluorescein-MultiCYP and 3FEME substrates, prepared in Opti-MEM serum-free medium and complete medium 2 at final concentrations of 10 µM, 50 µM, and 100 µM respectively, were added, and the cells were incubated at 37°C for 3 hours. After the reaction, the cell supernatant was mixed with D-luciferin detection reagent at a 1:1 ratio and incubated at 37°C in the dark for 30 minutes. The fluorescence signal was then detected, and the CYP4V2 enzyme activity was calculated based on the fluorescence signal intensity.

[0211] The reagent preparation and operation steps in this example are the same as those described in Examples 2-3. After the culture is completed, the cell culture plate is removed, the supernatant is removed, and fluorescein-MultiCYP substrate (final concentration: 10 µM, 50 µM, 100 µM) prepared with 50 μL of Opti-MEM serum-free medium is added to each well. The plate is then incubated in a 37℃±2℃ CO2 incubator for 3 hours.

[0212] Experimental results ( Figure 6 The results showed that, under three different substrate concentration gradients (10 μM, 50 μM, and 100 μM), the viral transduction efficiency of the HEK293-AAVR cell group was significantly higher than that of the wild-type HEK293 cell group. In summary, engineered cell lines expressing the AAVR receptor can significantly improve viral transduction efficiency and the width of the downstream detection signal window by enhancing viral vector binding and internalization efficiency.

[0213] Example 6. Specificity study of CYP4V2 protein activity detection by fluorescent substrate method

[0214] To evaluate whether the fluorescent substrate method can specifically detect the activity of CYP4V2 protein, we selected two recombinant AAV viruses that express two other enzymatically active proteins different from CYP4V2: AAV-GCDH (expressing glutaryl-coA dehydrogenase, GCDH) and AAV-GBA1 (expressing glucocerebrosidase, GCase), as control groups. The specific experimental design is as follows: AAV-CYP4V2, AAV-GCDH, and AAV-GBA1 viruses with infection multiples (MOIs) of 4.8E5, 2.4E5, 1.2E5, 6E4, 3E4, 1.5E4, and 7.5E3 were co-incubated with HEK293-AAVR cells. After transduction for 72 hours, the medium was changed and the cells were cultured for another 48 hours. Fluorescein-MultiCYP (final concentration: 100 µM) prepared in serum-free Opti-MEM medium was added, and the cells were incubated at 37°C for 3 hours. After the reaction, the cell supernatant was mixed with D-luciferin detection reagent at a 1:1 ratio and incubated at 37°C in the dark for 30 minutes. The fluorescence signal was then detected, and the CYP4V2 enzyme activity was calculated based on the fluorescence signal intensity.

[0215] The reagent preparation and operation steps in this example are the same as those described in Examples 2-3. After the culture is completed, the cell culture plate is removed, the supernatant is removed, and fluorescein-MultiCYP (final concentration: 100 µM) prepared with 50 μL of Opti-MEM serum-free medium is added to each well. The plate is then incubated in a 37℃±2℃ CO2 incubator for 3 hours.

[0216] Experimental results ( Figure 7 The results showed that in the AAV-CYP4V2 group, the detection signal value increased with increasing MOI, reaching a saturation value at MOI=2.4E5. For AAV-GCDH and AAV-GBA1, the detection signal values ​​did not show a significant trend under different MOI conditions, and the detection signals approached zero. These results indicate that this method can specifically detect the activity of CYP4V2 protein (AAV-CYP4V2 group), while showing no significant response to non-target enzyme active proteins (AAV-GCDH and AAV-GBA1 groups).

[0217] Example 7. Study on the time-signal response relationship of CYP4V2 protein activity detected by fluorescence substrate method in different substrate incubation and culture systems.

[0218] To evaluate the time-signal response relationship of CYP4V2 protein activity detection by fluorescence under different reaction culture conditions, we screened two preferred experimental conditions based on the previous experiments: 1) 100 µM fluorescein-MultiCYP / Opti-MEM serum-free medium; 2) 10 µM fluorescein-MultiCYP / complete medium. The following experimental design was adopted: AAV-CYP4V2 virus with an infection multiplicity (MOI) of 4.8E5 was co-incubated with HEK 293-AAVR cells, transduced for 72 hours, and cultured for another 48 hours after changing the medium. Fluorescein-MultiCYP (final concentration: 100 µM) prepared in Opti-MEM serum-free medium or 10 µM prepared in complete medium 2 was added to each well, and incubated for 0 hours, 1 hour, 2 hours, and 3 hours. After the reaction, the cell supernatant was mixed with D-luciferin detection reagent at a 1:1 ratio and incubated at 37°C in the dark for 30 minutes. The fluorescence signal was then detected, and the CYP4V2 enzyme activity was calculated based on the fluorescence signal intensity.

[0219] The reagent preparation and operation steps in this example are the same as those described in Examples 2-3. After the culture is completed, remove the cell culture plate, remove the supernatant, and add 50 μL of fluorescein-MultiCYP prepared in serum-free Opti-MEM medium (final concentration: 100 µM) or fluorescein-MultiCYP prepared in complete medium 2 (final concentration: 10 µM) to each well. Incubate in a 37℃±2℃ CO2 incubator for 0 hours, 1 hour, 2 hours, and 3 hours.

[0220] The experimental results are shown in Table 5. Figure 8 Under serum-free medium conditions of 100 µM fluorescein-MultiCYP / Opti-MEM, a good linear relationship was observed between different incubation times and the detection signal value (R² = 0.983); under conditions of 10 µM fluorescein-MultiCYP / complete medium, the linear relationship between different incubation times and the detection signal value was relatively weaker (R² = 0.822). In summary, the combination of 100 µM fluorescein-MultiCYP / Opti-MEM serum-free medium exhibited a better linear relationship (R² = 0.983), therefore, this method uses this condition for substrate incubation.

[0221] Table 5. Results of incubation in different culture media

[0222]

[0223] Example 8. Study on the time-signal response relationship of CYP4V2 protein activity detected by fluorescence substrate method under different substrate incubation time conditions.

[0224] To evaluate the signal response of different substrate incubation times to the detection of CYP4V2 protein activity using a fluorescence substrate assay, we selected two different substrate incubation times, 3 hours and 18 hours, for preliminary research. The experimental design was as follows: AAV-CYP4V2 virus with infection multiplicity (MOI) of 4.8E5 and 1E5 was co-incubated with HEK 293-AAVR cells, respectively, for 72 hours after transduction. After medium replacement, the cells were cultured for another 48 hours. Fluorescein-MultiCYP (final concentration: 100 µM) prepared in serum-free Opti-MEM medium was added to each well, and the cells were incubated for 3 hours and 18 hours, respectively. The cell supernatant after the reaction was mixed with D-luciferin detection reagent at a 1:1 ratio, and incubated at 37°C in the dark for 30 minutes. The fluorescence signal was then detected, and the CYP4V2 enzyme activity was calculated based on the fluorescence signal intensity.

[0225] The reagent preparation and operation steps in this example are the same as those described in Examples 2-3. After the culture is completed, the cell culture plate is removed, the supernatant is removed, and fluorescein-MultiCYP (final concentration: 100 µM) prepared with 50 μL of serum-free Opti-MEM medium is added to each well. The plate is then incubated in a 37℃±2℃ CO2 incubator for 3 hours or 18 hours.

[0226] Experimental results ( Figure 9 The results showed that, under different MOI conditions, the detection windows (difference between AAV-CYP4V2(+) and AAV-CYP4V2(-)) after substrate incubation for 3 hours and 18 hours were compared: When MOI=4.8E5: the detection window for 3 hours of incubation was 6051, and the detection window for 18 hours of incubation was 2674; when MOI=1E5: the detection window for 3 hours of incubation was 1059, and the detection window for 18 hours of incubation was 617. In summary, under both MOI conditions, the detection window obtained by substrate incubation for 3 hours was significantly higher than that obtained by incubation for 18 hours.

[0227] Based on the previously discovered advantage of the 3-hour incubation window, to further determine the optimal kinetic relationship between the incubation time of the luciferin-MultiCYP substrate and the fluorescence signal, we adopted the following experimental design: AAV-CYP4V2 virus with infection multiplicity (MOI) of 2E5, 1.2E5, 1E5, and 0.5E5 was co-incubated with HEK 293-AAVR cells. After transduction for 72 hours, the medium was changed, and the cells were cultured for another 48 hours. Luciferin-MultiCYP (final concentration: 100µM) prepared in serum-free Opti-MEM medium was added to each well, and incubation was performed for 0 min, 45 min, 135 min, and 180 min, respectively. The cell supernatant after the reaction was mixed with D-luciferin detection reagent at a 1:1 ratio, and incubated at 37°C in the dark for 30 minutes before fluorescence detection.

[0228] The reagent preparation and operation steps in this example are the same as those described in Examples 2-3. After the culture is completed, the cell culture plate is removed, the supernatant is removed, and 50 μL of fluorescein-MultiCYP prepared in serum-free Opti-MEM medium (final concentration: 100 µM) is added to each well. The plate is then placed in a CO2 incubator at 37℃±2℃ and incubated for 0 minutes, 45 minutes, 135 minutes, and 180 minutes.

[0229] The response relationship between different incubation times and detection signal values ​​was investigated under four different MOI conditions (2E5, 1.2E5, 1E5, 0.5E5). Results ( Figure 10 The results show that under all tested MOI conditions, the detection signal value increased with increasing incubation time, and reached saturation at approximately 135 minutes of incubation. When the incubation time was extended to 180 minutes, the signal value decreased. These results indicate that, under the experimental parameters used, the detection signal value reached its maximum peak at approximately 135 minutes of substrate incubation under different MOI conditions.

[0230] Example 9. Study on MOI-signal response relationship of CYP4V2 protein activity detected by fluorescence substrate method under different multiplicity of infection (MOI)

[0231] To evaluate the MOI-signal response relationship of fluorescence detection of CYP4V2 protein activity under different MOI conditions, we selected a series of different MOIs and co-cultured HEK 293-AAVR cells to detect CYP4V2 protein activity in the cell supernatant. The specific experimental design is as follows: AAV-CYP4V2 virus with MOIs of 4.8E5, 2.4E5, 1.2E5, 6E4, 3E4, 1.5E4, 7.5E3, and 0 was co-incubated with HEK 293-AAVR cells. After transduction for 72 hours, the medium was changed and the cells were cultured for another 48 hours. Fluorescein-MultiCYP (final concentration: 100 µM) prepared in serum-free Opti-MEM medium was added to each well, and the cells were incubated at 37°C for 3 hours. After the reaction, the cell supernatant was mixed with D-luciferin detection reagent at a 1:1 ratio and incubated at 37°C in the dark for 30 minutes. The fluorescence signal was then detected, and the CYP4V2 enzyme activity was calculated based on the fluorescence signal intensity.

[0232] The reagent preparation and operation steps in this example are the same as those described in Examples 2-3. After the culture is completed, the cell culture plate is removed, the supernatant is removed, and 50 μL of fluorescein-MultiCYP prepared in serum-free Opti-MEM medium (final concentration: 100 µM) is added to each well. The plate is then incubated in a CO2 incubator at 37℃±2℃ for 3 hours.

[0233] By detecting signal values ​​under different MOI conditions and plotting MOI-signal relationship curves ( Figure 11 The results showed that the detection signal value peaked at MOI = 2.4E5; when MOI increased to 4.8E5, the signal value tended to stabilize and then declined; within the MOI range of 0–4.8E5, the linear relationship between the detection signal value and MOI was relatively weak (R² = 0.778); within the MOI range of 7.5E3–2.4E5, the detection signal value and MOI showed a good linear relationship (R² = 0.996). Therefore, 7.5E3–2.4E5 is the ideal MOI range for enzyme activity studies.

[0234] Repeatability analysis was performed on 7 different MOIs (Table 6). When three independent determinations were performed under the condition of MOI=1.2E5, the standard deviation (SD) was 14.9 and the coefficient of variation (CV%) was 0.5, indicating that the method has good repeatability under this condition.

[0235] Table 6. Summary of MOI Linear Relationship Results

[0236]

Claims

1. A method for detecting the enzyme activity of CYP4V2 in vitro, comprising the following steps: a) The luciferin precursor was incubated with HEK293 cells expressing CYP4V2. The incubation system includes a culture medium, which is an Opti-MEM medium without serum, and the final concentration of the luciferin precursor in the culture medium is 50-100 μM; b) After incubation, the cell supernatant is collected and the luciferin content in the cell supernatant is detected; wherein, the cells expressing CYP4V2 are cells transfected with an adeno-associated virus (AAV) vector containing nucleic acid encoding CYP4V2, the AAV infects the cells multiple times at 1E5-4.8E5, and the cells expressing CYP4V2 are also artificially modified to overexpress the adeno-associated virus receptor AAVR, the amino acid sequence of which is shown in SEQ ID NO:

1.

2. The method according to claim 1, wherein in step a), the luciferin precursor is incubated with the cells expressing CYP4V2 for 2-4 hours.

3. The method according to claim 1, wherein the adeno-associated virus infects the cells multiple times at 2.4E5-4.8E5.

4. The method according to claim 1, wherein the preparation of cells expressing CYP4V2 comprises: After incubating the cells with the AAV vector containing the nucleotide sequence encoding CYP4V2 for 60-84 hours, the medium was changed, and the cells were cultured for another 36-60 hours.

5. According to claim 4, the AAV vector is incubated with the cells for 72 hours, the medium is changed, and the cells are cultured for another 48 hours.

6. The method according to any one of claims 1-5, wherein in step b), the method for detecting the fluorescein content comprises: The cell supernatant was co-incubated with a fluorescein detection reagent, and the fluorescence signal of the co-incubated solution was detected.

7. The method according to claim 6, wherein the incubation of the cell supernatant with the fluorescein detection reagent is carried out under light-protected conditions.

8. The method according to claim 6, wherein the cell supernatant is incubated with the fluorescein detection reagent at 37°C.

9. The method according to claim 6, wherein the cell supernatant is incubated with the fluorescein detection reagent for 20-40 minutes.

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