A method for screening genes related to lysosomal storage disease based on a lysosome overload model of caenorhabditis elegans and application thereof

Abstract

The application provides a method for screening lysosomal storage disease related genes based on a Caenorhabditis elegans lysosome overload model and application thereof, the method constructs a transgenic nematode strain containing an intestinal specific promoter nhx-2p driven CPL-1 and red fluorescent protein wrmScarlet fusion expression vector, induces intestinal cell secretion CPL-1::wrmScarlet fusion protein to body cavity fluid and is endocytosed in the body cavity cell and accumulated in the lysosome to form a quantifiable overload phenotype, then adopts feeding RNA interference technology to knock down gene expression, and observes the change of red fluorescence signal intensity of the body cavity cell under the stereoscopic fluorescence microscope to screen candidate genes for promoting or relieving lysosome accumulation, meanwhile, the model can be used for drug screening, has the advantages of observing under a low power lens, high throughput, quantification and convenient operation on the level of live animals, and solves the long-existing key technical bottleneck in the field of lysosomal storage disease.

Description

Technical Field

[0001] This invention relates to the fields of genetics and disease modeling, specifically to a coelomic cell lysosomal overload model constructed using Caenorhabditis elegans, and a method for high-throughput screening and identification of genes related to lysosomal storage diseases using this model. Background Technology

[0002] Lysosomal storage diseases (LSDs) are a group of inherited metabolic disorders caused by gene mutations leading to lysosomal dysfunction and the accumulation of macromolecules within cells due to their inability to degrade. Most LSDs are severe and lack a cure. Affected children often die young due to severe neurodegenerative changes, recurrent infections, or cardiopulmonary failure. Current treatments have very limited effectiveness for these predominantly neurological severe diseases. Identification of LSD-related genes currently relies mainly on gene sequencing of clinical patients, but this method struggles to discover new modified genes or genes with unknown functions. Traditional mammalian models (such as gene knockout mice) are costly and time-consuming, making large-scale forward genetic screening difficult. *C. elegans* possesses unique advantages, including complete transparency, abundant genetic tools, a short lifespan, and suitability for high-throughput manipulation. Its cellular functions are highly conserved evolutionarily, making it an ideal model for studying the function of organelles such as lysosomes. However, there are currently no reports of using induced coelomic cell lysosomal overload phenotypes for systematic gene screening.

[0003] In the prior art, the animal models of *C. elegans* are commonly found in: CN115067283B Obesity model, CN112544571B Establishment and application of *C. elegans* model for evaluating the safety and efficacy of sunscreens, and CN112471084B A method for constructing and applying a *C. elegans* high-glucose insulin resistance animal model.

[0004] Furthermore, in other animal models of the same disease, high magnification is required to observe the relevant phenotypes. In other words, there is currently a lack of ideal models in the field that can stably, specifically, and intuitively and sensitively report lysosomal storage function. Although existing cell models can be used for high-throughput screening, they cannot simulate the complex tissue interactions in vivo and there are currently no lysosomal storage phenotypes that can be observed under low magnification. On the other hand, animal models such as zebrafish and mice cannot be used for large-scale screening due to the complexity of phenotype reading, low throughput, and high cost.

[0005] Many core phenotypes of lysosomal dystrophins (LSDs), such as neurological degeneration, visceral enlargement, and lysosomal swelling, are difficult to detect in miniature model organisms. Furthermore, adding exogenous lysosomal aggregates to cell models cannot directly induce a clear lysosomal storage phenotype under low magnification. Therefore, there is currently a lack of an animal model that can conveniently, sensitively, and intuitively detect the lysosomal storage phenotype. Although various methods for labeling lysosomes exist, developing a stable, specific, and quantitative fluorescent reporter model that reflects this functional state of lysosomal storage remains a challenge and a cutting-edge direction in the field.

[0006] Therefore, there is an urgent need in this field for a novel phenotypically intuitive and quantifiable model for inducing lysosomal storage disease in *C. elegans*. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a method for screening genes related to lysosomal storage diseases based on a lysosomal overload model of *C. elegans*, and its application. Through genetic manipulation, functional overload or storage of lysosomes in six coelomocytes of *C. elegans* is specifically induced, resulting in a quantifiable phenotype. The specific implementation of the genetic manipulation is as follows: utilizing the highly active secretory function of *C. elegans* intestinal cells and the highly active endocytic function of coelomocytes, the lysosomal-localized hydrolase cathepsin L (CPL-1) is specifically expressed in intestinal cells, causing it to be secreted and released into the coelom via the fusion of the lysosomal membrane and the intestinal plasma membrane; subsequently, CPL-1 is endocytosed by the coelomocytes and completely accumulated in LMP-1-labeled lysosomes. Figure 2 Specifically, the method involved constructing the Is[nhx-2p::CPL-1::wrmScarlet] strain by driving CPL-1::wrmScarlet expression using the gut-specific promoter nhx-2p for subsequent screening. Its core effect and advantage lies in the fact that it is the first time that a lysosomal storage phenotype can be directly observed under low magnification and subjected to large-scale genetic or drug screening at the live animal level, solving a long-standing key technical bottleneck in this field.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A method for screening genes related to lysosomal storage diseases based on the lysosomal overload model of *C. elegans* includes the following steps:

[0010] 1) Construct a fusion expression vector of CPL-1 driven by the intestinal-specific promoter nhx-2p and red fluorescent protein wrmScarlet, and transform it into Caenorhabditis elegans to obtain a stable transgenic line;

[0011] 2) By inducing intestinal cells to secrete CPL-1::wrmScarlet fusion protein, it is absorbed by coelomic cells through coelomic fluid and accumulates in lysosomes, forming a lysosomal overload phenotype.

[0012] 3) An RNA interference feeding method was used, in which RNAi bacteria carrying different target gene fragments were fed to nematodes to knock down the expression of the corresponding genes.

[0013] 4) Observe the changes in the intensity of red fluorescence signal in coelomic cells under a stereofluorescence microscope, and determine the functional status of lysosomes based on the differences in fluorescence signal intensity;

[0014] 5) Genes corresponding to enhanced fluorescence signals were identified as candidate genes for promoting lysosomal accumulation, and genes corresponding to weakened signals were identified as candidate genes for alleviating or repairing lysosomal function.

[0015] Preferably, the *C. elegans* strain is an optimized strain based on the wild-type N2.

[0016] Furthermore, the method for constructing *C. elegans* strains expressing the CPL-1::wrmScarlet fusion protein for screening lysosomal storage disease-related genes includes the following steps:

[0017] S1. Construction of recombinant plasmid: Using the PBSK vector as a backbone, the promoter sequence of the *C. elegans* nhx-2 gene, the full-length sequence or functional fragment sequence encoding the CPL-1 protein, and the coding sequence of the *wrmScarlet* fluorescent protein reporter gene were operatively ligated to obtain the recombinant expression cassette *nhx-2p::CPL-1::wrmScarlet*.

[0018] S2. Microinjection transformation: Using wild-type Caenorhabditis elegans N2 strain adults (D1 stage) as the target, the recombinant plasmid obtained in step S1 was introduced into the gonadal germ cell region using gonadal microinjection technology;

[0019] S3. The P0 generation of Caenorhabditis elegans that survived the injection were cultured, and transgenic individuals carrying extrachromosomal arrays were screened from their F1 generation offspring by observing the signal of the wrmScarlet reporter gene. Subsequently, the obtained strains were irradiated using an RS-2000 Pro X-ray irradiation system (Rad Source) to induce the stable integration of exogenous transgenes into the genome. Finally, genetically stable transgenic Caenorhabditis elegans strains were obtained through passage culture and fluorescence screening.

[0020] Preferably, the CPL-1 fusion protein is released into the coelomic fluid via a non-classical protein secretion pathway through nematode intestinal cells, is internalized by coelomic cells at six fixed locations and enriched in their lysosomes, thereby inducing the formation of a reproducible lysosomal overload phenotype.

[0021] Preferably, the lysosomal overload phenotype is obtained by directly observing the intensity and distribution of red fluorescence signals in coelomic cells using a low-power fluorescence microscope.

[0022] Preferably, to ensure consistent induction, the nematodes are subjected to fluorescence signal collection on the third day of adulthood, and each treatment group contains at least fifty synchronized nematodes.

[0023] Preferably, RNA interference feeding uses a solid IPTG plate culture system, with each plate containing approximately fifty nematodes and corresponding RNAi bacterial suspensions. Fluorescence signals are detected after 5 days of incubation.

[0024] Preferably, the selected candidate genes are further examined using confocal microscopy to clarify the lysosomal storage status of coelomic cells.

[0025] Preferably, the fluorescence signal intensity is calculated using image analysis software to determine the average gray value, thereby quantitatively assessing the degree of lysosomal accumulation and establishing a three-level grading standard of strong, medium, and weak fluorescence.

[0026] A second aspect of the present invention provides a lysosomal overload model of Caenorhabditis elegans obtained based on the method described above. This model can exhibit the accumulation of fluorescent signals in coelomic cell lysosomes in vivo and has the characteristics of visualization, quantification and high-throughput detection.

[0027] A third aspect of the present invention provides a method for drug screening using the Caenorhabditis elegans lysosome overload model, wherein synchronized transgenic nematodes are exposed to a solution of the test compound for 72 hours, and the changes in fluorescence signal intensity of coelomic cells are observed to screen for candidate compounds that can reduce the lysosome overload phenotype.

[0028] A fourth aspect of this invention provides a screening system based on a lysosomal overload model of *C. elegans*, which can be used for screening genes related to lysosomal storage diseases, verifying lysosomal functional regulatory pathways, and developing drugs for lysosomal homeostasis regulation. This system uses changes in lysosomal fluorescence signals in coelomic cells as a unified detection indicator, achieving rapid and stable in vivo screening.

[0029] The beneficial effects of this invention are:

[0030] 1) Quantitative classification of lysosomal overload (strong / weak / moderate), achieving both intuitiveness and throughput;

[0031] 2) By artificially inducing specific cell overload through transcellular pathways (intestinal secretion → coelomic endocytosis), continuous secretion of intestinal cells and continuous endocytosis of coelomic cells were achieved in live animals. This can efficiently and stably induce lysosomal accumulation in vivo, and the resulting lysosomal accumulation phenotype is significantly better than the effect achieved by exogenous inducers in the currently disclosed technologies.

[0032] 3) It overcomes the limitations of traditional LSD animal models, which rely on cumbersome tissue sections, staining, or high-power microscopy for phenotypic observation, enabling direct, rapid, and non-destructive phenotypic observation of live nematodes under a low-power microscope. This makes ultra-high-throughput genetic and pharmacological screening possible, greatly improving the efficiency of discovering new genes and lead compounds;

[0033] In summary, this invention provides a powerful research tool that perfectly balances screening throughput, cost, ease of operation, and in vivo physiological relevance. It offers an unparalleled platform for the rapid discovery of novel LSD-related genes and the development of innovative therapies, and is expected to greatly accelerate the basic research and drug development process for lysosomal storage diseases. Attached Figure Description

[0034] Figure 1 This is a schematic diagram illustrating the technical principle of inducing lysosomal storage in coelomic cells through intestinal-specific expression of CPL-1::wrmScarlet.

[0035] Figure 2 Map showing the specific localization of CPL-1::wrmScarlet in lysosomes of coelomic cells;

[0036] Figure 3 For the screening process flowchart;

[0037] Figure 4 This is a phenotypic comparison diagram observed in Example 1;

[0038] Figure 5 This is a phenotypic comparison diagram observed in Example 2. Detailed Implementation

[0039] The present invention will be further described below through specific embodiments. To make the inventive objectives, technical solutions, and beneficial technical effects of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments. It should be understood that the embodiments described in this specification are merely for explaining the present invention and are not intended to limit the present invention.

[0040] Unless otherwise stated, all instruments and reagents used in the examples are commercially available or synthesized using conventional methods and can be used directly without further processing, and all instruments used in the examples are commercially available.

[0041] Example 1: Determination of the gene cup-5 (R13A5.1 corresponding to ORF 11010H4) that induces LSDs

[0042] Step 1: Remove the 96-well motherboard numbered 11010 from the -80°C ultra-low temperature freezer.

[0043] Dip the pipette tip into the bacterial clone in the corresponding well of row H, column 4 of the mother plate and inoculate it into a centrifuge tube containing approximately 800 μL of LB liquid medium (supplemented with 50 μg / ml ampicillin and 50 μg / ml tetracycline). Incubate at 37°C for 12-16 hours (overnight) to allow the bacteria to grow to saturation.

[0044] Step 2: Preparation of RNAi plates and induction of dsRNA expression

[0045] Prepare solid NGM agar plates with a diameter of 5 cm containing carbenicillin (50 μg / ml) and IPTG (2 mM) in advance.

[0046] Use a pipette to draw 350 μl of bacterial culture and spread it evenly onto an NGM plate, labeling it (11010-H4). Incubate the plate at room temperature for 24 h to induce the production of nematode gene knockdown dsRNA.

[0047] Step 3: Synchronize nematodes

[0048] Under standard culture conditions (20 °C), large numbers (more than 1000) of Is[nhx-2p::CPL-1::wrmScarlet] nematodes for selection were cultured on solid NGM plates. Day-old adult nematodes were washed off the NGM plates with M9 buffer and collected in 15 ml centrifuge tubes. After centrifugation to remove the M9 buffer, the nematodes were treated with 5 ml of bleach (2 ml sodium hypochlorite (bleach), 1 ml 5M sodium hydroxide, and 2 ml dd H2O). Purified embryos were collected by centrifugation and washing. The embryos were incubated in sterile M9 buffer at 20 °C with shaking for approximately 18 h to obtain a large number of L1-stage larvae with fully synchronized age.

[0049] Step 4: Inoculate with nematodes

[0050] The synchronized L1 larvae were resuspended in M9 buffer, and the nematode density was adjusted. Approximately 50 L1 larvae were inoculated onto 11010-H4 plates using a pipette. Nematodes inoculated onto HT115 bacterial plates containing the empty vector pL4440 served as a control.

[0051] Step 5: Cultivation

[0052] The inoculated plates were placed in a 20°C incubator. On the third day of culture, adult worms were eluted with M9 and transferred to new RNAi plates to avoid starvation.

[0053] Step 6: Observe under a low-power stereofluorescence microscope

[0054] On the fifth day of nematode culture (third day after the nematodes reach adulthood), the fluorescence signal of six coelomic cells was directly observed under the red channel of a stereofluorescence microscope on a 11010-H4 plate. The fluorescence signal of six coelomic cells from nematodes grown on a control plate was used as a control. Figure 3 In study A), the fluorescence signal of nematode coelomic cells on the 11010-H4 plate was found to be significantly stronger than that in the control group. Figure 3 (B vs A). This indicates that the gene knocked down by RNAi bacteria on the 11010-H4 plate is a candidate gene for inducing LSDs.

[0055] Step 7: Slide preparation and phenotypic observation under high magnification

[0056] Nematodes on 11010-H4 plates were fixed onto 3% agarose pads and images were randomly taken after anesthesia with 10 mM levamisole hydrochloride. The accumulation of lysosomal substrates in coelomic cells was observed and photographed using a Zeiss LMS900 laser confocal microscope equipped with a 63x oil immersion microscope. Figure 4 (D and C). Quantitative analysis further showed that the fluorescence signal in the lysosomes of nematode coelomic cells on the 11010-H4 plate was significantly stronger than that in the control group (D and C). Figure 4 (E). The gene number 11010-H4 can be found in the ORF RNAi library as R13A5.1. The gene number R13A5.1 can be found on the WormBase online website as cup-5.

[0057] Example 2: Determination of the gene ppk-1 (F55A12.3 corresponding to ORF 11203G1) for alleviating LSDs

[0058] Step 1: Remove the 96-well master plate (numbered 11203G1) from the -80°C cryogenic freezer. Dip a pipette tip into the bacterial clone in the corresponding well of row G, column 1, and inoculate it into a centrifuge tube containing approximately 800 μL of LB liquid medium (supplemented with 50 μg / ml ampicillin and 50 μg / ml tetracycline). Incubate at 37°C for 12–16 h (overnight) to allow the bacteria to reach saturation.

[0059] Step 2: Preparation of RNAi plates and induction of dsRNA expression

[0060] Prepare solid NGM agar plates with a diameter of 5 cm containing carbenicillin (50 μg / ml) and IPTG (2 mM) in advance.

[0061] Use a pipette to draw 350 μl of bacterial culture and spread it evenly onto an NGM plate, labeling it (11203-G1). Incubate the plate at room temperature for 24 h to induce the production of nematode gene knockdown dsRNA.

[0062] Step 3: Synchronize nematodes

[0063] Under standard culture conditions (20 °C), large numbers (more than 1000) of Is[nhx-2p::CPL-1::wrmScarlet] nematodes for selection were cultured on solid NGM plates. Day-old adult nematodes were washed off the NGM plates with M9 buffer and collected in 15 ml centrifuge tubes. After centrifugation to remove the M9 buffer, the nematodes were treated with 5 ml of bleach (2 ml sodium hypochlorite (bleach), 1 ml 5M sodium hydroxide, and 2 ml ddH2O). Purified embryos were collected by centrifugation and washing. The embryos were incubated in sterile M9 buffer at 20 °C with shaking for approximately 18 h to obtain a large number of L1-stage larvae with fully synchronized age.

[0064] Step 4: Inoculate with nematodes

[0065] The synchronized L1 larvae were resuspended in M9 buffer, and the nematode density was adjusted. Approximately 50 L1 larvae were inoculated onto a 11041-E2 plate using a pipette. Nematodes inoculated onto an HT115 bacterial plate containing the empty vector pL4440 served as a control.

[0066] Step 5: Cultivation

[0067] The inoculated plates were placed in a 20 °C incubator. On the third day of culture, adult worms were eluted using M9 and transferred to new RNAi plates.

[0068] Step 6: Observe under a low-power stereofluorescence microscope

[0069] On the fifth day of nematode culture (third day after the nematodes reach adulthood), the fluorescence signal of six coelomic cells was directly observed under the red channel of a stereofluorescence microscope on an 11203-G1 plate. The fluorescence signal of six coelomic cells from nematodes grown on a control plate was used as a control. Figure 4 In study A), the fluorescence signal of nematode coelomic cells on the 11203-G1 plate was found to be significantly weaker than that in the control group. Figure 4 (B vs A). This indicates that the corresponding gene knocked down by RNAi bacteria on the 11010-E2 plate is a candidate gene for alleviating LSDs.

[0070] Step 7: Slide preparation and phenotypic observation under high magnification

[0071] Nematodes on 11203-G1 plates were fixed onto 3% agarose pads and anesthetized with 10 mM levamisole hydrochloride before random image capture. Lysosomal substrate accumulation in coelomic cells was observed and imaged using a Zeiss LMS900 laser confocal microscope equipped with a 63× oil immersion lens. Figure 5 (D and C). Quantitative analysis further showed that the fluorescence signal of nematode coelomic cells on the 11203-G1 plate was significantly weaker than that in the control group (D and C). Figure 5 (E). The gene number 11203-G1 can be found in the ORF RNAi library as F55A12.3. The gene number F55A12.3 can be found on the WormBase online website as ppk-1.

[0072] Comparative Example 1:

[0073] This invention relates to a nematode RNAi screening method based on the CPL-1::wrmScarlet reporter system, which offers significant advantages in efficiency and cost-effectiveness compared to traditional mammalian cell CRISPR-Cas9 screening. Traditional methods suffer from bottlenecks such as long processing times (6-10 weeks), high costs (US$50,000-150,000 per screening), and reliance on expensive equipment (such as flow cytometry sorters costing millions of dollars) due to their technical complexity. This invention utilizes the strong fluorescence signal generated by CPL-1::wrmScarlet during lysosomal accumulation, simplifying phenotypic detection to direct low-power microscopic observation. This reduces the screening cycle to 6 days and the cost to the thousands of dollars, while avoiding the time-consuming 1-2 weeks and tens of thousands of dollars required by traditional methods for next-generation sequencing (NGS) and complex bioinformatics analysis. This invention enables rapid, intuitive, and high-throughput screening of candidate genes with low technical and financial barriers, providing a new and efficient platform for lysosomal storage disease research.

[0074] Comparative Example 2:

[0075] While using the Drosophila model for genome-wide screening of lysosomal storage diseases can reduce the cost of a single hybridization to a few yuan, the throughput is low and the results are not ideal. Furthermore, the cost of single-gene screening is skyrocketing, reaching at least ten times that of Caenorhabditis elegans.

[0076] One of the most important genetic systems used in Drosophila is the GAL4 / UAS system. Its core principle for screening genes related to lysosomal storage diseases lies in using tissue-specific GAL4 drivers to express LSD-causing genes in Drosophila with labeled lysosomes, constructing a disease model exhibiting phenotypes such as shortened lifespan and impaired motor function. Then, by hybridizing this strain with a genome-wide UAS-RNAi library, specific genes are systematically knocked down, and genetic modifiers that enhance or inhibit the aforementioned disease phenotypes are screened. Although such behavioral or macroscopic morphological phenotypes are easy to assess, they cannot directly reflect changes in the microscopic pathological state of lysosomes, and therefore cannot accurately assess the specific impact of gene perturbation on the degree of storage. High-magnification confocal microscopy is necessary to image lysosomes in order to directly assess changes in the number, volume, and degree of lysosome storage.

[0077] The efficiency difference is significant: For example, based on the CPL-1::wrmScarlet reporter gene model, direct observation of the lysosomal storage status of a single sample under a dissecting microscope takes only about 1 minute. In contrast, traditional methods based on the GAL4 / UAS system typically require more than 20 minutes per sample to obtain direct lysosomal phenotypic images (including sample preparation and image acquisition) using a high-power fluorescence microscope, resulting in an overall efficiency reduction of about 20 times.

[0078] Comparative Example 3:

[0079] While zebrafish models offer unique value in accurately predicting drug efficacy and toxicity in vivo due to their complex organ systems (such as the functional liver and vascularized nervous system) and complete pharmacokinetic environment, enabling genome-wide screening for lysosomal storage diseases, zebrafish have inherent limitations in genetic screening throughput compared to *C. elegans*. Zebrafish have a longer sexual maturation period of 3-4 months, more than 30 times that of *C. elegans* (approximately 3 days); their daily rearing costs are significantly higher, exceeding those of *C. elegans* by hundreds of times; and their genetic manipulation relies on microinjection, resulting in a single-session throughput (hundreds of embryos) that is 1-2 orders of magnitude lower than the batch-processable RNAi feeding technology for *C. elegans*. Therefore, zebrafish are not suitable for primary functional screening at the genome-wide scale; their core advantage lies in validating high physiological relevance during the preclinical drug development stage.

[0080] Comparative Example 4:

[0081] Genome-wide screening for lysosomal storage diseases using mouse models is extremely rare and almost impossible in routine scientific research. The core requirements for genome-wide screening are high throughput, high efficiency, and low cost, all of which mouse models suffer from fatal flaws. Compared to the bottlenecks faced by mouse models, such as long reproductive cycles (6-8 weeks to sexual maturity), limited litter size (usually 8-12 offspring / litter), and high costs (tens of thousands of yuan) for constructing and maintaining single-mutant strains, the *C. elegans* model used in this invention has significant advantages in these key indicators: its lifespan is short, maturing and producing offspring in only about 3 days; its reproductive capacity is high, with a single individual producing hundreds of eggs in its lifetime; genetic manipulation is simple and extremely low-cost, with the cost of constructing and maintaining single-gene mutant strains being only a fraction of that of mouse models. These quantitative advantages enable the nematode-based screening system to achieve orders-of-magnitude improvements in time, cost, and throughput, fundamentally overcoming the inherent limitations of mouse models in large-scale genetic screening. This provides an efficient, economical, and reliable in vivo platform for the development of genes and drugs related to lysosomal storage diseases.

[0082] The comparison between Examples 1-2 and Comparative Examples 1-4 illustrates that while existing cell models can perform high-throughput screening, they cannot simulate the complex tissue interactions in vivo, and currently lack lysosomal storage phenotypes observable under low magnification. Animal models such as Drosophila melanogaster, horsefish, and mice are unsuitable for large-scale screening due to complex phenotype readings, low throughput, and high costs. The CPL-1::wrmScarlet reporter gene model provided by this invention offers a breakthrough advantage: it enables rapid and intuitive observation of the lysosomal storage state of live samples under a low-magnification dissecting microscope for the first time, requiring only about 1 minute for a single sample. The throughput is approximately 20 times higher than traditional methods relying on high-magnification imaging, while significantly reducing time and economic costs. This provides an efficient solution for screening genes and drugs related to lysosomal storage diseases, making it the preferred choice for primary screening at the whole-genome level.

[0083] In summary, the technical solution of this invention overcomes the limitations of traditional LSD animal models, which rely on cumbersome tissue sections, staining, or high-power microscopy for phenotypic observation. It enables direct, rapid, and non-destructive phenotypic observation of live nematodes under a low-power microscope. This makes ultra-high-throughput genetic and pharmacological screening possible, greatly improving the efficiency of discovering new genes and lead compounds.

[0084] Combining the characteristics of nematodes—small size, short lifespan, and extremely low feeding costs—with mature RNAi feeding and screening technologies, this model can complete the screening of genes or large-scale compound libraries across the entire genome at the lowest cost and fastest speed, making it far more cost-effective than other animal models.

[0085] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for screening genes related to lysosomal storage disease based on a Caenorhabditis elegans model of lysosomal overload, characterized in that, Includes the following steps: 1) Construct a fusion expression vector of CPL-1 driven by the intestinal-specific promoter nhx-2p and red fluorescent protein wrmScarlet, and transform it into Caenorhabditis elegans to obtain a stable transgenic line; 2) By inducing intestinal cells to secrete CPL-1::wrmScarlet fusion protein, it is absorbed by coelomic cells through coelomic fluid and accumulates in lysosomes, forming a lysosomal overload phenotype. 3) The RNA interference method was used to feed nematodes RNAi bacteria carrying different target gene fragments to knock down the expression of the corresponding genes. 4) Observe the changes in the intensity of red fluorescence signal in coelomic cells under a stereofluorescence microscope, and determine the functional status of lysosomes based on the differences in fluorescence signal intensity; 5) Genes corresponding to enhanced fluorescence signals were identified as candidate genes for promoting lysosomal accumulation, and genes corresponding to weakened signals were identified as candidate genes for alleviating or repairing lysosomal function. 2.The method for screening lysosomal storage disease related genes based on the Caenorhabditis elegans lysosome overload model according to claim 1, wherein, The CPL-1 fusion protein is released into the coelomic fluid via the classical secretory pathway from nematode intestinal cells. It is then endocytosed by coelomic cells at six fixed locations and enriched in their lysosomes, inducing a reproducible lysosomal overload phenotype. 3.The method for screening lysosome storage disease related genes based on the Caenorhabditis elegans lysosome overload model according to claim 1, wherein, The lysosomal overload phenotype was obtained by directly observing the intensity and distribution of red fluorescence signals in coelomic cells using a low-power fluorescence microscope. 4.The method for screening lysosome storage disease related genes based on the Caenorhabditis elegans lysosome overload model according to claim 1, wherein, To ensure consistent induction, fluorescence signals were collected from the nematodes on the third day after they became adults, and each treatment group contained at least fifty synchronized nematodes.

5. The method for screening genes related to lysosomal storage diseases based on the lysosomal overload model of *C. elegans* according to claim 1, characterized in that, RNA interference feeding was performed using a solid IPTG plate culture system, with each plate containing at least fifty synchronized nematodes. Fluorescence signals were detected after 5 days of incubation.

6. The method for screening genes related to lysosomal storage diseases based on the lysosomal overload model of *C. elegans* according to claim 1, characterized in that, Candidate genes screened under stereofluorescence microscopy were further examined using confocal microscopy to clarify the lysosomal storage in coelomic cells.

7. The method for screening genes related to lysosomal storage diseases based on the lysosomal overload model of *C. elegans* according to claim 1, characterized in that, The fluorescence signal intensity was calculated using image analysis software to determine the average gray value, quantitatively assess the degree of lysosomal accumulation, and establish a three-level grading standard of strong, medium, and weak fluorescence signals.

8. A method for drug screening using a *C. elegans* lysosomal overload model obtained by the method according to any one of claims 1-7, characterized in that, Synchronized transgenic nematodes were incubated in a solution of the test compound for 72 hours. Changes in fluorescence signal intensity in coelomic cells were observed to screen for candidate compounds that could weaken or enhance the lysosomal overload phenotype.

9. The application of the *C. elegans* lysosomal overload model obtained by the method of any one of claims 1-7 in screening genes related to lysosomal storage diseases, verifying lysosomal functional regulatory pathways, and developing drugs for regulating lysosomal homeostasis, characterized in that... By using changes in lysosomal fluorescence signals in coelomic cells as a unified detection indicator, a rapid and stable in vivo screening system was established.

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