Skin organoid HPV infection model based on human embryonic stem cell source as well as construction method and application of skin organoid HPV infection model

Abstract

The invention provides a skin organoid HPV infection model based on a human embryonic stem cell source and a construction method and application thereof, and relates to the technical field of molecular biology, the construction method comprises the following steps: integrating an HPV genome to an AAVS1 site of genome DNA of a human embryonic stem cell to obtain the human embryonic stem cell carrying the HPV genome, and performing induced differentiation and activation of the HPV genome to obtain the human embryonic stem cell carrying the HPV genome. A skin-like organ HPV infection model is formed, stable heredity and controllable expression of an HPV genome are achieved, virus particle assembly is achieved through induced differentiation, establishment of a virus replication environment, activation of HPV early gene expression, driving of HPV genome replication and induction of late gene expression, and the natural infection state of HPV is simulated. The technical problem that in the prior art, an infection model cannot simulate the life cycle of HPV dependent terminal differentiation is solved, and an irreplaceable technical platform is provided for research of an HPV pathogenic mechanism and research and development of antiviral drugs and vaccines.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to a skin organoid HPV infection model based on human embryonic stem cells, its construction method, and its application. Background Technology

[0002] Human papillomavirus (HPV) is a small, double-stranded DNA virus whose infection is closely related to a variety of human diseases. It is a major pathogenic factor for various epithelial cancers, including cervical cancer, oropharyngeal cancer, and anal cancer. Although HPV research has been conducted for decades, understanding its complete life cycle, carcinogenic mechanisms, and the development of effective antiviral drugs still faces significant challenges. The core bottleneck lies in the lack of robust, reproducible, and physiologically relevant experimental models that can highly simulate the natural infection process in the human body.

[0003] Because HPV cannot efficiently infect and replicate in traditional cell culture systems, the commonly used HPV research model is an immortalized keratinocyte cell line (such as HaCaT) in which HPV genes (usually key "early gene regions," such as E6 and E7) are introduced. While these models are easy to operate, their cell sources are mostly cancerous or immortalized cells with unstable genomes, and they cannot fully simulate the layering, differentiation, and complete life cycle of normal epithelial tissue. More importantly, they usually require the use of retroviruses or lentiviruses to randomly integrate HPV gene fragments into the host genome. This random integration cannot control the copy number and integration site, leading to abnormal viral gene expression and failing to accurately reproduce the replication and pathogenesis process of the virus in its natural chromosomal environment. Furthermore, random integration results in uncontrollable viral gene expression and uneven copy numbers, severely affecting the reproducibility of experiments and the reliability of data.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] One of the objectives of this invention is to provide a method for constructing a skin organoid HPV infection model based on human embryonic stem cells, so as to solve the technical problem that existing infection models cannot simulate the HPV-dependent terminal differentiation life cycle.

[0006] The second objective of this invention is to provide a skin organoid HPV infection model based on human embryonic stem cells.

[0007] A third objective of this invention is to provide a skin organoid HPV infection model prepared by the above-described construction method, or the application of the above-described skin organoid HPV infection model in screening or evaluating anti-HPV drugs.

[0008] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: In a first aspect, the present invention provides a method for constructing a skin organoid HPV infection model based on human embryonic stem cells, comprising integrating the HPV genome into the AAVS1 site of the genomic DNA of human embryonic stem cells to obtain human embryonic stem cells carrying the HPV genome, and induced differentiation to activate the HPV genome to form a skin organoid HPV infection model.

[0009] Furthermore, the induced differentiation includes the following steps: S1. Inducing human embryonic stem cells carrying the HPV genome to differentiate into ectoderm-like cells, wherein the ectoderm-like cells include epidermal ectoderm and neural crest ectoderm; S2 induces neural crest cells in the brain to differentiate into dermal fibroblasts; S3 induces the development and maturation of sensory nerve endings and hair follicles, activates the HPV genome, and obtains a skin organoid HPV infection model.

[0010] Furthermore, integrating the HPV genome into the AAVS1 site of the genomic DNA of human embryonic stem cells involves introducing a homologous recombinant plasmid containing the HPV genome, a plasmid expressing sgRNA, and a plasmid expressing Cas9 or Cas12 into human embryonic stem cells. The sgRNA is a specific sgRNA targeting the AAVS1 site, and the sequence of the sgRNA is shown in SEQ ID NO.4.

[0011] Furthermore, the homologous recombination plasmid includes an HPV genome, and has an upstream homologous arm upstream of the HPV genome and a downstream homologous arm downstream of the HPV genome; the upstream and downstream homologous arms are used to integrate the HPV genome into the AAVS1 site of the genomic DNA of human embryonic stem cells; The nucleotide sequence of the upstream homologous arm is shown in SEQ ID NO.5, and the nucleotide sequence of the downstream homologous arm is shown in SEQ ID NO.6.

[0012] Furthermore, the HPV genome includes HPV1 genome, HPV5 genome, or HPV16 genome; Preferably, the plasmid expressing sgRNA and the plasmid expressing Cas9 or Cas12 are the same plasmid.

[0013] Secondly, the present invention provides a skin organoid HPV infection model based on human embryonic stem cells, which is prepared using the above-described construction method.

[0014] Furthermore, in the described skin organoid HPV infection model, the HPV genome is in an activated state.

[0015] Furthermore, the activated state includes at least one of early HPV gene initiation expression, HPV genome replication, late gene expression, capsid protein synthesis, and complete HPV viral particle assembly.

[0016] Thirdly, the present invention provides a skin organoid HPV infection model prepared by the above-described construction method or the application of the above-described skin organoid HPV infection model in screening or evaluating anti-HPV drugs.

[0017] Furthermore, the anti-HPV drug includes at least one of the following: neutralizing antibodies targeting capsid proteins, viral entry inhibitors, small molecule capsid binders, small molecule substances targeting key viral replication enzymes, immunomodulators, or therapeutic vaccines.

[0018] This invention provides a method for constructing a skin organoid HPV infection model based on human embryonic stem cells. The method integrates the HPV genome into the AAVS1 site of the genomic DNA of human embryonic stem cells, enabling the human embryonic stem cells to stably carry the HPV genome. This allows for stable inheritance and controllable expression of the HPV genome. Through induced differentiation, a viral replication environment is established, activating early HPV gene expression, driving HPV genome replication, inducing late-stage gene expression, and achieving viral particle assembly. The constructed skin organoid HPV infection model simulates the natural HPV infection state, solving the technical problem that existing infection models cannot simulate the HPV life cycle dependent on terminal differentiation. This provides an irreplaceable technical platform for research on HPV pathogenesis mechanisms, and the development of antiviral drugs and vaccines. Attached Figure Description To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0019] Figure 1 The image shows the pAAVS1-IRES-HPV expression plasmid provided in Example 1 of this invention. Figure 2 A map of the pLentiV2-Cas9-U6-AAVS1-sgRNA expression plasmid provided in Example 1 of this invention; Figure 3 The mRNA expression levels of early and late HPV1 genes in HPV1-infected skin organoids at different differentiation stages, as provided in Example 3 of this invention; Figure 4The mRNA expression levels of early and late HPV5 genes in HPV5-infected skin organoids at different differentiation stages, as provided in Example 3 of this invention; Figure 5 The mRNA expression levels of early and late HPV 5 genes in HPV16-infected skin organoids at different differentiation stages, as provided in Example 3 of this invention; Figure 6 This is an observation diagram of the morphology of HPV1-infected skin organoids provided in Embodiment 3 of the present invention; Figure 7 This is an observation diagram of the morphology of HPV5-infected skin organoids provided in Embodiment 3 of the present invention; Figure 8 This is an image showing the morphology of viral particles in HPV16-infected skin organoids provided in Embodiment 3 of the present invention. Figure 9 The results of the sensitivity test of HPV1-infected skin organoids against HPV compounds provided in Example 3 of the present invention; Figure 10 The results of the sensitivity test of HPV5-infected skin organoids against HPV compounds provided in Example 3 of the present invention; Figure 11 The results of the sensitivity test of HPV16-infected skin organoids against HPV compounds provided in Example 3 of the present invention. Detailed Implementation

[0020] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0021] Generally, the nomenclature and techniques used in cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization, together with those described herein, are those well-known and commonly used in the art. Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well-known in the art and described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature, laboratory procedures, and techniques used in analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry, together with those described herein, are those well-known and commonly used in the art.

[0022] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] This invention provides a method for constructing a skin organoid HPV infection model based on human embryonic stem cells, comprising integrating the HPV genome into the AAVS1 site of the genomic DNA of human embryonic stem cells to obtain human embryonic stem cells carrying the HPV genome, inducing differentiation, activating the HPV genome, and forming a skin organoid HPV infection model.

[0024] By integrating the HPV genome into the AAVS1 site of the genomic DNA of human embryonic stem cells, human embryonic stem cells can stably carry the HPV genome, enabling stable inheritance and controllable expression of the HPV genome. After induced differentiation, a viral replication environment is established, activating early HPV gene expression, driving HPV genome replication, inducing late gene expression, and achieving viral particle assembly. The constructed skin organoid HPV infection model simulates the natural HPV infection state, solving the technical problem that existing infection models cannot simulate the HPV life cycle dependent on terminal differentiation. This provides an irreplaceable technical platform for research on HPV pathogenesis mechanisms, the development of antiviral drugs, and vaccines.

[0025] In some specific implementations, the induced differentiation includes the following steps: S1. Inducing human embryonic stem cells carrying the HPV genome to differentiate into ectoderm-like cells, wherein the ectoderm-like cells include epidermal ectoderm and neural crest ectoderm; S2 induces neural crest cells in the brain to differentiate into dermal fibroblasts; S3 induces the development and maturation of sensory nerve endings and hair follicles, activates the HPV genome, and obtains a skin organoid HPV infection model.

[0026] This method induces human embryonic stem cells carrying the HPV genome to differentiate into ectoderm-like cells, establishing an epidermal lineage and suppressing non-epidermal lineages to achieve HPV genome silencing. It also induces neural crest cells to differentiate into dermal fibroblasts, constructing mesenchymal components within organoids and providing necessary paracrine signals (such as FGFs and IGFs) to support epithelial structure maturation before terminal epidermal differentiation, keeping HPV in a latent state. The development and maturation of nerve endings and hair follicles form highly layered epithelial tissue, producing cells expressing KRT1 / KRT10 (spike layer) and filaggrin / loricrin (granular layer). These cells express transcription factors required for HPVLCR, allowing the HPV genome to be activated sequentially according to the order of early gene expression, driving HPV genome replication, and inducing late gene expression, thus achieving viral particle assembly. This method achieves coordinated regulation of HPV viral replication and host differentiation timing.

[0027] In some specific embodiments, integrating the HPV genome into the AAVS1 site of the genomic DNA of human embryonic stem cells includes introducing a homologous recombinant plasmid containing the HPV genome, a plasmid expressing sgRNA, and a plasmid expressing Cas9 or Cas12 into human embryonic stem cells; wherein the sgRNA is a specific sgRNA targeting the AAVS1 site, and the sequence of the sgRNA is shown in SEQ ID NO.4.

[0028] In some specific embodiments, the homologous recombination plasmid includes an HPV genome, and has an upstream homologous arm upstream of the HPV genome and a downstream homologous arm downstream of the HPV genome; the upstream and downstream homologous arms are used to integrate the HPV genome into the AAVS1 site of the genomic DNA of human embryonic stem cells; the nucleotide sequence of the upstream homologous arm is shown in SEQ ID NO.5, and the nucleotide sequence of the downstream homologous arm is shown in SEQ ID NO.6.

[0029] In some specific implementations, the HPV genome includes the HPV1 genome, the HPV5 genome, or the HPV16 genome.

[0030] In some specific implementations, the plasmid expressing sgRNA and the plasmid expressing Cas9 or Cas12 are the same plasmid.

[0031] In some specific embodiments, the screening marker gene includes a puromycin resistance gene.

[0032] According to another aspect of the present invention, a skin organoid HPV infection model based on human embryonic stem cells is also provided, which is prepared using the above-described construction method.

[0033] In some specific implementations, the HPV genome is in an activated state in the skin organoid HPV infection model.

[0034] The activated state includes at least one of the following: early HPV gene initiation expression, HPV genome replication, late gene expression, capsid protein synthesis, and complete HPV viral particle assembly.

[0035] According to another aspect of the present invention, a skin organoid HPV infection model prepared by the above-described construction method is also provided, or the application of the above-described skin organoid HPV infection model in screening anti-HPV drugs is also provided.

[0036] In some specific embodiments, the anti-HPV drug includes at least one of the following: a neutralizing antibody targeting the capsid protein, a viral entry inhibitor, a small molecule capsid binder, a small molecule substance targeting a key enzyme in viral replication, an immunomodulator, or a therapeutic vaccine.

[0037] The present invention will be further illustrated below by way of examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0038] Example 1: Construction of hESC engineered cell line Using CRISPR / Cas9-mediated homologous recombination technology, the complete HPV1 and HPV5 genomes were precisely integrated into the AAVS1 site of the human embryonic stem cell (hESC) genome, respectively, thereby constructing stable hESC seed cells carrying the HPV1 genome, the HPV5 genome, and the HPV16 genome. The HPV1, HPV5, and HPV16 genomes were all synthesized by Suzhou Hongxun Biotechnology Co., Ltd., and each genome contains an early region, a late region, and an upstream regulatory region. The nucleotide sequence of the HPV1 genome is shown in SEQ ID NO.1, the HPV5 genome in SEQ ID NO.2, and the HPV16 genome in SEQ ID NO.3. The specific steps for each genome are as follows: 1. Construction of gene knock-in vector Target selection: The AAVS1 site in the human genome was selected, and sgRNA targeting this site was designed to ensure high cleavage efficiency and low off-target effects. The sgRNA sequence is shown in SEQ ID NO.4.

[0039] Homologous arms: A 550bp left homologous arm (SEQ ID NO.5) and a 550bp right homologous arm (SEQ ID NO.6) are added on both sides of the HPV genome sequence. The homologous arm sequences are homologous to the genome sequences on both sides of the AAVS1 site target sequence.

[0040] The left and right homologous arms were cloned into the IRES vector, and then the HPV genome was cloned into the IRES vector containing the homologous arms, resulting in the homologous recombination plasmid IRES vector containing the HPV genome, named pAAVS1-IRES-HPV, with the structure shown below. Figure 1 As shown, the HPV genome is located between the two homologous arms of the insertion site. The specific sequences of the homologous recombinant plasmid IRES containing the HPV genome include: the left homologous arm sequence of the target gene, the HPV genome, and the right homologous arm sequence of the target gene.

[0041] The sgRNA sequence used for gene editing was cloned into the pLentiV2-Cas9-U6-sgRNA plasmid expression vector to obtain the pLentiV2-Cas9-U6-AAVS1-sgRNA expression plasmid, as shown in the diagram. Figure 2 As shown.

[0042] 2. Construction, screening, and genotyping of hESC monoclonal cell lines carrying the HPV genome. pLentiV2-Cas9-U6-AAVS1-sgRNA and pAAVS1-IRES-HPV were mixed at a plasmid molar ratio of 1:2 and transfected into undifferentiated, highly viable pluripotent H9-hESC cells using an ECM 830 cell electroporator at 300 mA for 4 ms. After 48 hours of electroporation, cells that were not successfully transfected were selected using 1 μg / ml puromycin for one day. The puromycin-resistant cell pool was digested into single cells and seeded at low density in 96-well plates. Single cell clones were picked using limiting dilution and expanded. After 20 weeks of culture, the picked single-clone cells were cultured in 24-well plates. Genomic DNA was extracted from the cells, followed by PCR gel electrophoresis and Sanger sequencing to identify positive single clones, thus obtaining the hESC-AAVS1-IRES-HPV single-clone cell line with the AAVS1 gene locus integrated into the HPV genome.

[0043] Example 2: Differentiation and culture of HPV skin organoid models Using the cell line obtained in Example 1, skin organoids were induced and differentiated to construct an HPV skin organoid model, including the following three stages: Phase 1: Inducing hESC-AAVS1-IRES-HPV cell line to differentiate into ectoderm-like cells (Day 0-5), the steps are as follows: (1) Two days before differentiation begins, hESCs are digested and counted, diluted with E8 medium to a concentration of 7000 cells / 100 μL, and the cells are seeded into a low-adhesion U-shaped 96-well plate to condense into cell spheres (Embryonic Body). (2) On day 0 of differentiation, the agglutinated cell spheres obtained in step (1) were resuspended in the first differentiation medium and transferred to a new low-adhesion U-shaped 96-well plate and cultured for 5 days to obtain ectoderm-like cells (epidermal ectoderm and neural crest ectoderm cells). The first differentiation medium was prepared by adding E6 medium as the basal medium, and adding FGF2 at a final concentration of 6 ng / mL, BMP4 at a final concentration of 4 ng / mL, SB-431542 at a final concentration of 8 μM, and a matrix gel at a final volume fraction of 1%.

[0044] Phase 2: Inducing brain neural crest cells to differentiate into dermal fibroblasts (Day 6-11), the steps are as follows: (1) On day 6 of differentiation, add 25 μL of the second differentiation medium to each of the above culture wells for further culture. The second differentiation medium was prepared by adding E6 medium as the basal medium to a final concentration of 200 ng / mL FGF2 and a final concentration of 1.5 μM LDN-193189.

[0045] (2) On the 8th day of differentiation, add 75 μL of fresh E6 medium to each of the above culture wells for culture.

[0046] (3) On the 10th day of differentiation, 100 μL of culture medium was aspirated from each of the above culture wells and 100 μL of fresh E6 culture medium was added again for culture.

[0047] Phase 3: Inducing the development and maturation of sensory nerve endings and hair follicles (Days 12-140), the steps are as follows: (1) On day 12 of differentiation, the organoid culture obtained in stage 2 was resuspended in the third differentiation medium and transferred to a low-adhesion 24-well cell culture plate, which was then placed on a shaker rotating at 65 rpm for suspension culture. The third differentiation medium was a mixture of Advanced DMEM / F12, Neurobasal, and Defined Keratinocyte-SFM in a 1:1:1 volume ratio as the basal medium, with the following added to a final concentration of 5 μM RA, 0.5% N2, 1% B27, 0.1 mM β-mercaptoethanol, 1% GlutaMAX, and 1.5% Matrigel.

[0048] (2) The fresh fourth differentiation medium was replaced every 3 days thereafter, and the culture was continued until day 140 to obtain the HPV organoid model.

[0049] The fourth culture medium was a mixture of Advanced DMEM / F12, Neurobasal, and Defined Keratinocyte-SFM in a volume ratio of 1:1:1 as the basal medium. The following solutions were added to this mixture to a final concentration of 5 μM RA, 0.5% N2 (v / v), 1% B27 (v / v), 0.1 mM β-mercaptoethanol, and 1% GlutaMAX (v / v).

[0050] Example 3: Validation of HPV-infected skin organoids This embodiment systematically verifies whether the HPV skin organoid model differentiated and cultured in Example 2 can truly simulate the natural infection process of HPV in human skin. The natural infection process includes the maintenance of the viral genome, differentiation-dependent viral gene expression, and the production of viral particles.

[0051] 1. Timing analysis of viral gene expression: RNA was extracted from organoids in the early (undifferentiated) and mature (fully differentiated) stages of culture, including Day 0 (ES), Day 1, Day 34, and Day 46. The mRNA expression levels of early and late HPV genes were detected by qRT-PCR. The results are as follows: Figures 3-5 As shown, the expression level of the viral genome gradually increases with the differentiation and maturation of skin organoids, realizing the coordinated regulation of HPV virus replication and host differentiation timing.

[0052] 2. Virus particle assembly and morphology verification: Transmission electron microscopy was used to observe mature organoid sections, and the results are as follows: Figures 6-8 As shown in the figure (scale bar is 500nm), icosahedral virus particles with a diameter of about 55nm were found in keratinocytes, proving that the model can support the complete viral life cycle and successfully achieve viral assembly.

[0053] 3. Model Functional Validation: Drug sensitivity testing was performed using a known anti-HPV compound (podophyllotoxin, Podofilox) at a concentration of 40 μM for 144 hours. The results are as follows: Figures 9-11 As shown, qRT-PCR was used to verify the downregulation of E6 expression or the reduction of L1 protein expression, demonstrating that this model can be used for drug screening.

[0054] This invention utilizes CRISPR / Cas9 technology to target the AAVS1 site of H9 human embryonic stem cell lines (hESCs). By knocking in the whole genome sequences of different HPV types, HPV-hESC cell lines are constructed. The differentiation of these cell lines is induced using a 3D skin organoid culture method to construct an HPV-infected skin organoid model. This model can be applied to study the mechanism of HPV infection in the skin at the organoid level and to screen anti-HPV drugs.

[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for constructing a skin organoid HPV infection model based on human embryonic stem cells, characterized in that, This includes integrating the HPV genome into the AAVS1 site of the genomic DNA of human embryonic stem cells to obtain human embryonic stem cells carrying the HPV genome, which are then induced to differentiate, activate the HPV genome, and form a skin organoid HPV infection model.

2. The construction method according to claim 1, characterized in that, The induced differentiation includes the following steps: S1. Inducing human embryonic stem cells carrying the HPV genome to differentiate into ectoderm-like cells, wherein the ectoderm-like cells include epidermal ectoderm and neural crest ectoderm; S2 induces neural crest cells in the brain to differentiate into dermal fibroblasts; S3 induces the development and maturation of sensory nerve endings and hair follicles, activates the HPV genome, and obtains a skin organoid HPV infection model.

3. The construction method according to claim 1, characterized in that, The AAVS1 site for integrating the HPV genome into the genomic DNA of human embryonic stem cells involves introducing a homologous recombinant plasmid containing the HPV genome, a plasmid expressing sgRNA, and a plasmid expressing Cas9 or Cas12 into human embryonic stem cells. The sgRNA is a specific sgRNA targeting the AAVS1 site, and the sequence of the sgRNA is shown in SEQ ID NO.

4.

4. The construction method according to claim 3, characterized in that, The homologous recombination plasmid includes an HPV genome and has an upstream homologous arm upstream of the HPV genome and a downstream homologous arm downstream of the HPV genome; the upstream and downstream homologous arms are used to integrate the HPV genome into the AAVS1 site of the genomic DNA of human embryonic stem cells. The nucleotide sequence of the upstream homologous arm is shown in SEQ ID NO.5, and the nucleotide sequence of the downstream homologous arm is shown in SEQ ID NO.

6.

5. The construction method according to any one of claims 1 to 4, characterized in that, The HPV genome includes at least one of the HPV1 genome, HPV5 genome, or HPV16 genome; Preferably, the plasmid expressing sgRNA and the plasmid expressing Cas9 or Cas12 are the same plasmid.

6. A skin organoid HPV infection model based on human embryonic stem cells, characterized in that, It is prepared by the construction method described in any one of claims 1 to 5.

7. The skin organoid HPV infection model according to claim 6, characterized in that, In the skin organoid HPV infection model, the HPV genome is in an activated state.

8. The skin organoid HPV infection model according to claim 7, characterized in that, The activation state includes at least one of the following: early HPV gene initiation expression, HPV genome replication, late gene expression, capsid protein synthesis, and complete HPV viral particle assembly.

9. The application of the skin organoid HPV infection model prepared by the construction method according to any one of claims 1 to 5 or the skin organoid HPV infection model according to any one of claims 6 to 8 in screening or evaluating anti-HPV drugs.

10. The application according to claim 9, characterized in that, The anti-HPV drug includes at least one of the following: neutralizing antibodies targeting capsid proteins, viral entry inhibitors, small molecule capsid binders, small molecule substances targeting key viral replication enzymes, immunomodulators, or therapeutic vaccines.

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