DNAH5 gene mutant cell as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a DNAH5 gene mutant cell as well as a preparation method and application thereof. In order to explore and research the action mechanism of DNAH5 gene mutation in primary ciliary dyskinesia, a single-base editing tool is further utilized to try to repair gene mutation sites and evaluate the curative effect, and an effective cell treatment model is provided for primary ciliary dyskinesia caused by DNAH5 gene mutation. The DNAH5, c.7915C to T cell line provided by the invention can simulate primary ciliary dyskinesia diseases, is closer to clinical phenotypes, and provides an accurate and effective in-vitro research material for gene therapy of DNAH5 single gene mutation, and a new possible and effective method is established for clinical treatment of the primary ciliary dyskinesia in the future, and the DNAH5, c.7915C to T cell line provided by the invention can simulate the primary ciliary dyskinesia diseases and is closer to clinical phenotypes.

Description

Technical Field

[0001] This application belongs to the field of biotechnology, specifically relating to a DNAH5 gene mutant cell, its preparation method, and its application. Background Technology

[0002] Primary ciliary dyskinesia (PCD) is a rare autosomal recessive genetic disorder caused by abnormalities in the structure and / or function of cilia. Cilia play important functions in the respiratory tract, reproductive system, and middle ear, including clearing mucus and propelling fluids. PCD patients often present with chronic respiratory infections, sinusitis, bronchiectasis, male infertility, and approximately 50% of patients also have situs inversus (Kartagener syndrome). DNAH5 (Dynein Axonemal Heavy Chain 5) is the most common gene mutation in PCD, and new mutants are constantly being discovered. There is a strong correlation between the mutated genotype and the ciliary dyskinesia phenotype. The DNAH5 gene, located on human chromosome 5p15.2, encodes the heavy chain protein of the outer dynein arm (ODA). The dynein arm is a key component of the axoneme, generating mechanical force through ATP hydrolysis to drive the cilia's movement. Mutations in the DNAH5 gene can lead to the loss or dysfunction of the external motor arm, weakening or eliminating ciliary movement and reducing mucus clearance efficiency, thus triggering typical symptoms of PCD, such as neonatal respiratory distress, chronic cough, and recurrent otitis media. Visceral transposition (Kartagener syndrome) is also relatively common. Approximately 30% of PCD cases are caused by DNAH5 mutations, making it one of the most common known pathogenic genes. It follows an autosomal recessive inheritance pattern, requiring patients to carry two pathogenic alleles (homozygous or compound heterozygous mutations). Carriers (heterozygotes) are usually asymptomatic. Reported mutations include nonsense mutations, frameshift mutations, splice site mutations, and large deletions, most of which result in protein truncation or loss of function.

[0003] Encoding dynein axonemal heavy chain 5, DNAH5 is a core component of the outer dynein arm in the "9+2" ultrastructure of cilia, providing power for ciliary movement. DNAH5 is the most common pathogenic gene for primary ciliary dyskinesia (PCD), far exceeding other genes (such as DNAH11 and CCDC39). DNAH5 mutations lead to impaired ciliary motility, loss of respiratory mucus clearance, recurrent infections, bronchiectasis, and progressive decline in lung function. 50% of patients also have situs inversus (Kartagener syndrome), affecting the establishment of the left-right axis during embryonic development.

[0004] Traditional clinical testing methods mainly include: whole-exome sequencing or targeted PCD gene panel screening for DNA H5 gene mutations; transmission electron microscopy (TEM) to observe ciliary cross-sections to confirm the absence of the external driving arm; or nasal nitric oxide (nNO) testing to determine whether a patient has PCD. Therefore, current treatment for PCD primarily focuses on symptomatic management, such as airway clearance and antibiotic treatment for infections.

[0005] Therefore, further research on DNAH5 mutations is necessary, which will help genetic counseling and future precision treatment exploration, and provide detection methods and means for subsequent DNAH5 gene therapy. Summary of the Invention

[0006] Based on this, one embodiment of this application provides a DNAH5 gene mutant cell, its preparation method, and its application.

[0007] This application provides a DNAH5 gene mutant cell with c.7915C>T, which leads to nonsense mutation in the formation of the stop codon and causes abnormal protein function.

[0008] In one embodiment, the cell type includes the human normal lung epithelial cell line BEAS-2B cells.

[0009] Another aspect of this application provides a method for preparing DNAH5 gene mutant cells, including: using gene editing to induce the c.7915C>T mutation.

[0010] In one embodiment, the gene editing method includes CRISPR-Cas9.

[0011] In one embodiment, the method includes: designing an sgRNA targeting the c.7915 site of the DNAH5 gene; inserting the sgRNA into a vector carrying Cas9 to construct a targeting vector.

[0012] A donor vector containing upstream and downstream homologous sequences of the DNAH5c.7915C>T mutation site was constructed; and the target vector and the donor vector were introduced into cells to prepare DNAH5 gene mutant cells.

[0013] In one embodiment, the nucleotide sequence of the sgRNA is shown in SEQ ID NO.1-SEQ ID NO.2.

[0014] In one embodiment, the vector carrying Cas9 includes a lentiviral vector.

[0015] In one embodiment, the lentivirus vector includes the LentiCRISPRv2 vector.

[0016] In one embodiment, the method of introducing cells includes electroporation.

[0017] In one embodiment, the parameters for the electrotransfer process include a program using the Lonza4D nuclear transfer instrument DS-150.

[0018] In one embodiment, the process of introducing cells further includes steps of drug screening and cloning.

[0019] In one embodiment, the drugs used for drug screening include puromycin.

[0020] This application also provides the use of the aforementioned DNAH5 gene mutant cells as model cells in the identification and / or testing of drugs.

[0021] The drug is used for the prevention and / or treatment of primary ciliary dyskinesia and / or complications associated with primary ciliary dyskinesia.

[0022] This application also provides an sgRNA, the nucleotides of which are shown in SEQ ID NO.1-SEQ ID NO.2.

[0023] Another aspect of this application provides a combined product comprising sgRNA as shown in SEQ ID NO.1-SEQ ID NO.2 and exogenous DNA containing the DNAH5c.7915C>T mutation site.

[0024] In one embodiment, the combined product further includes the Cas9 protein or a nucleic acid fragment encoding it.

[0025] This application aims to explore the role of DNAH5 gene mutations in the development of primary ciliary dyskinesia. Using gene editing tools, it attempts to repair gene mutation sites and evaluate the therapeutic effects, providing an effective cell therapy model for primary ciliary dyskinesia caused by DNAH5 gene mutations. The DNAH5 (c.7915C to T) cell line provided in this application more closely resembles the phenotype of clinical primary ciliary dyskinesia cells, providing accurate and effective in vitro research materials for gene therapy of DNAH5 single gene mutations. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 Screening and preparation of cell lines with DNAH5 disease-related point mutations (c.7915C to T); Figure 1 A: Schematic diagram of the c.7915 site of the human DNA H5 gene; Figure 1 B: Schematic diagram of the process for constructing the CRISPR / Cas9-mediated c.7915C>T mutant cell line; Figure 1 The genome sequencing peak diagram of the C:DNAH5 c.7915T mutant cell line clearly shows the homozygous C>T mutation at the c.7915 site; Figure 1 D: Detection of relative expression of DNAH5 in wild-type and DNAH5 c.7915T mutant;

[0028] Figure 2 For the screening and preparation of DNAH5 immunogen; Figure 2 A in the sequence represents the amino acid sequence of the human DNA H5 immunogen segment. Figure 2 B: Schematic diagram of the three-dimensional structure of the human DNA H5 immunogen segment; Figure 2 C: Construction map of human DNA H5 expression vector; Figure 2 D: DNAH5 antigen purification and Coomassie Brilliant Blue assay; lanes 1-2 are 1 mg / mL bovine serum albumin (BSA) and DNAH5, respectively;

[0029] Figure 3 Preparation and application of DNAH5 polyclonal antibodies; Figure 3 A in the text refers to the detection of DNAH5 antibody titer. Figure 3 B: DNAH5 antibody purification and Coomassie Brilliant Blue assay; Figure 3 C in the diagram represents the detection of exogenous proteins using immunoblotting. Lanes 1-3 contain 100ng, 50ng, and 10ng of DNAH5 antigen, respectively, with purified antibodies used as primary antibodies. Lane 4 contains 100ng of antigen, with unpurified serum used as the primary antibody. Lane 5 contains 100ng of antigen and a blank antibody control. Figure 3 D: Immunoblot detection of endogenous proteins; lanes 1-2 contain 5 μg and 2.5 μg of cell lysis buffer, respectively. Figure 3 E in the text: Immunofluorescence detection of DNAH5 co-localization with mitochondria; Figure 3 F in the text refers to the detection of co-localization of DNAH5 with the endoplasmic reticulum using immunofluorescence. Detailed Implementation

[0030] The present application will be further described in detail below with reference to the embodiments and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0031] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0032] the term

[0033] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0034] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0035] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0036] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0037] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0038] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0039] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0040] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0041] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0042] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0043] In this application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0044] All references to documents mentioned in this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the inventive purpose and / or technical solution of this application, all cited documents are incorporated herein by reference in their entirety and for all purposes. When citing documents in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. When citing documents in this application, examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0045] This application has identified a case of PCD with a mutation in the DNAH5 gene through previous research. This case is a biallelic homozygous mutation at position 7915, in which cytosine C is mutated to thymine T (c.7915C>T) (each parent carries one allele mutation). This mutation leads to a nonsense mutation in the stop codon, which encodes a corresponding change in amino acid (p.Q2639X), thereby causing abnormal protein function.

[0046] CRISPR / Cas9 base editing technologies (such as BE3 and ABE) can achieve precise single-base replacement (such as C→T or A→G) without double-strand DNA breaks. They are particularly suitable for correcting point mutations and avoid the risk of random insertion / deletion caused by traditional CRISPR cutting. This has a significant advantage in repairing high-frequency point mutations in PCD (such as some DNA H5 mutations).

[0047] Encoding dynein axonemal heavy chain 5, DNAH5 is a core component of the outer dynein arm in the "9+2" ultrastructure of cilia, providing power for ciliary movement. DNAH5 is the most common pathogenic gene for primary ciliary dyskinesia (PCD), accounting for 25.4% of patients in China (similar to Caucasian populations), significantly higher than other genes (such as DNAH11 and CCDC39). DNAH5 mutations lead to impaired ciliary motility, loss of airway mucus clearance, and recurrent infections, bronchiectasis, and progressive decline in lung function. 50% of patients also have visceral transposition (Kartagener syndrome), affecting the establishment of the left-right axis during embryonic development. CRISPR-based single-base editing technology, through precise repair of pathogenic mutations, not only holds promise for overcoming the current lack of a cure for PCD but also pioneers a new paradigm for gene therapy of hereditary respiratory diseases. This study focuses on a nonsense mutation in DNAH5 (e.g., c.7915T, p.Q2639X) that introduces a premature stop codon, leading to complete loss of protein function (classified as a pathogenic variant by the ACMG guidelines). To screen for efficient single-base editing systems to restore DNAH5 against high-frequency single-base mutations (c.7915A to G), this study first prepared a DNAH5 c.7915T mutant cell line, providing an effective tool for subsequent gene editing.

[0048] This application provides a DNAH5 gene mutant cell with c.7915C>T, resulting in a nonsense mutation in the stop codon and a corresponding change in the encoded amino acid (p.Q2639X), leading to abnormal protein function. According to the ACMG guidelines, this mutation is a zero-effect mutation, resulting in loss of gene function, and is preliminarily determined to be a pathogenic mutation. The clinical manifestations of the case include situs inversus, dyspnea, and abnormal ciliary electron microscopy structure.

[0049] In some embodiments, the cell type includes the human normal lung epithelial cell line BEAS-2B cells.

[0050] BEAS-2B is an epithelial cell line isolated from the bronchus of a healthy person without cancer. These cells are immortalized by infection and transformation with adenovirus 12-SV40 hybrid virus (Ad12SV40), and are positive for keratin and SV40 large T antigen staining. These cells can still undergo squamous differentiation under serum, making them suitable for screening chemical and biological agents that induce or affect differentiation and tumorigenesis, as well as serving as a suitable transfection host.

[0051] Another aspect of this application provides a method for preparing DNAH5 gene mutant cells, including: using gene editing to induce the c.7915C>T mutation.

[0052] In some of these embodiments, the gene editing method includes CRISPR-Cas9.

[0053] This application provides a method for preparing DNAH5 gene mutant cells, including:

[0054] Design an sgRNA targeting the c.7915 site of the DNAH5 gene; insert the sgRNA into a vector carrying Cas9 to construct a targeting vector.

[0055] A donor vector containing upstream and downstream homologous sequences of the DNAH5 c.7915C>T mutation site was constructed; and the target vector and the donor vector were introduced into cells to prepare DNAH5 gene mutant cells.

[0056] In some embodiments, the nucleotide sequence of the sgRNA is shown as SEQ ID NO.1-SEQ ID NO.2.

[0057] sgRNA1: GAGCTATTGGATAAACGAATGG (SEQ ID NO. 1).

[0058] sgRNA2: AGCTATGTGGATAAACGAATGGG (SEQ ID NO. 2).

[0059] In some embodiments, the length of the upstream homologous sequence of the DNAH5 c.7915C>T mutation site is 450bp-550bp.

[0060] In some embodiments, the length of the downstream homologous sequence of the DNAH5 c.7915C>T mutation site is 450bp-550bp.

[0061] In some embodiments, the vector carrying Cas9 is a lentiviral vector.

[0062] In some embodiments, the lentiviral vector includes the Lenti CRISPRV2 vector. LentiCRISPRv2 is a powerful and widely used tool, particularly well-suited for establishing stable and efficient gene knockout cell lines in a variety of mammalian cell lines. Its core advantages lie in the efficient delivery and stable integration of lentiviruses, and its built-in puromycin screening.

[0063] In some embodiments, the method of introducing cells includes electroporation.

[0064] In some embodiments, the parameters for the electrotransfer process include a program using the Lonza4D nuclear transfer instrument DS-150.

[0065] In some embodiments, the instruments used for electroporation include: SG Cell Line 4D- XKit.

[0066] This application also provides a method for preparing the DNAH5 gene mutant cells, which further includes drug screening and cloning steps after cell introduction.

[0067] In some embodiments, the drugs used for drug screening include puromycin. Puromycin is an aminoglycoside antibiotic produced by *Streptomyces niger*. It disrupts peptide transport on ribosomes, causing immature chain termination during translation, thereby inhibiting protein synthesis. Puromycin is a potent translation inhibitor in both prokaryotic and eukaryotic cells. The puromycin N-acetyltransferase gene (pac) in *Streptomyces* can induce resistance to puromycin. Puromycin acts rapidly, causing cell death quickly even at low concentrations.

[0068] This application also provides a method for preparing DNAH5 gene mutant cells, which yields DNAH5 gene mutant cells.

[0069] This application also provides the use of the DNAH5 gene mutant cells in the identification and / or testing of drugs.

[0070] The drug is used for the prevention and / or treatment of primary ciliary dyskinesia and / or complications associated with primary ciliary dyskinesia.

[0071] This application also provides an sgRNA, the nucleotides of which are shown in SEQ ID NO.1-SEQ ID NO.2.

[0072] Another aspect of this application provides a combined product comprising sgRNA as shown in SEQ ID NO.1-SEQ ID NO.2, and exogenous DNA containing the DNAH5 c.7915C>T mutation site.

[0073] In one embodiment, the combined product of this application further includes the Cas9 protein or a nucleic acid fragment encoding it.

[0074] This application also provides a detection primer set, the nucleotide sequences of which are shown in SEQ ID NO.3-SEQ ID NO.4.

[0075] This application also provides a detection method, including detecting the expression level of DNAH5 c.7915.

[0076] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0077] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0078] It should be understood that in the various embodiments of this application, the order of the above-mentioned processes does not imply the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0079] Example 1

[0080] This embodiment provides a method for constructing a DNAH5 gene mutant cell line, including the following steps:

[0081] In this embodiment, two vectors were used to establish the DNAH5-MUT BHT-101 cell line using CRISPR / Cas9 technology: a targeting vector (Cas9-sgRNA vector) and a donor vector.

[0082] The targeting vector can co-express sgRNA and Cas9 targeting the DNAH5 gene.

[0083] The donor vector contains two parts: 1) Up and Down homologous arms - which are identical to the sequences upstream and downstream of the break site, respectively; 2) the DNA fragment of the negative selection gene mCherry to be recombined and enter the target site.

[0084] I. Construction of a DNAH5 disease-related point mutation (c.7915C to T) cell line

[0085] This application analyzed the location of the human DNA H5 c.7915 site on the genome and designed a single-stranded guide RNA (sgRNA) near this location (e.g., ...). Figure 1 As shown in A), the sgRNA target sequence needs to cover the region near the mutation site (usually 20 bp) and ensure that the PAM sequence (NGG) is located at the 3' end of the target site.

[0086] The sequence is as follows:

[0087] sgRNA1: GAGCTATTGGATAAACGAATGG (SEQ ID NO. 1).

[0088] sgRNA2: AGCTATGTGGATAAACGAATGGG (SEQ ID NO. 2).

[0089] The sgRNA was inserted into the lenti crisperV2 vector carrying Cas9 to construct the targeting vector. At the same time, 500 bp upstream and downstream of the DNAH5c.7915 site were cloned into the expression vector as donor vectors for gene mutation.

[0090] BEAS-2B cells were cultured in 6-well plates with high-glucose DMEM cell culture medium (10% serum). BEAS-2B cells in the logarithmic growth phase were digested with trypsin, centrifuged after digestion, resuspended in PBS, and 5 × 10⁶ cells were collected after cell counting. 5 Electroporation was performed on individual cells. Electroporation was performed using an SG Cell Line 4D- X Kit: Thoroughly mix cells with electroporation buffer and replenishment solution, add 1 μg donor vector and 1 μg target vector. After thorough mixing, electroporate normal human lung epithelial cells BEAS-2B using a Lonza 4D DS-150 transducer. Gently transfer the cell suspension to 6-well plates containing 2 mL of pre-warmed DMEM medium for culture.

[0091] Puromysin was added for drug screening 24 hours later, and cloning was performed 72 hours later. When the monoclonal cells expanded to 6-well plates, a portion of the cells were taken to extract the genome and sent to the company for first-generation sequencing to identify whether the DNA H5 c.7915 site mutation was successful. Figure 1 (B in the middle).

[0092] The primer sequences for genome amplification and sequencing are as follows:

[0093] forword: TGGGTATGAGCTGTGCCAGGTGAC (SEQ ID NO. 3).

[0094] reverse: GGTATGTCATTGCGTCCACCA (SEQ ID NO. 4).

[0095] The mutant cell line that was successfully sequenced in the first generation was amplified and named DNAH5 c.7915T mutant. Figure 1(As shown in C in the diagram). Because the DNAH5 mutation results in the premature generation of a stop codon within the gene, leading to decreased mRNA stability, this application will next detect the DNAH5 mRNA level using reverse transcription PCR. First, WT and mutant cells were seeded in 6-well plates, and RNA was extracted using TransZol UP (Transgen, ET111-01-V2) after 24 hours. Finally, quantitative RT-PCR was used to detect the expression of DNAH5 mRNA in the cells.

[0096] The primers used for qPCR detection are as follows:

[0097] forword: AACTTGGCTCTCTAGGAGGGG (SEQ ID NO. 5).

[0098] reverse: GCAAACGTCTCACACTGTTGA (SEQ ID NO. 6).

[0099] The reaction system is shown in Table 1 below:

[0100] Table 1

[0101] DNA template (RNA extraction product reverse transcription) 100ng 2×Universal SYBR Green Fast qPCR Mix 5μL SEQ ID NO.5 (10μM) 0.4μL SEQ ID NO.6 (10μM) 0.4μL DW To 10μL

[0102] The reaction conditions are shown in Table 2 below:

[0103] Table 2

[0104]

[0105] qPCR analysis revealed that DNAH5 mutation significantly inhibited DNAH5 expression. Figure 1 (as shown in D).

[0106] Based on the relative expression detection of DNAH5 in wild type and DNAH5 c.7915T mutant, this application prepared a DNAH5 gene c.7915C>T mutant cell line.

[0107] In the early stages of our treatment, we treated a case of PCD with a DNAH5 gene mutation. This case involved a biallelic homozygous mutation at position 7915, where cytosine C was mutated to thymine T (c.7915C>T). Each of the parents carried one allele mutation, which resulted in a nonsense mutation in the stop codon, altering the encoded amino acid (p.Q2639X) and leading to abnormal protein function.

[0108] According to the ACMG guidelines, this variant is a zero-efficacy variant, leading to loss of gene function, and is preliminarily identified as a pathogenic variant. The patient presented with total situs inversus, dyspnea, and abnormal ciliary electron microscopy. Targeting this mutation, we successfully prepared a DNAH5 gene c.7915C>T mutant cell line. Obtaining this cell line provides an effective tool for screening single-base editing systems for PCD disease and verifies the feasibility of gene editing therapy for this type of disease.

[0109] Example 2: Preparation and Detection of DNAH5 Polyclonal Antibody

[0110] Mutations in DNAH5 can lead to a significant decrease in DNAH5 protein levels, affecting the function of DNAH5. Since currently available antibodies cannot meet the needs of detecting human DNAH5 protein, this application aims to prepare polyclonal antibodies against DNAH5 through screening, providing a detection method and means for subsequent DNAH5 gene therapy.

[0111] I. Screening and preparation of DNAH5 immunogen

[0112] To detect the expression of human DNAH5, this application prepared an antibody against human DNAH5. DNAH5 is a ciliated dynamin heavy chain with a molecular weight of approximately 529 kDa, which is very large, making it difficult to purify the full-length DNAH5 protein in vitro for immunization. Therefore, this application chose to use a local peptide fragment of DNAH5 (…). Figure 2 The DNAH5 immunogen (shown as AB in the diagram) was used for immunization. The DNAH5 immunogen was cloned and inserted into the His-tagged pET28a vector via enzyme digestion and ligation. Figure 2 (C in the middle).

[0113] The sequence of the local peptide is shown in SEQ ID NO.7.

[0114] VTNOIISACKAYITNNGTASIWNOPODVVEEKILSAIKLKOEYOLCFHKTKOKLKONPNAKOFDFSEMYIFGKFETFHRRLAKIIDIFTTLKTYSVLODSTIEGLEDMATKYOGIVATIKKKEYNFL DORKMDFDODYEEFCKOTNDLHNELRKFMDVTFAKIONTNOALRMLKKFERLNIPNLGIDDKYOLILENYGADIDMISKLYTKOKYDPPLARNOPPIAGKILWAROLFHRIOOPMOLFOOHPAV(SEQ ID NO.7).

[0115] The successfully constructed prokaryotic expression vector pET-28a / His-DNAH5-His was used to electroporate 100 ng of BL21(DE3) competent cells. After culturing at 37°C until the colony diameter reached approximately 0.2 mm, several colonies were picked and individually scraped from each colony into 5 mL of kanamycin-resistant LB medium. The cells were then placed in a shaker at 37°C for approximately 3 hours. When the OD600 of the bacteria reached approximately 0.6, 2.4 mM IPTG was added for induction. Induction was performed for 6 hours. One tube was reserved as the uninduced culture without IPTG, and 0.5 mL of the uninduced culture was stored at 4°C for later use. After 6 hours of induction, 0.5 mL of the induced culture was centrifuged and added to 50 μL of the medium. The DW was suspended, and 50 μL of 2×SDS loading buffer was added. Denaturation was performed at 95℃ for 10 min. After SDS-PAGE electrophoresis, the cells were stained with Coomassie Brilliant Blue solution and incubated at room temperature for 4 h. The cells were then destained until colorless, and the expression of the target protein was observed by photography. Strains that had successfully induced the expression of the target protein were selected for subsequent large-scale induction. Inoculation was performed at a seed culture to culture medium ratio of 1:100, and 500 mL of the induction culture was used for large-scale purification. The bacterial pellet was collected by centrifugation, resuspended in 80 mL of 8M urea, and sonicated on ice for 5 min × 3 times until the bacterial solution was clear. The mixture was centrifuged at 7000 rpm for 10 min at 4℃, and the supernatant was retained while the pellet was discarded. The pellet was bound with Ni-TAT beads and eluted in a chromatography cabinet at 4℃ for 3 h using a gradient elution with 8M urea containing different concentrations of imidazole from low to high. After SDS-PAGE electrophoresis, the sample was stained with Coomassie Brilliant Blue, and bovine serum albumin was used as a control to detect the concentration and purity of the target protein, successfully obtaining the DNAH5 immunogen. Figure 2 (as shown in D).

[0116] II. Preparation and Detection of DNAH5 Antibody

[0117] Rabbits were immunized with the prepared DNAH5 immunogen. The antibodies were purified from rabbit serum using antigen affinity purification to obtain rabbit polyclonal antibodies against DNAH5. The titer of the DNAH5 polyclonal antibody was first detected by indirect enzyme-linked immunosorbent assay (ELISA). 4 μg / mL protein was coated onto a 96-well plate, and 100 μL was incubated overnight at 4°C. The plate was washed three times with PBST, dried, and the antibody was serially diluted from 1 μg / mL. Different concentrations of the antibody were added and incubated at 37°C for 1 h. The plate was washed three times with PBST, dried, and 100 μL of goat anti-rabbit IgG-HRP was added (1:2500 dilution). The plate was incubated at 37°C for 40 min. The plate was washed six times with PBST, dried, and 100 μL of ABTS was added for color development. The plate was incubated at 37°C for 10 min, and the data were read at a wavelength of 405 nm using a multi-functional microplate reader. The results showed that the obtained antibody titer was greater than 5 × 10⁻⁶. 5 ( Figure 3(A) 5 μg of the antibody to be tested was subjected to SDS-PAGE electrophoresis and stained with Coomassie Brilliant Blue to determine the purity of the antibody. The results showed that a relatively pure DNAH5 antibody was successfully obtained. Figure 3 (B in the middle).

[0118] Simultaneously, this application uses 100 ng, 50 ng, and 10 ng of antigen for electrophoresis. Using a transfer apparatus, wet transfer is performed at constant voltage (100V) for 90 min to transfer and immobilize the protein onto a PVDF membrane (BIO-RAD). The transferred PVDF membrane is then placed in 5% skim milk dissolved in 1×PBST and blocked at room temperature for 2 h, followed by overnight incubation at 4°C. The surface moisture of the PVDF membrane is then aspirated, and 1 μg / μL anti-DNAH5 antibody is added. Figure 3 Lanes C1-3 of the sample were used for electrophoresis with 100 ng of DNAH5, and the samples were then separated using unpurified serum. Figure 3 Lane C 4) and blank ( Figure 3 Incubate the PVDF membrane overnight at 4°C in lane C (4). Wash three times with 1×PBST on a decolorizing shaker for 8 min each time. Dilute the HRP-labeled goat anti-rabbit IgG with 1×PBST according to the specified ratio, place the PVDF membrane in the diluted secondary antibody, and incubate on a decolorizing shaker for 45 min. Wash three times with 1×PBST on a decolorizing shaker for 5 min each time. Mix ECL chromogenic solutions A and B in a 1:1 ratio, add the mixture to the PVDF membrane after surface moisture has been absorbed, and place it in the dark for 5 min. Then, photograph the membrane using a fully automated chemiluminescence imaging analysis system.

[0119] The results showed that the purified antibody could recognize DNAH5 well.

[0120] To further confirm whether the obtained antibody could recognize human DNAH5, BEAS-2B cells were collected from a 6 cm dish with pre-chilled PBS and centrifuged in a 15 mL centrifuge tube at 4000 rpm for 3 min at 4 °C. The cells were resuspended in a 1.5 mL lysis buffer containing enzymes (150 mM NaCl, 20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1% NP-40, 1 μg / mL Aprotinin, 1 μg / mL Lepstatin A, 1 μg / mL Leupeptin, 200 mM NaF, 200 mM Na3NO4) and lysed at 4 °C for 10 min. Afterwards, the cells were centrifuged at 12000 g for 10 min at 4 °C. The supernatant was collected, and the protein concentration was determined using a Bradford protein assay kit. Cells were then lysed with SDS sample buffer, and the sample was denatured by heating in a metal bath at 110 °C for 10 min. Then, 5 μg and 2.5 μg of cell lysis buffer were taken for SDS-PAGE electrophoresis. The protein was transferred and immobilized on a PVDF membrane (BIO-RAD) using a constant voltage (100V) wet transfer for 90 min. After transfer, the PVDF membrane was cut to a suitable size, placed in an antibody incubation cassette, and washed once with PBST. Then, the PVDF membrane was blocked with 5% (w / v) skim milk at room temperature for 1 h. After milk blocking, it was washed three times with PBST, 5 min each time. After washing, the PVDF membrane was incubated with primary antibody at 37°C for 1 h or overnight at 4°C. After primary antibody incubation, it was washed three times with PBST, 5 min each time. The membrane was then incubated with HRP-conjugated secondary antibody (1 / 10,000) at room temperature for 60 min. After secondary antibody incubation, it was washed three times with PBST, 5 min each time. Finally, it was exposed and developed using ECL chemiluminescence solution (Beyotime). The results showed that the obtained antibody could recognize endogenous human DNAH5 protein (…). Figure 3 (as shown in D).

[0121] To further determine the application scope of the obtained antibodies, this application placed circular coverslips in 6-well plates or 3.5 cm dishes, added cells, and after 24 hours of cell attachment, washed the 6-well plates or 3.5 cm dishes once with room temperature PBS, added 4% paraformaldehyde at room temperature, and fixed for 15 minutes. After fixation, washed three times with room temperature PBS, 5 minutes each time. The coverslips containing cells were then inverted onto anti-DNAH5 and anti-Tom20 (mitochondrial markers) or anti-Sec61β (endoplasmic reticulum markers) prepared with 10% FBS and 0.1% saponin, and incubated overnight at 4°C or for 1 hour at room temperature. After washing three times with PBS, incubated with fluorescently labeled secondary antibodies of different species at room temperature for 45-60 minutes. After washing three times with PBS, washed once with ultrapure water. Finally, the cell slides were fixed onto glass slides using mounting medium (SBA, Southern Biotech). The fixed slides were imaged using a Nikon A1 laser scanning confocal microscope.

[0122] The results showed that DNAH5 was located in the cytoplasm, did not co-localize with mitochondria, and partially co-localized with the endoplasmic reticulum. Figure 3 (as shown in EF).

[0123] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. DNAH5 gene mutant cells, characterized in that, It has c.7915C>T, which leads to nonsense mutations in the formation of the stop codon, causing abnormal protein function.

2. The cell according to claim 1, characterized in that, The cell types include the normal human lung epithelial cell line BEAS-2B cells.

3. A method for preparing DNAH5 gene mutant cells, characterized in that, include: Gene editing techniques were used to induce the c.7915C>T mutation.

4. The method for preparing DNAH5 gene mutant cells according to claim 3, characterized in that, The gene editing methods include CRISPR-Cas9.

5. The method for preparing DNAH5 gene mutant cells according to claim 4, characterized in that, include: Design sgRNA targeting the c.7915 site of the DNAH5 gene; The sgRNA was inserted into a vector carrying Cas9 to construct a targeting vector; A donor vector containing upstream and downstream homologous sequences of the DNAH5 c.7915C>T mutation site was constructed; and the target vector and the donor vector were introduced into cells to prepare DNAH5 gene mutant cells.

6. The method for preparing DNAH5 gene mutant cells according to claim 5, characterized in that, The nucleotide sequences of the sgRNA are shown in SEQ ID NO.1-SEQ ID NO.

2.

7. The method for preparing DNAH5 gene mutant cells according to any one of claims 3 to 6, characterized in that, The method satisfies one or more of the following conditions: (1) The vector carrying Cas9 includes a lentiviral vector; Optionally, the lentiviral vector includes the Lenti CRISPRv2 vector; (2) The method of introducing cells includes electroporation; Optionally, the parameters for the electroporation process include the use of the Lonza 4D nuclear transfer instrument DS-150 program; (3) After cell introduction, the steps also include drug screening and cloning; Optionally, the drugs used in the drug screening include puromycin.

8. The use of the DNAH5 gene mutant cells as described in claim 1 or 2 as model cells in the identification and / or testing of drugs; The drug is used for the prevention and / or treatment of primary ciliary dyskinesia and / or complications associated with primary ciliary dyskinesia.

9. An sgRNA, characterized in that, Its nucleotides are shown in SEQ ID NO.1-SEQ ID NO.

2.

10. A combination product, characterized in that, Includes sgRNAs with nucleotides such as those shown in SEQ ID NO.1-SEQ ID NO.2 and exogenous DNA containing the DNAH5 c.7915C>T mutation site; Optionally, the combined product may also include the Cas9 protein or a nucleic acid fragment encoding it.

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