Use of C17orf67 gene expression inhibition reagent in the preparation of drugs for treating acquired epidermolysis bullosa

Abstract

The present invention provides a use of a C17orf67 gene expression inhibitor in the preparation of a drug for treating acquired epidermolysis bullosa, belonging to the field of medicine. The present invention finds that knocking out the C17orf67 gene or reducing the expression of the C17orf67 polypeptide can significantly reduce subepidermal fissures, blisters, itching and inflammatory cell infiltration, treat acquired epidermolysis bullosa, and has broad application prospects.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and in particular relates to use of a reagent for inhibiting C17orf67 gene expression in preparing a drug for treating acquired epidermolysis bullosa. Background Art

[0002] Acquired epidermolysis bullosa (EBA) is an acquired skin disease characterized by blisters or separation of the skin or mucous membranes from underlying tissues after minor mechanical trauma, manifested by erythema, blisters, erosions, and crusted lesions; subepidermal fissures, inflammatory cell infiltration, and itching. This disease is relatively rare and belongs to a mechanical blistering dermatosis and is classified as an autoimmune disease. Patients often experience extreme itching where blisters and herpes appear. EBA mainly affects adults, while children and the elderly are less affected. In addition, female patients are more common than male patients, and there is usually no history of hereditary epidermolysis bullosa in the patient's family.

[0003] C17orf67, Human (339210), is an endogenous polypeptide gene whose function has not yet been reported in the literature. Only some genomics articles have analyzed it as a susceptibility gene for diseases such as thyroid hormone imbalance and otitis media.

[0004] There are currently no reports of C17orf67 being associated with epidermolysis bullosa acquisita. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing a drug for treating acquired epidermolysis bullosa.

[0006] The present invention provides use of an agent for inhibiting the expression of C17orf67 gene in preparing a medicine for treating epidermolysis bullosa acquisita.

[0007] Furthermore, the agent for inhibiting the expression of the C17orf67 gene is an agent for knocking out or knocking down the C17orf67 gene.

[0008] Furthermore, the reagent comprises the nucleotide sequence shown in SEQ ID NO.2 and / or SEQ ID NO.3.

[0009] Furthermore, the drug is a drug for reducing skin damage in epidermolysis bullosa acquisita.

[0010] Furthermore, the skin lesions include erythema, blisters, erosions and crusts.

[0011] Furthermore, the drug is a drug for reducing subepidermal clefts in epidermolysis bullosa acquisita.

[0012] Furthermore, the drug is a drug for reducing blisters in epidermolysis bullosa acquisita.

[0013] Furthermore, the drug is a drug for reducing inflammatory cell infiltration in epidermolysis bullosa acquisita.

[0014] Furthermore, the drug is a drug for reducing the itching of epidermolysis bullosa acquisita.

[0015] Furthermore, the epidermolysis bullosa acquired is bullous pemphigoid.

[0016] In summary, the present invention provides a use of a C17orf67 gene expression inhibitor in the preparation of a drug for treating acquired epidermolysis bullosa. - / - The authors constructed EBA mouse models of various genotypes and found that knocking out the C17orf67 gene or reducing the expression of the C17orf67 polypeptide could significantly reduce subepidermal fissures, blisters, itching, and inflammatory cell infiltration, and treat acquired epidermolysis bullosa, which has broad application prospects.

[0017] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0018] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Construction of a map for C17orf67 knockout mice

[0020] Figure 2 Figure 1 shows the identification of C17orf67 gene mice; A shows the genotype of mice identified by PCR, homozygous mice: 800bp; heterozygous mice: 360bp, 800bp. B shows the qPCR detection of WT mice and C17orf67 + / - Mouse and C17orf67 - / - Statistical graph of C17orf67 mRNA expression results in mice; n=12, one-way ANOVA; ***P<0.001.

[0021] Figure 3 C17orf67 - / -The mice had no significant difference in appearance and body weight compared with WT mice; A is an 8-week-old WT mouse and C17orf67 - / - Mouse appearance diagram; B is 8-week-old WT mouse and C17orf67 - / - Comparison of body weight of mice (C17orf67 KO).

[0022] Figure 4 Comparison of the severity of skin lesions in EBA mice; A is a flowchart for constructing the EBA mouse model; B is a comparison of the severity of skin lesions in WT mice and C17orf67 - / - Skin lesions of EBA mice at D14.

[0023] Figure 5 The skin lesion manifestations and severity scores of the four groups of mice (**P<0.01, ***P<0.001); A is the skin lesion manifestation diagram of each group of mice on D6 and D14 days of the experiment, and B is a line graph of the skin lesion manifestations and severity scores of the four groups of mice on different days.

[0024] Figure 6 For WT mice and C17orf67 - / - HE staining results of the mouse EBA model skin; A is the H&E staining result of the WT control group mouse skin; B is C17orf67 - / - The results of H&E staining of the skin of mice in the control group; C and D are the results of H&E staining of the skin of mice in the EBA modeling group, C is the results of H&E staining of the skin of mice in the EBA modeling group of WT mice: subepidermal blisters were formed, and more inflammatory cells were infiltrated; D is C17orf67 - / - H&E staining results of the mouse skin in the EBA modeling group: subepidermal fissures were formed and moderate amounts of inflammatory cells were infiltrated.

[0025] Figure 7 A is WT mice EBA modeling group mice and C17orf67 - / - Dermal-epidermal separation (DES) of EBA skin in mice EBA modeling group, B is the frequency of WT mice EBA modeling group and C17orf67 - / - Inflammatory cell infiltration in EBA of mice in the EBA modeling group; **P<0.01, ***P<0.001

[0026] Figure 8 The scratching situation of mice in each group; A is the statistical chart of the scratching times of mice in each group at different time points; B is the statistical chart of the scratching times of mice in each group at day 6, C17orf67 - / - The scratching frequency of mice in the EBA modeling group was significantly lower than that of mice in the WT EBA modeling group; (*P<0.05, **P<0.01).

[0027] Fig. 9:Intralesional injection of C17orf67 peptide at different concentrations caused different degrees of itching. **P<0.01, ***P<0.001 DETAILED DESCRIPTION

[0028] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0029] The C17orf67 polypeptide was customized by Wuhan Maisi Biotechnology Co., Ltd. The amino acid sequence of the C17orf67 polypeptide is SEQ ID NO.1:

[0030] SPILTEKQAKQLLRSRRQDRPSKPGFPDEPMREYMHHLLALEHRAEEQFL EHWLNPHCKPHCDRNRIHPV.

[0031] Chloroquine: CAS number: 50-63-5, Sigma, C6628.

[0032] Histamine: CAS number: 51-45-6, Sigma, H7125.

[0033] Example 1: Effect of C17orf67 on epidermolysis bullosa acquisita

[0034] 1. Experimental Methods

[0035] (I) Construction of C17orf67 knockout mice

[0036] 1. Construction method and its verification

[0037] Nanjing University-Nanjing Institute of Biomedicine was commissioned to design two gRNAs for the exons of the C17orf67 gene (chromosome 1) in C57BL / 6 mice, including gRNA1 (SEQ ID NO.2: AGTTGTATTCTTGACTGGAGTGG) and gRNA2 (SEQ ID NO.3: CAAGCTCTGTTGGCACTCTTTGG). CRISPR / Cas9 technology was used to construct C17orf67 knockout mice (C17orf67) by high-throughput electroporation of fertilized eggs. - / - Mouse)( Figure 1 ).

[0038] The mouse tail genomic DNA was extracted using a gene extraction kit, and the mouse genotype was identified using PCR technology. The genotype of the only amplified 800bp band was C17orf67. - / - Mouse ( Figure 2). At the same time, the WT mice and C17orf67 + / - and C17orf67 - / - The skin of the back of the neck was taken from mice, RNA was extracted, and qPCR was used to detect the expression of C17orf67 mRNA in the skin of mice with different genotypes. Figure 2 As shown, it can be seen that C17orf67 - / - The mRNA level of C17orf67 in mice is relative to that of C17orf67 + / - The expression of C17orf67 in WT mice was significantly decreased, indicating that C17orf67 was successfully constructed. - / - Mouse.

[0039] 2. C17orf67 - / - Mouse appearance and body weight detection

[0040] In WT mice, C17orf67 - / - The appearance and weight of the mice were monitored. Figure 3 A and Figure 3 B, C17orf67 - / - The appearance and body weight of mice were no different from those of WT mice.

[0041] The experimental results showed that C17orf67 was successfully constructed - / - Mice, and the growth and development of mice were not affected.

[0042] (II) Construction and validation of the epidermolysis bullosa acquired (EBA) mouse model

[0043] 1. Steps for constructing the EBA mouse model

[0044] COL7a1 antibody was used to induce a bullous pemphigoid (BP)-like EBA mouse model.

[0045] Mice aged 6-8 weeks were selected and divided into two groups: control group (Control) and EBA modeling group. The control group was divided into WT control group (WT+Control) and C17orf67 - / - The control group (C17orf67KO+Control), the EBA modeling group was divided into WT mouse EBA modeling group (WT+anti-COL7al) and C17orf67 - / - Mouse EBA model group (C17orf67 - / - +anti-COL7al), 8 mice in each group.

[0046] The hair on the back of the neck of each group of mice was shaved with a razor, with an area of ​​about 1 cm×1 cm.

[0047] EBA control group: mice in the EBA control group were subcutaneously injected with 100ug PBS on 0d, 2d, and 4d.

[0048] EBA modeling group: 100ug COL7a1 antibody / IgG antibody was diluted into 100uL PBS, and the EBA modeling group was subcutaneously injected with 100ug COL7a1 antibody on 0d, 2d, and 4d.

[0049] 2. Experimental results

[0050] 1. Record of skin lesions in mice

[0051] The skin lesions of the mice were recorded every other day. The skin lesions on the back and back of the neck of each group of mice were analyzed. The erythema, blisters, erosions, and scabs of the mice's skin were recorded.

[0052] The severity of skin lesions was scored as follows: 0: no lesions; 1: less than 20 lesions or less than 10% of the skin involved; 2: more than 20 lesions or 10-20% of the skin involved; 3: 20-40% of the skin involved; 4: 40-60% of the skin involved; 5: more than 60% of the skin involved.

[0053] The experimental results are as follows Figure 4 as well as Figure 5 As shown, from Figure 4 It can be seen that after 14 days, C17orf67 - / - The skin lesions of the control group mice were milder than those of the WT mice in the EBA modeling group. Results of the skin lesion severity scores on different days Figure 5 It can be seen that there is a significant difference in the severity of skin lesions on the back of the neck between the two groups of modeling mice (**P<0.01, ***P<0.001), C17orf67 - / - The severity score of skin lesions on the back of the neck in the EBA modeling group of mice was significantly lower than that in the WT mice EBA modeling group.

[0054] 2. Histopathological examination

[0055] The skin lesions of each group of mice were detected by H&E staining. Figure 6 shown.

[0056] Figure 6 A and Figure 6 B shows that the H&E staining results of the two control groups were normal. In the EBA modeling group, compared with the WT mice EBA modeling group, C17orf67 - / - The subepidermal cracks and blisters in the EBA modeling group were milder, and the inflammatory cell infiltration in the dermis was reduced.

[0057] The frequency of skin epidermal separation was used to quantitatively analyze the degree of subepidermal fissures and blisters. The results are as follows: Figure 7 As shown in A, it can be seen that C17orf67 - / - The frequency of subepidermal fissures and blisters in the EBA modeling group of mice was significantly lower than that in the EBA modeling group of WT mice (**P<0.01).

[0058] The infiltration of immune cells was detected by observing the distribution and morphology of immune cells in multiple areas of H&E sections of skin tissues of different groups. The results of inflammatory cell infiltration were as follows: Figure 7 As shown in B, it can be seen that C17orf67 - / - The inflammatory cell infiltration in the EBA modeling group of mice was significantly lower than that in the WT EBA modeling group of mice.

[0059] 3. Record of mouse scratching

[0060] The total number of scratching times of mice in each group was recorded every other day. The experimental results are shown in Figure 8 As shown in A; Figure 8 B is the scratching frequency of mice in each group on the 6th day.

[0061] Figure 8 A shows that the scratching of mice in the EBA modeling group was most obvious on the 6th day. As time went on, the number of scratching times and the total time of scratching in the EBA modeling group decreased, and the degree of itching in the mice was alleviated.

[0062] Figure 8 B shows that on day 6, C17orf67 - / - The frequency of scratching in the EBA model group of mice was significantly lower than that in the WT mice EBA model group.

[0063] The experimental results showed that knocking out the C17orf67 gene can significantly reduce the skin lesions of acquired epidermolysis bullosa as well as the cumulative area of ​​lesions, subepidermal fissures, blisters, inflammatory cell infiltration and itching.

[0064] (III) Study on the itch induced by C17orf67 peptide

[0065] 1. Different concentrations of C17orf67 peptide

[0066] 50uL of 0.9% saline, 0.3mM C17orf67 peptide, 1mM C17orf67 peptide, and 3mM C17orf67 peptide were intradermally injected into the back of the neck of C57BL / 6 mice, and videos were recorded for half an hour after the injection to record the number of times the mice scratched.

[0067] The experimental results are as follows Fig. 9As shown, it can be seen that with the increase of the injection concentration of C17orf67 polypeptide, the scratching of mice increased significantly. The experimental results showed that C17orf67 polypeptide can increase the itching degree of mice, providing a new perspective for the pathogenesis of acquired epidermolysis bullosa.

[0068] The experimental results showed that C17orf67 polypeptide can cause itching, and the degree of itching increases with the increase of C17orf67 polypeptide concentration.

[0069] In summary, the present invention provides a use of a C17orf67 gene expression inhibitor in the preparation of a drug for treating acquired epidermolysis bullosa. - / - Mice and EBA mouse models of various genotypes were constructed, and it was found that knocking out the C17orf67 gene or reducing the expression of the C17orf67 polypeptide can significantly reduce subepidermal fissures, blisters, itching and inflammatory cell infiltration, and treat acquired epidermolysis bullosa, which has broad application prospects.

Claims

1. Use of an agent for inhibiting the expression of the C17orf67 gene in the preparation of a drug for treating acquired epidermolysis bullosa; the agent for inhibiting the expression of the C17orf67 gene is an agent for knocking out or knocking down the C17orf67 gene; the agent for knocking out or knocking down the C17orf67 gene comprises the gRNA shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that: The drug is a drug for reducing skin damage in epidermolysis bullosa acquisita.

3. The use according to claim 2, characterized in that: The skin lesions include erythema, blisters, erosions and crusting.

4. The use according to claim 1, characterized in that: The drug is a drug for reducing subepidermal fissures in epidermolysis bullosa acquisita.

5. The use according to claim 1, characterized in that: The drug is a drug for reducing blisters in epidermolysis bullosa acquisita.

6. The use according to claim 1, characterized in that: The drug is a drug for reducing the infiltration of inflammatory cells in epidermolysis bullosa acquisita.

7. The use according to claim 1, characterized in that: The drug is a drug for reducing the itching of epidermolysis bullosa acquisita.

8. The use according to any one of claims 1 to 7, characterized in that: The epidermolysis bullosa acquired is bullous pemphigoid.

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