Application of huR and its coding gene in preparation of medicine for treating presbycusis

Abstract

This invention discloses the application of HuR and its encoding gene in the preparation of drugs for treating age-related hearing loss, belonging to the field of genetic engineering technology. It provides the application of the HuR encoding gene in the preparation of drugs for the prevention or treatment of age-related hearing loss. The nucleotide sequence of the HuR encoding gene is shown in SEQ ID NO.1. The amino acid sequence of HuR is shown in SEQ ID NO.2. This invention is the first to discover the use of HuR in the prevention or treatment of age-related hearing loss. The HuR encoding gene, after being packaged with adeno-associated virus (AAV), is injected into the inner ear through a round window, causing it to be widely overexpressed in various types of cells in the inner ear. This can resist or delay the onset of age-related hearing loss, thus providing a theoretical basis for the prevention and improvement of hearing loss in patients with age-related hearing loss in clinical practice.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, and in particular relates to the application of HuR and its encoding gene in the preparation of drugs for treating age-related hearing loss. Background Technology

[0002] Age-related hearing loss (ARHL), also known as presbyopia, is one of the most common and complex chronic diseases among the elderly, with its pathogenesis remaining unclear. In my country, hearing loss is one of the most common disabilities, with presbyopia accounting for more than half of all hearing loss cases. ARHL not only severely impacts patients' quality of life but also imposes a significant economic burden and pressure on society. Therefore, ARHL is not only a medical problem but also a social one. However, developing more effective treatment strategies for ARHL requires a comprehensive understanding of its underlying mechanisms.

[0003] The RNA-binding protein HuR is a classic regulator of RNA metabolism. Recent studies have shown that HuR plays an important role in age-related diseases. In cardiac tissue, HuR enhances the expression of the aging inducer P21, and HuR degradation successfully halts cardiac aging. Furthermore, HuR and its stress particles can promote osteogenesis during bone aging. However, the potential mechanism of action of HuR in ARHL remains unclear. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention proposes the application of HuR and its encoding gene in the preparation of drugs for treating age-related hearing loss. Overexpression of the HuR encoding gene in inner ear cells can effectively delay the onset of age-related hearing loss from its source without producing any toxic side effects, and can serve as an effective target for the prevention and treatment of age-related hearing loss.

[0005] To achieve the above objectives, the present invention provides the application of the HuR-encoded gene in the preparation of drugs for the prevention or treatment of age-related hearing loss, wherein the nucleotide sequence of the HuR-encoded gene is shown in SEQ ID NO.1.

[0006] Preferably, the drug prevents or treats age-related hearing loss by overexpressing the HuR encoding gene, thereby increasing the expression or activity of HuR; the amino acid sequence of the HuR is shown in SEQ ID NO.2.

[0007] The present invention also provides an overexpression adeno-associated virus vector, the overexpression adeno-associated virus vector comprising a HuR encoding gene; the nucleotide sequence of the HuR encoding gene is shown in SEQ ID NO.1.

[0008] The present invention also provides the application of the overexpressing adeno-associated virus vector in the preparation of drugs for the prevention or treatment of age-related hearing loss, wherein the overexpressing adeno-associated virus vector prevents or treats age-related hearing loss by overexpressing the HuR-encoded gene.

[0009] The present invention also provides the use of HuR in the preparation of drugs for the prevention or treatment of age-related hearing loss, wherein the amino acid sequence of HuR is shown in SEQ ID NO.2.

[0010] Preferably, by increasing the expression or activity of HuR, hair cell loss is reduced, and the number of senescent hair cells is decreased, thereby preventing or treating age-related hearing loss.

[0011] The present invention also provides a drug for preventing or treating age-related hearing loss, the drug comprising one of HuR, a HuR expression promoter, or a HuR activity promoter; the amino acid sequence of the HuR is shown in SEQ ID NO.2.

[0012] Preferably, the drug reduces hair cell loss and decreases the number of senescent hair cells by increasing the expression or activity of HuR, thereby preventing or treating age-related hearing loss.

[0013] Preferably, the drug further includes pharmaceutically acceptable excipients.

[0014] Compared with the prior art, the present invention has the following advantages and technical effects:

[0015] This invention is the first to discover the use of HuR in the prevention or treatment of age-related hearing loss. The HuR encoding gene, after being packaged with adeno-associated virus (AAV), is injected into the inner ear through a round window, causing it to be widely overexpressed in various types of cells in the inner ear. This can resist or delay the onset of age-related hearing loss, thus providing a theoretical basis for the prevention and improvement of hearing loss in patients with age-related hearing loss in clinical practice. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1This study outlines the construction and analysis of HuR expression vectors and the SAMP8 mouse model for treating age-related hearing loss. A is a schematic diagram of HuR expression vector construction, where AAV-ie-HA represents the empty vector and AAV-ie-HuR-HA represents the HuR overexpression vector. B is a schematic diagram of rescuing hearing in SAMP8 mice through round window injection of AAV-ie-HuR. C is a comparison of transfection efficiency between AAV-ie-CON and AAV-ie-HuR, where green fluorescence represents cells effectively transfected and red fluorescence represents phalloidin, with a scale bar of 50 μm. D is a statistical graph showing the percentage of HA-labeled supporting cells in AAV-ie-CON and AAV-ie-HuR, with "ns" indicating significance analysis. E is a statistical graph showing the percentage of HA-labeled hair cells in AAV-ie-CON and AAV-ie-HuR, with "ns" indicating significance analysis.

[0018] Figure 2The images show the ABR audiometry results and basement membrane immunofluorescence staining results of AAV virus-treated mice. In Figure A, the pure tone threshold and click threshold statistics for each frequency in mouse ABR audiometry are presented. CON represents the untreated control group, AAV-ie-CON represents the AAV-ie-CON injection group, and AAV-ie-HuR represents the AAV-ie-HuR injection group. * indicates a significant difference compared to AAV-ie-CON. Figure B shows representative ABR waveforms at P90 in mice. CON represents the untreated control group, AAV-ie-CON represents the AAV-ie-CON injection group, and AAV-ie-HuR represents the AAV-ie-HuR injection group. The colored line represents the lowest detected threshold level. Figure C shows the staining results of mouse basement membrane slices. CON represents the untreated control group, AAV-ie-CON represents the AAV-ie-CON injection group, and AAV-ie-HuR represents the AAV-ie-HuR injection group. Green fluorescence represents Myo7A, and red fluorescence represents phalloides. Cyclic peptides, scale bar 50 μm, D represents statistical analysis of mouse hair cell loss rate, CON in the figure represents the untreated control group, AAV-ie-CON represents the AAV-ie-CON injection group, AAV-ie-HuR represents the AAV-ie-HuR injection group, "*" and "ns" represent significance analysis compared with AAV-ie-CON, E represents staining results of mouse basement membrane aging markers, CON in the figure represents the untreated control group, AAV-ie-CON represents the AAV-ie-CON injection group, AAV-ie-HuR represents the AAV-ie-HuR injection group, green fluorescence represents P21, red fluorescence represents Myo7A, scale bar 20 μm, F represents statistical analysis of mouse basement membrane aging markers, CON in the figure represents the untreated control group, AAV-ie-CON represents the AAV-ie-CON injection group, AAV-ie-HuR represents the AAV-ie-HuR injection group, "*" and "ns" represent significance analysis compared with AAV-ie-CON. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] Example 1

[0025] 1. Construction of HuR overexpression vector:

[0026] A construct of an AAV2 inverted terminal repeat (ITR-flanked) sequence comprising a CAG promoter, a HuR coding gene, an HA sequence, a marmot hepatitis virus posttranscriptional regulatory element (WPRE) sequence, and an SV40 poly(A) sequence was designed. This structure was packaged into an AAV-ie capsid to prepare AAV-ie-HuR. The AAV-ie capsid is a synthetic capsid that exhibits potent transduction efficiency in various cell types of the inner ear without causing significant hearing loss.

[0027] The nucleotide sequence of the HuR-encoding gene is SEQ ID NO.1:atgtctaatggttatgaagaccacatggcggaagactgcaggga tgacattgggagaacgaatttaattgtcaactacctccctcagaacatgacccaagaggaactacgaagtctgttcagcagcattggcgaggttgaatctgcaaagcttattcgggataaagtagcaggacacagcttgggctacggttttgtgaactatgtgactgcaaaagatgcagagagagcaatcagcacactgaacggcttgagactccagtccaaaaccattaaggtgtcatatgctcgcccaagctcagaggtcatcaaagatgccaacttatacatcagtgggctcccaaggaccatgacacagaaggatgtggaagacatgttttctcggtttgggcgaatcatcaactccagggtccttgtggatcagaccacaggtttgtccagaggggttgcctttatccggtttgacaaacggtcagaagcagaagaggcaattaccagtttcaatggtcataaacccccaggttcctccgagcccatcacagtgaagtttgcagccaatcccaaccagaacaaaaacatggctctcctctcgcagctgtaccactcgcctgctaggcggtttggaggccctgtacaccaccaggcacagagattcaggttctcccctatgggtgtagatcacatgagtgggatttctggtgtcaatgtccccggcaatgcttcctcgggctggtgcatcttcatctacaaccttgggcaagacgccgatgaggggatcctctggcagatgtttggcccctttggtgcagttaccaatgtgaaagtgattcgtgatttcaacaccaacaagtgcaaagggtttggttttgtgaccatgacaaactatgaagaagctgcaatggccatagcaagtctgaacggctaccgcctgggggacaaaattttacaggtttccttcaaaaccaacaagtcccacaaa。.

[0028] Amino acid sequence of HuR SEQ ID NO.2: MSNGYEDHMAEDCRDDIGRTNLIVNYLPQNMTQE ELRSLFSSIGEVESAKLIRDKVAGHSLGYGFVNYVTAKDAERAISTLNGLRLQSKTIKVSYARPSSEVIKDANLYISGLPRTMTQKDVEDMFSRFGRIINSRVLVDQTTGLSRGVAFIRFDKRSEAEEAITSFNGHKPPGSSEPIT VKFAANPNQNKNMALLSQLYHSPARRFGGPVHHQAQRFRFSPMGVDHMSGISGVNVPGNASSGGWCIFIYNLGQDADEGILWQMFGPFGAVTNVKVIRDFNTNKCKGFGFVTMTNYEEAAMAIASLNGYRLGDKILQVSFKTNKSHK.

[0029] like Figure 1 Figure A shows a schematic diagram of the construction of the HuR overexpression vector.

[0030] 2. Establishment of a SAMP8 mouse model for treating age-related hearing loss:

[0031] SAMP8 mice exhibit progressive hearing loss similar to age-related hearing loss in humans. Therefore, wild-type SAMP8 mice at 3 days of age (P3) were selected and divided into three equal groups: an untreated control group (CON), an AAV-ie-CON injection group (AAV-ie-CON), and an AAV-ie-HuR injection group (AAV-ie-HuR). AAV-ie-HuR or AAV-ie-CON was injected into the left cochlea of ​​the newborn (P3) SAMP8 mice through a round window. The AAV titer was 2.0 × 10⁻⁶. 12 GC / mL, with a viral volume of 1.0 μL injected into each ear. Seven days post-injection, mouse cochleas were harvested to analyze transfection efficiency. HA-positive staining and quantitative analysis showed that AAV-ie-HuR transduced inner ear cells with very high efficiency.

[0032] 3. Audiological assessment:

[0033] (1) Hearing test: The auditory brainstem response (ABR) of AAV-injected SAMP8 mice was measured, and the hearing threshold levels of the untreated control group (CON), the AAV-ie-CON injection group (AAV-ie-CON), and the AAV-ie-HuR injection group (AAV-ie-HuR) were compared. The lower the ABR hearing threshold of HuR-overexpressing mice in each frequency band, the better the hearing condition.

[0034] (2) Basement membrane preparation staining: Cochlear basement membrane preparations were prepared from untreated control group (CON), AAV-ie-CON injection group (AAV-ie-CON), and AAV-ie-HuR injection group (AAV-ie-HuR) mice and stained with immunofluorescence. The loss of hair cells in the untreated control group (CON), AAV-ie-CON injection group (AAV-ie-CON), and AAV-ie-HuR injection group (AAV-ie-HuR) mice was compared using myoglobin VIIA (Myo7A), phalloidin, and 4',6-diamidinyl-2-phenylindole (DAPI). The number of senescent hair cells in the untreated control group (CON), AAV-ie-CON injection group (AAV-ie-CON), and AAV-ie-HuR injection group (AAV-ie-HuR) mice was compared using Myo7A, cell cycle-dependent protein kinase inhibitor 1A (P21), and DAPI. The number of hair cells was observed by observing green fluorescence (488nm) in the immunofluorescence staining results of the basement membrane smear.

[0035] ① Basement membrane preparation: sampling and fixation: mouse cochlea samples were taken and placed in 4% paraformaldehyde overnight at 4°C; washing: washed 3 times with PBS, 5 min each time; decalcification: the cochlea was placed in EDTA washing solution (10%) at room temperature for 5 h; washing: washed 3 times with PBS, 5 min each time; the basement membrane was carefully peeled off under a dissecting microscope, and the basement membrane was cut into 3 segments and placed in PBS.

[0036] ② Immunofluorescence staining permeation: 0.3% Triton X-100 permeation at room temperature for 15 min; washing: PBS wash 3 times, 5 min each time; blocking: 10% goat serum, room temperature for 1 h; primary antibody (rabbit Myo7A 1:400, rabbit HA 1:400, rabbit P211:400) incubated overnight at 4℃ on a shaker; washing: PBS wash 3 times, 5 min each time; secondary antibody (goat anti-rabbit, 1:400) incubated at room temperature in the dark for 1 h; washing: PBS wash 3 times, 5 min each time; 488-labeled Phalloidin (green, dilution 1:3000) incubated at room temperature in the dark for 15 min; washing: PBS wash 3 times, 5 min each time; DAPI mounting was performed to observe the loss of hair cells.

[0037] 4. Result determination:

[0038] The effect of HuR overexpression in the mouse cochlea on delaying age-related hearing loss was evaluated using the above methods. It can be determined that HuR overexpression can effectively delay the onset of age-related hearing loss.

[0039] like Figure 1Figure B shows a schematic diagram illustrating the process of rescuing hearing in SAMP8 mice via round-window injection of AAV-ie-HuR; as shown in Figure B. Figure 1 As shown in Figure C, this compares the transfection efficiency of AAV-ie-CON and AAV-ie-HuR. The green fluorescence represents HA-labeled cells that were effectively transfected, and the red fluorescence represents phalloidin. The figure shows that both AAV-ie-CON and AAV-ie-HuR can effectively transfect supporting cells and hair cells throughout the cochlea. Figure 1 China D and Figure 1 As shown in Figure E, the percentages of HA-labeled supporting cells and HA-labeled hair cells are displayed, indicating that AAV-ie-CON and AAV-ie-HuR have very high transduction efficiency in both hair cells and supporting cells.

[0040] like Figure 2 As shown in Figure A, the statistical results of pure tone thresholds and click thresholds at various frequencies in mouse ABR audiometry are presented. It can be seen that compared with the control group (CON), the AAV-ie-CON group showed no significant difference in thresholds at any frequency tested at P90, demonstrating the safety of the AAV carrier and surgical procedure. However, injection of AAV-ie-HuR at P3 significantly reduced the ABR threshold. Figure 2 As shown in Figure B, this is a representative ABR waveform at P90 in SAMP8 mice from the untreated control group (CON), the AAV-ie-CON injection group (AAV-ie-CON), and the AAV-ie-HuR injection group (AAV-ie-HuR). The colored line represents the lowest detected threshold level, indicating that the Click threshold of SAMP8 mice in the AAV-ie-HuR group is reduced. Figure 2 C and Figure 2 As shown in Figure D, this table presents the staining results of the basement membrane of SAMP8 mice in the untreated control group (CON), the AAV-ie-CON injection group (AAV-ie-CON), and the AAV-ie-HuR injection group (AAV-ie-HuR), as well as the statistical analysis results of hair cell loss rate. Green fluorescence represents Myo7A, and red fluorescence represents phalloidin, indicating that round window injection of AAV-ie-HuR effectively reduced hair cell loss in SAMP8 mice. Figure 2 China E and Figure 2 As shown in Figure F, the staining results and statistical analysis results of aging markers on the basement membrane of SAMP8 mice in the untreated control group (CON), AAV-ie-CON injection group (AAV-ie-CON), and AAV-ie-HuR injection group (AAV-ie-HuR) are presented. The green fluorescence is P21 and the red fluorescence is Myo7A, indicating that round window injection of AAV-ie-HuR significantly reduced senescent hair cells in SAMP8 mice.

[0041] This invention has been experimentally verified to effectively reduce senescent hair cells in mouse inner ear cells using AAV technology, thereby preventing or delaying the onset of age-related hearing loss without any side effects. This has high reference value for the clinical treatment of age-related hearing loss patients and is suitable for widespread application. Therefore, HuR can serve as an effective target for the prevention and treatment of age-related hearing loss, providing more treatment options for clinical prevention and treatment of age-related hearing loss, and has significant social and economic value.

[0042] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Use of a gene encoding HuR in the preparation of a medicament for preventing or treating presbycusis, characterized in that, The nucleotide sequence of the HuR coding gene is shown as SEQ ID NO. 1, and the drug can prevent or treat presbycusis by overexpressing the HuR coding gene, increasing the expression of HuR or improving the activity of HuR.

2. Use of overexpression of adeno-associated viral vector in the preparation of a medicament for preventing or treating presbycusis, characterized in that, The overexpression adeno-associated virus vector comprises a HuR coding gene, and the nucleotide sequence of the HuR coding gene is shown as SEQ ID NO. 1; the overexpression adeno-associated virus vector can prevent or treat presbycusis by overexpressing the HuR coding gene.

3. Use of HuR in the preparation of a medicament for preventing or treating presbycusis, characterized in that, The amino acid sequence of the HuR is shown as SEQ ID NO. 2, and the presbycusis can be prevented or treated by increasing the expression of HuR or improving the activity of HuR, reducing the loss of hair cells and reducing the number of aging hair cells.

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