Application of Stab2 protein or its encoded nucleic acid in the preparation of drugs for treating hearing loss

Abstract

This invention discloses the application of Stab2 protein or its encoded nucleic acid in the preparation of drugs for treating hearing loss, belonging to the fields of biomedicine and gene therapy. This invention aims to address the lack of effective repair methods for cochlear band synapse damage in existing technologies. The drug mainly comprises a recombinant viral vector (preferably a lentiviral vector) carrying the Stab2 coding sequence, which, after preparation, is suitable for delivery to the inner ear via round window injection. Experiments show that overexpression of Stab2 protein in the cochlea can significantly promote the regeneration of inner hair cell band synapses after noise damage, effectively increasing the number of synapses. Simultaneously, it can significantly reduce the auditory brainstem response threshold and increase the amplitude of the ABR I wave, which represents nerve conduction function. This invention, by repairing damaged synaptic connections, can effectively treat noise-induced hearing loss and latent hearing loss, improve tinnitus, hyperacusis, and speech discrimination disorders, and has good prospects for clinical translation.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and gene therapy, specifically to the application of Stab2 protein or its encoded nucleic acid in the preparation of drugs for treating hearing loss. Background Technology

[0002] Hearing loss is one of the most common sensory disorders worldwide. According to the World Health Organization, there are many causes of deafness, among which noise exposure is one of the leading causes of sensorineural hearing loss in adults.

[0003] For a long time, hearing research has focused primarily on the survival and regeneration of cochlear hair cells. However, recent studies have found that in the early stages of noise exposure or during aging, before the hair cells die, the synaptic connections between the hair cells in the cochlea and the neurons of the spiral ganglion—the band synapses—are often the first to be damaged. This loss of synapses is often insidious; patients may appear normal in routine audiogram tests, but experience difficulty distinguishing speech in noisy environments. This is known as "latent hearing loss."

[0004] Current clinical interventions (such as hearing aids) can only amplify sound and cannot repair broken synaptic connections; while existing gene therapy research focuses primarily on hair cell regeneration genes (such as Atoh1), with relatively little research on targets specifically for the repair of synapses. Therefore, identifying key molecules that can specifically promote synapse regeneration is of significant clinical importance for fundamentally restoring hearing function. Summary of the Invention

[0005] To address the shortcomings of existing technologies, current treatments for sensorineural hearing loss (such as hearing aids and cochlear implants) primarily focus on amplifying sound signals or replacing them with electrical stimulation, failing to fundamentally repair damaged neural structures. In particular, there is a lack of effective biological treatments for damage or loss of the banded synapses in the cochlear hair cells caused by noise exposure. Damage to the banded synapses not only leads to an elevated hearing threshold but is also often accompanied by tinnitus, hyperacusis, and decreased speech discrimination ability in noisy environments—perceptual abnormalities (i.e., implicit hearing loss)—significantly impacting patients' quality of life.

[0006] Therefore, the primary objective of this invention is to provide the application of the Stab2 protein or its encoded nucleic acid in the preparation of drugs for treating hearing loss, thereby fundamentally rescuing hearing loss and improving related perceptual abnormalities by restoring the number and function of synapses.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] This invention provides the use of the Stab2 protein or its encoded nucleic acid in the preparation of a medicament for treating hearing loss.

[0009] Preferably, the amino acid sequence of the Stab2 protein is as shown in SEQ ID NO: 1, or the nucleotide sequence encoding the nucleic acid is as shown in SEQ ID NO: 2.

[0010] Preferably, the hearing loss mainly refers to hearing damage caused by noise exposure, especially hearing loss involving the loss or dysfunction of the band synapses of hair cells in the cochlea.

[0011] Preferably, the drug comprises a recombinant viral vector carrying a Stab2-encoded nucleic acid.

[0012] Preferably, the recombinant viral vector is selected from: lentiviral vector, adeno-associated virus vector, or adenovirus vector.

[0013] More preferably, the recombinant viral vector is a lentivirus vector. The lentivirus vector can efficiently infect cochlear cells and stably express the Stab2 protein therein.

[0014] Preferably, the recombinant viral vector contains a CMV promoter capable of driving specific or constitutive expression of the Stab2 gene in cochlear cells.

[0015] Preferably, the drug is formulated for administration via a round window injection. Direct delivery of the drug into the inner ear lymphatic fluid using microinjection ensures effective contact and action of the drug on the inner hair cells of the cochlea.

[0016] Preferably, the viral vector titer in the drug is 0.5 × 10⁻⁶. 8 ~5×10 8 TU / mL.

[0017] Preferably, the volume of the drug administered at one time is 1 to 5 μL, more preferably 2 μL, to ensure an effective therapeutic concentration is achieved without damaging the fine structures of the inner ear.

[0018] The present invention also provides a pharmaceutical composition for treating hearing loss, comprising a therapeutically effective amount of a gene therapy vector encoding the Stab2 protein and pharmaceutically acceptable excipients.

[0019] Preferably, the gene therapy vector comprises a promoter capable of driving the expression of the Stab2 gene in mammalian cochlear cells and the coding sequence of the Stab2 gene.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. This invention is the first to discover the crucial repair role of the Stab2 protein in the auditory system. Experimental results show that overexpression of the Stab2 protein in damaged cochlea can significantly promote the regeneration of band synapses in inner hair cells. Immunofluorescence counting shows that the number of synapses in the treatment group was significantly increased compared to the untreated group, and could even recover to near-normal levels.

[0022] 2. This invention not only achieves structural regeneration but also functional restoration. Auditory brainstem response (ABR) test results showed that after Stab2 gene therapy, the hearing thresholds of experimental animals were significantly improved at multiple frequencies (e.g., 4, 8, 16, 24, and 32 kHz). More importantly, the amplitude of the ABR I wave, representing the function of the auditory nerve synapse, was significantly increased. This directly proves that the newly regenerated synapse has normal physiological function and can effectively transmit sound signals from hair cells to the auditory nerve.

[0023] 3. This invention can effectively improve various perceptual abnormalities caused by synaptic lesions, including but not limited to reducing tinnitus symptoms, relieving auditory hypersensitivity, and improving speech discrimination ability in noisy environments, overcoming the problem that traditional hearing aids cannot solve the problem of "being able to hear but not being able to hear clearly".

[0024] 4. The circular window injection method used in this invention, combined with a preferred lentiviral vector and a specific dosage, has shown good transfection efficiency and biosafety in animal models, with no obvious damage to the cochlear structure, and has good potential for clinical translation. Attached Figure Description

[0025] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some schematic diagrams of certain embodiments of the present invention, and therefore should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 The hearing threshold results of the Auditory Brainstem Response (ABR) in Example 4;

[0027] Figure 2 The amplitude results of the auditory brainstem response (ABR) wave I in Example 4;

[0028] Figure 3 The results are from the immunofluorescence analysis in Example 5. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Of course, the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0030] Unless otherwise specified, all chemical reagents and materials in this invention are purchased from the market or synthesized from raw materials purchased from the market.

[0031] The present invention will be further described below through specific embodiments.

[0032] Example 1: Construction and preparation of Stab2 overexpression lentiviral vector

[0033] Target gene acquisition: Using mouse cochlear cDNA as a template, specific primers were designed to amplify the full-length coding sequence of the Stab2 gene. The nucleotide sequence of the Stab2 gene is shown in SEQ ID NO: 2, and the corresponding amino acid sequence of the Stab2 protein is shown in SEQ ID NO: 1.

[0034] Vector construction: The amplified Stab2 fragment was cloned into the lentiviral shuttle plasmid pLV4ltr Puro-CMV-c3xFLAG-Stab2. This plasmid contains the CMV promoter, which drives efficient expression of Stab2.

[0035] Virus packaging: The constructed overexpression plasmid and packaging plasmid (psPAX2, pMD2.G) were co-transfected into HEK293T cells. Cell supernatants were collected at 48 h and 72 h post-transfection, concentrated by ultracentrifugation, and resuspended in PBS.

[0036] Titer determination: The titer of the concentrated Stab2 overexpression lentivirus (LV-Stab2) was determined to be 5 × 10⁻⁶. 8 TU / mL. Lentiviral virus carrying only the empty vector (LV-Vector) was also prepared as a control.

[0037] Example 2: Establishment and grouping of an animal model of noise-induced hearing loss

[0038] Experimental animals: Eight-week-old CBA / J mice with normal hearing were selected.

[0039] Modeling method: Mice were placed in a specially designed soundproof box and exposed to broadband white noise with an intensity of 100 dB SPL for 2 hours to induce permanent hearing threshold shift and significant band synapse loss.

[0040] The experiment was randomly divided into two groups:

[0041] Control group: LV-Vector was injected through the round window 24 hours after noise exposure.

[0042] Stab2 overexpression group: LV-Stab2 was injected via round window 24 hours after noise exposure.

[0043] Example 3: Round window injection drug delivery surgery

[0044] Mice were anesthetized by intraperitoneal injection, the hair behind the ears was shaved off, and the mice were disinfected.

[0045] The skin behind the ear was incised, the muscle bluntly dissected, and the ossicular bullae exposed. Under a microscope, the bony walls of the ossicular bullae were carefully drilled open to expose the round window niche.

[0046] Using a glass microelectrode connected to a microinfusion pump, puncture the circular window membrane and slowly inject 2 μL of viral fluid (LV-Stab2 or LV-Vector) at a rate of 200 nL / min.

[0047] The needle was left in place for 10 minutes to prevent leakage of the medication. The round window was then sealed with muscle tissue, and the skin was sutured. The mice were closely monitored for recovery after the operation.

[0048] Example 4: Auditory Brainstem Response (ABR) Testing and Functional Assessment

[0049] Fourteen days after viral injection, ABR tests were performed on mice in each group.

[0050] Hearing threshold: Figure 1 The hearing threshold results are from the Auditory Brainstem Response (ABR). Figure 1 A represents the ABR threshold of mice before noise exposure; Figure 1 B represents the ABR threshold in mice one day after noise exposure; Figure 1 C represents the ABR threshold in mice 14 days after noise exposure. Detection results ( Figure 1 The results showed that the hearing threshold of mice in the control group was significantly increased at frequencies from 4 to 32 kHz; while the hearing threshold of mice in the Stab2 overexpression group was significantly lower than that of the control group, indicating that hearing sensitivity was significantly restored.

[0051] I-wave amplitude: Under 32kHz, 90dB SPL sound intensity stimulation, the I-wave amplitude in the control group was significantly reduced (suggesting impaired nerve conduction); in contrast, the I-wave amplitude in the Stab2 overexpression group recovered and was significantly higher than that in the control group. Figure 2 This directly confirms that Stab2 overexpression effectively restores the signal transmission function between inner hair cells and the auditory nerve.

[0052] Example 5: Immunofluorescence analysis of the number of cochlear ribbon synapses

[0053] After the ABR test, mouse cochlear tissue was taken, fixed, decalcified, and microscopically dissected to extract the basement membrane.

[0054] Staining: Immunofluorescence double staining was performed using anti-CtBP2 antibody (to label the presynaptic zona plexus) and anti-Myo7a antibody (to label hair cells).

[0055] Counting: The number of CtBP2 positive spots at the base of the inner hair cells was observed under a confocal microscope.

[0056] Results: Images were acquired using a laser scanning confocal microscope (Nikon). Immunofluorescence results ( Figure 3 This indicates that overexpression of Stab2 can significantly increase the number of band synapses, rescuing band synapse loss caused by noise exposure.

[0057] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. The use of Stab2 protein or its encoded nucleic acid in the preparation of a drug for treating hearing loss; the amino acid sequence of the Stab2 protein is shown in SEQ ID NO: 1, or the nucleotide sequence of the encoded nucleic acid is shown in SEQ ID NO: 2; the hearing loss is noise-induced hearing loss.

2. The application according to claim 1, characterized in that, The drug contains a recombinant viral vector carrying Stab2-encoded nucleic acid.

3. The application according to claim 2, characterized in that, The recombinant viral vector is selected from: lentiviral vector, adeno-associated virus vector, or adenovirus vector.

4. The application according to claim 3, characterized in that, The recombinant viral vector contains a CMV promoter capable of driving specific or constitutive expression of the Stab2 gene in cochlear cells.

5. The application according to any one of claims 1 to 4, characterized in that, The drug is formulated into a dosage form suitable for round window injection; the viral vector titer in the drug is 0.5 × 10⁻⁶. 8 ~5×10 8 TU / mL.

6. The application according to claim 1, characterized in that, The drug has one or more of the following functions: (1) Promotes the regeneration of banded synapses in the hair cells of the cochlea; (2) Increase the number of band synapses; (3) Increase the amplitude of wave I in auditory brainstem response; (4) Improve perceptual abnormalities caused by hearing loss, wherein the perceptual abnormalities are selected from tinnitus, speech discrimination disorder or hyperacusis.

7. The application according to claim 1, characterized in that, The medication for treating hearing loss includes: (a) A therapeutically effective amount of a recombinant viral vector encoding the Stab2 protein; and (b) Pharmaceutically acceptable carriers or excipients; The titer of the recombinant viral vector is 0.5 × 10⁻⁶. 8 ~5×10 8 .

8. The application according to claim 7, characterized in that, The unit dose volume of the drug is 1~5 μL.

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