Method for synergistically regulating proliferation and differentiation of inner ear stem cells by GPA, Espin and DKK3 and application of method in hair cell regeneration

Abstract

The invention relates to the technical field of stem cells, and discloses a method for cooperatively regulating proliferation and differentiation of inner ear stem cells by GPA, Espin and DKK3 and application of the method in hair cell regeneration, and the method comprises the following steps: step 1, optimizing a cooperative regulation strategy for proliferation and differentiation of the inner ear stem cells into hair cells; 2, studying the effect and mechanism of the multi-gene synergistic effect on proliferation and differentiation of inner ear stem cells and functional maturation and survival of newborn hair cells in a living body injury model; and 3, researching the recovery conditions of the functions of newborn hair cells and the auditory function of the mouse in a living body injury model after AAV-mediated multi-gene coordinated regulation. According to the method for cooperatively regulating and controlling proliferation and differentiation of the inner ear stem cells by GPA, Espin and DKK3 and application of the method in hair cell regeneration, AAV-ie (AAV-InnerEar) is a novel adeno-associated virus, the virus can safely and efficiently infect the mouse inner ear stem cells, and an effective way is provided for regulating and controlling regeneration of the inner ear stem cells into hair cells through gene therapy.

Description

Technical Field

[0001] The present invention relates to the field of stem cell technology, specifically to a method for collaboratively regulating the proliferation and differentiation of inner ear stem cells by GPA, Espin and DKK3, and its application in hair cell regeneration. Background Art

[0002] Deafness is the most common sensory impairment in the world. According to the World Health Organization, 1.5 billion people worldwide, or 20% of the total population, suffered from hearing loss in 2021, with the majority living in low- and middle-income countries. In China, statistics from 2010 showed that 20.54 million people, or a quarter of all people with disabilities, were diagnosed with deafness.

[0003] Currently, there are 1.5 billion people with hearing loss worldwide, including over 20 million in China, with 30,000 new cases of congenital deafness each year. Sensorineural hearing loss is primarily caused by irreversible damage to inner ear hair cells, and current clinical treatment options are limited. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for synergistically regulating the proliferation and differentiation of inner ear stem cells by GPA, Espin and DKK3 and its application in hair cell regeneration, so as to solve the problems raised in the above background technology.

[0005] To achieve the above objectives, the present invention provides the following technical solutions: a method for synergistically regulating the proliferation and differentiation of inner ear stem cells by GPA, Espin, and DKK3 and its application in hair cell regeneration, comprising the following steps:

[0006] Step 1: Optimize the coordinated regulation strategy of inner ear stem cell proliferation and differentiation into hair cells;

[0007] Step 2: Study the effects and mechanisms of multi-gene synergy on the proliferation and differentiation of inner ear stem cells and the functional maturation and survival of newly born hair cells in an in vivo injury model;

[0008] Step 3: Study the function of newborn hair cells and the recovery of mouse hearing function after AAV-mediated multi-gene coordinated regulation in an in vivo injury model.

[0009] Preferably, in step 1, siRNA targeting Dkk3 is designed, the knockdown efficiency of each siRNA is verified in hair cell lineage HE-OC1 cells in vitro, and the siRNA with the highest knockdown efficiency is selected for use.

[0010] Preferably, in step 1, GPA, Espin and Dkk3 are constructed into a CAG-driven AAV-ie packaging plasmid system to construct an overexpression virus system.

[0011] Preferably, the step 1 utilizes an siRNA and AAV-ie mediated overexpression virus system to introduce siRNA or gene into flow-sorted Lgr5-positive inner ear stem cells, and performs suspension division culture and adherent differentiation culture in vitro.

[0012] Preferably, in step 2, a neomycin-induced hearing loss model is constructed.

[0013] Preferably, in step 2, AAV-ie-CAG-GPA, AAV-ie-CAG-Espin and AAV-ie-CAG-DKK3 or related AAV-ie-U6-shRNA are co-introduced into mice injured by neomycin using the semicircular canal injection method, and EDU is injected intraperitoneally from the 2nd to the 7th day after surgery to mark dividing cells.

[0014] Preferably, the step three is to detect the morphology and structure of new hair cells after gene therapy.

[0015] Preferably, the step three detects the electrophysiological characteristics of newly generated hair cells after gene therapy.

[0016] The present invention also provides the application of GPA, Espin and DKK3 in synergistically regulating the proliferation and differentiation of inner ear stem cells in hair cell regeneration, clarifies the structure and function of new hair cells and the degree of auditory function recovery after gene therapy synergistically regulated by GPA, Espin and DKK3, and lays a theoretical and experimental foundation for the clinical application of Lgr5+ inner ear stem cells to regenerate hair cells and restore auditory function.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. This method of synergistically regulating the proliferation and differentiation of inner ear stem cells by GPA, Espin, and DKK3 and its application in hair cell regeneration. AAV-ie (AAV-InnerEar) is a new adeno-associated virus that can safely and efficiently infect mouse inner ear stem cells, providing an effective way to regulate the regeneration of inner ear stem cells into hair cells through gene therapy.

[0019] 2. This method of synergistically regulating the proliferation and differentiation of inner ear stem cells with GPA, Espin, and DKK3 and its application in hair cell regeneration will clarify the structure and function of newly generated hair cells and the degree of auditory function recovery after synergistic gene therapy with GPA, Espin, and DKK3, laying a theoretical and experimental foundation for the clinical application of Lgr5+ inner ear stem cells to regenerate hair cells and restore auditory function. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a schematic diagram of the technical roadmap of the present invention;

[0021] Figure 2 This is a schematic diagram showing that Dkk3 of the present invention affects the spheroidization ability of Lgr5-positive inner ear stem cells;

[0022] Figure 3 Schematic diagram of the neomycin-induced deafness mouse model of the present invention;

[0023] Figure 4 This is a schematic diagram of the AAV-ie-mediated multi-factor GPA coordinated regulation of inner ear hair cell regeneration of the present invention. DETAILED DESCRIPTION

[0024] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0025] See also Figure 1-4 , the present invention provides the following technical solutions:

[0026] The method for synergistically regulating the proliferation and differentiation of inner ear stem cells by GPA, Espin, and DKK3 and its application in hair cell regeneration comprises the following steps:

[0027] Step 1: Optimize the coordinated regulatory strategy for inner ear stem cell proliferation and differentiation into hair cells, design siRNA targeting Dkk3, verify the knockdown efficiency of each siRNA in hair cell lineage HE-OC1 cells in vitro, select the siRNA with the highest knockdown efficiency for use, and simultaneously construct GPA, Espin, and Dkk3 into the CAG-driven AAV-ie packaging plasmid system to construct an overexpression virus system. Then, use the siRNA and AAV-ie-mediated overexpression virus system to introduce siRNA or gene into flow-sorted Lgr5-positive inner ear stem cells, and perform suspension division culture and adherent differentiation culture in vitro

[0028] Step 2: To investigate the effects and mechanisms of multi-gene synergistic effects on the proliferation and differentiation of inner ear stem cells and the functional maturation and survival of newly generated hair cells in an in vivo injury model, a neomycin-induced hearing loss model was first established. AAV-ie-CAG-GPA, AAV-ie-CAG-Espin, and AAV-ie-CAG-DKK3 or related AAV-ie-U6-shRNA were co-introduced into neomycin-injured mice via semicircular canal injection. Simultaneously, EDU was injected intraperitoneally from day 2 to day 7 after surgery to label dividing cells.

[0029] Step 3. Study the function of newborn hair cells and the recovery of mouse hearing function after AAV-mediated multi-gene coordinated regulation in an in vivo injury model, and detect the morphology and structure of newborn hair cells and the electrophysiological characteristics of newborn hair cells after gene therapy.

[0030] When in use, the gene knockdown system: design siRNA targeting Dkk3, verify the knockdown efficiency of each siRNA in hair cell lineage HE-OC1 cells in vitro, and select the siRNA with the highest knockdown efficiency for use.

[0031] Gene overexpression system: GPA, Espin, and Dkk3 were constructed into the CAG-driven AAV-ie packaging plasmid system to construct an overexpression virus system.

[0032] Based on the results of the first step, siRNA and AAV-ie-mediated overexpression viral systems were used to introduce siRNA or genes into flow-sorted Lgr5-positive inner ear stem cells, which were then cultured in suspension and adherent differentiation in vitro. Inner ear stem cell sphere formation and sphere re-passage experiments were conducted to examine the effects of individually or synergistically regulating the expression levels of GPA, Espin, and Dkk3 on the proliferation and differentiation capacity of inner ear stem cells. EdU immunostaining was used to assess the proliferation of Lgr5-positive inner ear stem cells. Direct differentiation experiments were conducted to examine the effects of GPA, Espin, and Dkk3 on the differentiation of inner ear stem cells into hair cells or other supporting cells. The hair cell marker myosin7a was used to assess the ability of Lgr5-positive inner ear stem cells to regenerate hair cells, and the supporting cell markers Sox2 and P27 were used to assess the ability of Lgr5-positive inner ear stem cells to differentiate into other supporting cells.

[0033] Based on the work in Part I, an AAV-IE-mediated coordinated gene overexpression or knockdown system was constructed based on the actual positive or negative effects of the gene. A trace amount of AAV virus was injected through the semicircular canals into the inner ears of mice with neomycin-induced hair cell damage to evaluate the effects of coordinated regulation of GPA, Espin, and DKK3 on the proliferation and differentiation of Lgr5-positive inner ear stem cells and the functional maturation and survival of newly generated hair cells.

[0034] Construct a neomycin-induced hearing loss model: Neomycin (200 mg / kg) was injected into the abdominal cavity of P8 mice for 7 consecutive days. Lgr5-EGFP-CreERT2 / + / Rosa26-tdTomato / + transgenic mice were selected to facilitate the subsequent tracking of inner ear stem cells.

[0035] Continued gene therapy experiments.

[0036] Based on the results of the first part of the experiment, AAV-ie-CAG-GPA, AAV-ie-CAG-Espin, and AAV-ie-CAG-DKK3, or the related AAV-ie-U6-shRNA, were delivered via semicircular canal injection into mice following neomycin injury. EdU was administered intraperitoneally from day 2 to day 7 postoperatively to label dividing cells. Four weeks after viral injection, Lgr5-positive inner ear stem cells (IRIS) proliferation, differentiation, and hair cell regeneration were assessed. IRIS proliferation was assessed using EdU, while IRIS hair cell markers myosin7a and parvalbumin were used to measure IRIS hair cell numbers, and supporting cell markers p27 and Sox2 were used to measure supporting cell numbers. Morphological changes in IRIS hair cells were assessed using scanning electron microscopy, and the ratio of inner to outer hair cells in newly formed hair cells was determined using the inner hair cell marker otoferlin and the outer hair cell marker prestin.

[0037] After gene therapy, the morphology and structure of newborn hair cells were detected: the cilia morphology of newborn hair cells was detected by using the hair cell cilia markers F-actin and Espin and scanning electron microscopy; FM-143 was used to detect whether tdTomato-positive newborn hair cells had MET channels; the presynaptic marker Ctbp2 and the postsynaptic marker Shank1 were used to detect the synaptic regeneration of tdTomato-positive newborn hair cells; the apoptosis of tdTomato-positive newborn hair cells was detected by using the cell apoptosis marker Caspase3 or TUNEL.

[0038] Detect the electrophysiological characteristics of newborn hair cells after gene therapy: Use patch clamp technology and whole-cell extracellular recording method to record the electrophysiological changes of tdTomato-positive newborn hair cells.

[0039] Recovery of hearing in mice: In in vivo experiments, ABR and DPOAE were used to assess the auditory function of mice older than P30. After gene therapy, the mice underwent auditory function assessment at 1 month, 3 months, and 6 months, and the hearing thresholds were tested under 4, 8, 16, 24, and 32 kHz short pure tones to verify the auditory function restoration and long-term effects that can be achieved by using the AAV-mediated gene therapy system for hearing loss treatment.

[0040] Although the embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for synergistically regulating the proliferation and differentiation of inner ear stem cells using GPA, Espin, and DKK3, characterized by: The following steps are involved: Step 1: Optimize the coordinated regulation strategy of inner ear stem cell proliferation and differentiation into hair cells; Step 2: Study the effects and mechanisms of multi-gene synergy on the proliferation and differentiation of inner ear stem cells and the functional maturation and survival of newly born hair cells in an in vivo injury model; Step 3: Study the function of newborn hair cells and the recovery of mouse hearing function after AAV-mediated multi-gene coordinated regulation in an in vivo injury model.

2. The method for synergistically regulating the proliferation and differentiation of inner ear stem cells by GPA, Espin and DKK3 according to claim 1, characterized in that: In the step 1, siRNAs targeting Dkk3 are designed to verify the knockdown efficiency of each siRNA in hair cell lineage HE-OC1 cells in vitro, and the siRNA with the highest knockdown efficiency is selected for use.

3. The method of claim 1 wherein GPA, Espin, and DKK3 synergistically regulate the proliferation and differentiation of inner ear stem cells, wherein: In the step 1, GPA, Espin and Dkk3 are constructed into the CAG-driven AAV-ie packaging plasmid system to construct an overexpression virus system.

4. The method for synergistically regulating the proliferation and differentiation of inner ear stem cells by GPA, Espin and DKK3 according to claim 1, characterized in that: The step 1 utilizes an siRNA and AAV-ie mediated overexpression virus system to introduce siRNA or gene into flow-sorted Lgr5-positive inner ear stem cells, and performs suspension splitting culture and adherent differentiation culture in vitro.

5. The method for synergistically regulating the proliferation and differentiation of inner ear stem cells by GPA, Espin and DKK3 according to claim 1, characterized in that: The second step is to construct a neomycin-induced hearing loss model.

6. The method of claim 1 wherein GPA, Espin and DKK3 synergistically regulate the proliferation and differentiation of inner ear stem cells, wherein: In step 2, AAV-ie-CAG-GPA, AAV-ie-CAG-Espin and AAV-ie-CAG-DKK3 or related AAV-ie-U6-shRNA are co-introduced into mice injured by neomycin using the semicircular canal injection method, and EDU is injected intraperitoneally from the second to the seventh day after surgery to mark dividing cells.

7. The method of claim 1 wherein GPA, Espin and DKK3 synergistically regulate the proliferation and differentiation of inner ear stem cells, characterized in that: The third step is to detect the morphology and structure of new hair cells after gene therapy.

8. The method of claim 1 wherein GPA, Espin and DKK3 synergistically regulate the proliferation and differentiation of inner ear stem cells, wherein: The third step is to detect the electrophysiological characteristics of the newly generated hair cells after gene therapy.

9. Application of GPA, Espin, and DKK3 to synergistically regulate inner ear stem cell proliferation and differentiation in hair cell regeneration, characterized by: The method comprises the GPA, Espin and DKK3 synergistically regulating the proliferation and differentiation of inner ear stem cells as described in any one of claims 1 to 8, clarifying the structure and function of newly generated hair cells and the degree of recovery of auditory function after gene therapy synergized by GPA, Espin and DKK3, and laying a theoretical and experimental foundation for the clinical application of Lgr5+ inner ear stem cells to regenerate hair cells and restore auditory function.

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