CN117448381A - Gene editing activation Atoh1 transcription promotes vestibular hair cell regeneration and repair vestibular function - Google Patents
Gene editing activation Atoh1 transcription promotes vestibular hair cell regeneration and repair vestibular function Download PDFInfo
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- CN117448381A CN117448381A CN202311401734.6A CN202311401734A CN117448381A CN 117448381 A CN117448381 A CN 117448381A CN 202311401734 A CN202311401734 A CN 202311401734A CN 117448381 A CN117448381 A CN 117448381A
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Abstract
The application relates to the field of gene editing, in particular to activating Atoh1 transcription to promote vestibular hair cell regeneration and repair vestibular functions. The present application provides a gene editing system comprising: DNA editing element and coding gene thereof, CRISPR nuclease and coding gene thereof, gene regulation element and coding gene thereof, and vector, wherein the DNA editing element targets Atoh1, and the gene regulation element is used for activating Atoh1. The application confirms that the treatment of vestibular dysfunction caused by hair cell injury can be successfully realized in vivo by utilizing the CRISPR system to up-regulate Atoh1 through vestibular electrophysiological detection and vestibular functional behavioural detection.
Description
Technical Field
The application relates to the field of gene editing, in particular to activating Atoh1 transcription to promote vestibular hair cell regeneration and repair vestibular functions.
Background
Vestibular dysfunction is often manifested as dizziness, imbalance or dizziness, which are very common in elderly people. The results of the group queues show that the prevalence rate of dizziness and unbalance is 20-30% in the old group (age not less than 65 years). The prevalence of dizziness and unbalance was found to rise sharply with age, with this proportion exceeding 50% in community residents over 80 years old. In elderly community outpatients with ages of more than or equal to 65 years, 17% take dizziness as a complaint. Vestibular dysfunction affects the pace, somatic control ability of the elderly, increasing the risk of elderly falling. Vestibular dysfunction causes anxiety and panic, serious people cannot care for life, and great burden is imposed on families and society.
Vestibular receptors of the inner ear provide vestibular sensory information of head position and movement, and clinically 60-70% of vestibular dysfunction is caused by abnormal function of the vestibular organs of the inner ear. Therefore, patients with vestibular dysfunction are first diagnosed in the otorhinolaryngology department and occupy 15% of the outpatient service of the otorhinolaryngology department, which is one of the most common diseases of the otorhinolaryngology department. According to the international classification committee for vestibular disorders of the B ar ny association: vestibular dysfunction disorders include peripheral vestibular disorders affecting the vestibular labyrinth of the inner ear, and related disorders affecting the conduction pathways of the labyrinth to the brain and the vestibular cortex. Among them, bilateral vestibular disease (Bilateral Vestibulopathy, BVP), acute unilateral peripheral vestibular disease, meniere's disease, benign paroxysmal positional vertigo, and the like account for the vast majority of peripheral vestibular diseases. BVP accounts for 6.7% of dizziness or dizziness cases, patients have definite vestibular dysfunction, and the patients are intensively represented by walking or standing unsteady, and can also have indirect symptoms such as social limitation, concentration failure, space memory decline and the like, so that the overall quality of life is seriously influenced. According to the B ar ny association consensus, hair cell death by ototoxic drugs is the leading cause of BVP.
No clinically effective therapeutic drug exists for vestibular dysfunction, and the main therapeutic strategy is rehabilitation: central compensation and vision and body sense substitution are promoted through rehabilitation training. After unilateral vestibular function is lost, the vestibular function can be partially restored through central compensation; BVP, in turn, causes permanent vestibular dysfunction and vibration hallucinations, which are difficult to regulate through the central system. Regenerative therapy may be the last hope to radical cure BVP.
Vestibular hair cells are transducer cells that convert mechanical stimulus into an electrical signal, and function of the vestibular hair cells is to sense a change in position of the head and then stimulate the vestibular cochlear nerve, thereby allowing the center to sense position. Mammalian vestibular hair cells and supporting cells are derived from the same progenitor cells, and congenital anomalies, ototoxic drugs, viral diseases, degenerative changes and the like can all cause the deletion of vestibular hair cells and cause vestibular dysfunction. Most of the supporting cells lose transdifferentiation capacity in one week after birth, only a very small number of the supporting cells retain limited regeneration capacity in adults, and hair cells spontaneously regenerated in adulthood are immature and lack afferent nerve connection, so vestibular function cannot be restored by spontaneous regeneration. Vestibular dysfunction can be effectively treated if regenerated vestibular hair cells can be made functional and can be connected to vestibular nerves, thereby replacing missing and damaged hair cells. Atoh1 gene is a key gene for vestibular hair cell transdifferentiation, but exogenous overexpression of Atoh1 cannot realize vestibular function recovery, and continuous overexpression of Atoh1 is caused due to lack of an intranuclear regulatory mechanism in extranuclear overexpression, so that the maturation and even apoptosis of new hair cells are finally affected. In recent years, CRISPR gene editing tools show strong gene modification capability, and dmas 9 without nucleic acid cleavage activity is combined with a transcriptional regulatory element to activate a gene promoter, so that the CRISPR gene editing tools are novel means for activating endogenous expression of genes. The CRISPR gene activation technology can activate Atoh1 gene transcription in vivo, promote vestibular hair cell regeneration and development maturation, and realize vestibular dysfunction treatment caused by ototoxic drugs.
Atoh1 is a key transcription factor determining the fate of hair cells, belongs to the bHLH (basic Helix-Loop-Helix) family, and is critical for hair cell development. Ectopic expression of Atoh1 results in ectopic hair cell formation, and whether the expression level of Atoh1 increases during inner ear development determines whether pre-sensory cells will form hair cells. Unlike cochlear hair cells, hair cells of the mammalian vestibule have some regenerative capacity. This low level of regenerative capacity, while not capable of spontaneously restoring impaired vestibular function, theoretically increases the likelihood of successful gene therapy.
Exogenous overexpression of Atoh1 has been a major research direction in regeneration-related studies directed to vestibular hair cells to date. The Cheng team uses Atoh1 over-expression transgenic mice to realize the over-expression of Atoh1 in Plp1 positive support cells, and can observe the obvious regeneration of vestibular hair cells and the partial reconstruction of vestibular functions after vestibular injury [ Sayyid ZN, wang T, chen L, jones SM, cheng ag. Atoh1 Directs Regeneration and Functional Recovery of the Mature Mouse Vestibular system. Cell rep.2019;28 312-24e4], confirming that single up-regulation of Atoh1 can promote the functional reconstruction of the vestibule of mice. Atoh1 upregulation was demonstrated to enhance vestibular hair cell regeneration. Other researchers found that ectopic neohair cells could be seen in the inner ear of wild-type mice by local injection of Atoh1 over-expression virus to the inner ear; a significant increase in hair cell number was also seen in the vestibule following drug injury, with exogenous overexpression of Atoh1 suggesting that vestibular hair cell regeneration may be promoted. However, several studies have demonstrated that overexpression of exogenous Atoh1 fails to achieve restoration of vestibular function [ Qian X, ma R, wang X, xu X, yang J, chi F, ren d. Simultaneous genetamin-mediated damage and Atoh1overexpression promotes hair Cell regeneration in the neonatal mouse utricle. Exp Cell res.2021;398 (1) 112395.Guo JY,He L,Chen ZR,Liu K,Gong SS,Wang GP.AAV8-mediated Atoh1overexpression induces dose-dependent regeneration of vestibular hair cells in adult mice. Neurosci Lett.2021;747:135679.Taylor RR,Filia A,Paredes U,Asai Y,Holt JR,Lovett M,Forge A.Regenerating hair cells in vestibular sensory epithelia from humans.Elife.2018;7.]. Segil found that Atoh1mRNA was expressed only in the E12.5-P4 period in the inner ear of mice, and mRNA could not be detected in the inner ear after adult, suggesting that proper regulation of Aoh 1 was required for hair cell development and maturation. However, it is not clear how the changes in the spatial and temporal expression of Atoh1 are regulated. With the deep research of chromatin accessibility and post-transcriptional modification, the synergistic effect between the Atoh1 enhancer and the promoter may be the key to the regulation of hair cell regeneration by Atoh1.
Disclosure of Invention
In view of the above-described drawbacks of the prior art, the inventors of the present application studied that sustained overexpression of exogenous Atoh1 would result in apoptosis of hair cells, which may be the main reason why no article has been reported to achieve restoration of vestibular function in mammals by gene therapy. The transcription of Atoh1 has a self-regulating mechanism, and short-term activation can be realized under physiological conditions. Viral overexpression is a common strategy for gene up-regulation, but viral overexpression strategies do not allow restoration of vestibular function.
The application aims to provide a gene editing and activating Atoh1 transcription to promote vestibular hair cell regeneration and repair vestibular functions, a CRISPR activation system is adopted in a bilateral vestibular dysfunction mouse model caused by ototoxic drugs, the expression level of Atoh1 is regulated and controlled to rise, and the recovery of the vestibular hair cell number and functions is realized.
To achieve the above and other related objects, a first aspect of the present application provides a gene editing system comprising: DNA editing element and coding gene thereof, CRISPR nuclease and coding gene thereof, gene regulation element and coding gene thereof, and vector, wherein the DNA editing element targets Atoh1, and the gene regulation element is used for activating Atoh1.
In a second aspect, the present application provides a pharmaceutical composition comprising the aforementioned gene editing system, and a pharmaceutically acceptable carrier.
A third aspect of the present application provides the use of the aforementioned gene editing system or the aforementioned pharmaceutical composition in the preparation of a product having any one or more of the following functions:
1) Upregulating mRNA expression levels of Atoh 1;
2) Inducing hair cells to regenerate and mature;
3) Treating vestibular dysfunction caused by vestibular hair cell injury.
Compared with the prior art, the beneficial effects of this application are:
1. by in vitro MEF transfection and inner ear local injection, the expression level of Atoh1mRNA is detected by RT-PCR and RNAscope, and the successful up-regulation of Atoh1 in vitro and in vivo by using a CRISPR system is confirmed.
2. The mice were dissected and the oval sacs were removed at a time after the administration of the model mice for vestibular dysfunction. By observing the number, morphology, cilia structure, nerve synapse connection and the like of the hair cells through immunofluorescence technology, it is confirmed that the hair cells can be successfully induced to regenerate and mature in vivo by utilizing a CRISPR system.
3. Functional assays were performed at a time after administration of mice model of vestibular dysfunction. Through vestibular electrophysiology detection and vestibular functional behavioural detection, it is confirmed that the treatment of vestibular dysfunction caused by ototoxic drugs can be successfully realized in vivo by up-regulating Atoh1 by using a CRISPR system.
Drawings
FIG. 1 is a flow chart of the experimental design of the present application.
FIG. 2 is a diagram showing insertion examples of the genotype mouse dCAS9-SPH in example 1 of the present application.
FIG. 3 is a graph showing the expression level of Atoh1 in example 1 of the present application.
FIG. 4 is a plasmid map of the plasmid vector in example 2 of the present application.
FIG. 5 shows the proportion of Gfp +Sox2+ positive cells in the total number of cells supported in the different vectors of example 2 of the present application.
Fig. 6 is a diagram of vestibular immunofluorescence in example 3 of the present application.
FIG. 7 is a diagram showing information on viral plasmids in example 4 of the present application.
FIG. 8 is a graph showing the expression level of Atoh1 in example 4 of the present application.
FIG. 9 is a vestibular immunofluorescence of the number of hair cells in example 4 of the present application.
Fig. 10 is a vestibular immunofluorescence of example 4 of the present application, fig. 4A shows a primary hair cell maturation marker, fig. 4D shows a secondary hair cell maturation marker, fig. 4G shows a hair cell nerve connection, and fig. 4J shows a hair cell cilia.
Fig. 11 is a chart of cortical potential and behavioural outcome statistics in example 4 of the present application.
Fig. 12 is a view of vestibular immunofluorescence and laser confocal microscopy of example 5 of the present application.
Fig. 13 is a confocal microscope image of example 6 of the present application.
Fig. 14 is a behavioral experiment of example 6 of the present application.
FIG. 15 shows the information of the double viral system vector in example 7 of the present application.
Fig. 16 is a graph of vestibular electrophysiology function for different virus ratios in example 7 of the present application.
Fig. 17 is a laser confocal microscope image in example 7 of the present application.
Fig. 18 is a functional diagram of vestibular behavioural in example 7 of the present application.
Detailed Description
In order to make the objects, technical solutions and advantageous effects of the present application clearer, the present application is further described below with reference to examples. It should be understood that the examples are presented by way of illustration only and are not intended to limit the scope of the application. The test methods used in the following examples are conventional, unless otherwise indicated, and other advantages and effects of the present application will be readily apparent to those skilled in the art from the disclosure herein.
The inventor of the application finds that the gene editing activates Atoh1 transcription to promote vestibular hair cell regeneration and repair vestibular functions through a great deal of research and study, and completes the application on the basis.
In one aspect, the present application provides a gene editing system comprising: DNA editing element and coding gene thereof, CRISPR nuclease and coding gene thereof, gene regulation element and coding gene thereof, and vector, wherein the DNA editing element targets Atoh1, and the gene regulation element is used for activating Atoh1.
In the gene editing system provided herein, the DNA editing element encoding gene is selected from the group consisting of SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. or SEQ ID NO: 4.
In the gene editing system provided herein, the CRISPR nuclease is selected from Cas9, cas12, cas13 protein family, or variants thereof; preferably, the CRISPR nuclease variant is selected from dCas9, cas9Nickase, spRY; still further, the CRISPR nuclease is selected from dCas9.
In the gene editing system provided by the application, the gene regulatory element SPH comprises Suntag, p65 and HSF1, the gene regulatory element VPR is used in a double-virus system, and the gene regulatory element is used for activating a target gene and is specifically embodied for activating Atoh1.
In the gene editing system provided herein, the vector is selected from an adenovirus vector or an adeno-associated virus vector, further, the adeno-associated virus vector is selected from AAV1, AAV2, or AAV8. Adeno-Associated Virus (AAV) is a small DNA-containing particle isolated from simian adenovirus that can replicate only when co-cultured with adenovirus, and is about 25nm in diameter, consisting of capsid protein and a single-stranded DNA genome of 4.7kb in length, without an envelope. Has good infection effect on skeletal muscle, retina, liver cells, heart smooth muscle cells, neuron cells, islet B cells, joint synovial cells and the like.
In a specific embodiment of the present application, the delivery gene in the AAV vector may be EGFP, RFP.
In a specific embodiment of the present application, the delivery gene in the AAV vector may also be a combination of one or more of dCas9C (C-terminus of dCas 9), dCas9N (N-terminus of dCas 9), gRNA, cre, EGFP.
AAV vectors of the present application are selected from AAV1, AAV2, or AAV8 serotype vectors. The infection efficiency and the diffusion capability of different serotypes at different parts of an organism are different, and the proper serotypes are important in selecting the proper AAV serotypes by combining factors such as characteristics of cells and each AAV serotype, whether genes can realize efficient and stable expression or not, and even the final research result.
The injection mode of AAV vectors greatly affects the infection efficiency in animals, and common injection modes are as follows: tracheal intubation, tail intravenous injection, intraperitoneal injection, enema, brain stereotactic injection, in-situ injection, etc. Tissue-specific gene regulation can be achieved by selecting an appropriate injection mode, and for a specific part, a local in-situ injection mode is generally adopted, such as brain stereotactic injection, muscle site-specific injection, liver parenchymal injection, myocardial in-situ injection, original eye injection, intra-articular injection and the like. In a specific embodiment of the present application, the AAV vector is injected into the ear tissue through the round window of the inner ear.
In a preferred embodiment of the present application, the DNA editing element encodes a gene selected from the group consisting of SEQ ID NO:1, crispr nuclease is selected from dCas9 and vector is selected from AAV8.
In the gene editing system provided by the application, the gene editing system further comprises a Cre enzyme for tracing or a coding gene thereof. In a specific embodiment of the present application, the AAV packages a fluorescent gene such as EGFP, and when the gene editing system is successfully infected, the cells express EGFP fluorescence. Furthermore, the animal model selects dCAS9-SPH mice controlled by loxp sites, AAV packages Cre enzyme to combine with the DNA editing element, when the gene editing system infects cells with loxp, the Cre enzyme recognizes the loxp sites, the target genes are positioned under the guidance of the DNA editing element, and the dCAS9 system expression is activated.
In the gene editing system provided by the application, the dCAS9 comprises an N-terminal dCAS9N and a C-terminal dCAS9C, and further, the dCAS9N and the dCAS9C are positioned on different vectors; further, the dCas9N and the DNA editing element encoding genes are located on the same vector.
In one embodiment of the present application, as shown in FIG. 15, the genome length of the AAV itself is limited to no more than 4.7kb, and if packaging elements are required, the length limitation of the AAV itself is exceeded when packaging the packaged elements into one AAV vector, so dCAS9 is split into two AAV vectors for packaging separately, one AAV vector contains CMV promoter and gRNA for initiation thereof, and the other AAV vector contains CAGmini promoter and dCas9C for initiation thereof.
Specific embodiments of the present application include the following:
(1) The sgRNA plasmid was constructed in an in vitro design, transfection screening was performed using dCS 9-SPH mouse MEF, AAV vectors were screened in vivo by inner ear injection, and levels of vestibular Atoh1 upregulation were tested in vivo using dCS 9-SPH mice.
(2) And constructing an IDPN injury vestibular hair cell model. After activating Atoh1 by using CRISPRa, the newly generated dCAS9-SPH mice observe the regeneration condition of vestibular hair cells after IDPN injury by immunofluorescence and scanning electron microscopy, and the source of the hair cells is clarified by using pedigree tracking. Vestibular electrophysiology and vestibular functional behavioural tests are used to detect changes in vestibular function.
(3) Adult dCas9-SPH mice were subjected to crispla activation with Atoh1, and then observed for vestibular hair cell regeneration after IDPN injury by immunofluorescence and scanning electron microscopy. Vestibular electrophysiology and vestibular functional behavioural tests are used to detect changes in vestibular function.
(4) And (3) dividing dCAS9 into an N end and a C end by adopting a gene cutting strategy, constructing a double-virus system, packaging the N end and the sgRNA into one AAV, and packaging the C end into one AAV independently. After the inner ear of an adult wild mouse activates Atoh1 by using a double virus system, the regeneration condition of vestibular hair cells after IDPN injury is observed through immunofluorescence and a scanning electron microscope. Vestibular electrophysiology and vestibular functional behavioural tests are used to detect changes in vestibular function.
In another aspect, the present application provides a pharmaceutical composition comprising the aforementioned gene editing system, and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier should be compatible with the gene editing system, i.e., capable of blending therewith without substantially reducing the efficacy of the pharmaceutical composition. Materials that can be used as carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methyl cellulose, ethyl cellulose and methyl cellulose; tragacanth powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyols such as malondiol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifying agents, such as Tween; wetting agents, such as sodium lauryl sulfate; a colorant; a flavoring agent; tabletting, stabilizing agent and antioxidant; a preservative; non-thermal raw water; isotonic saline solution; and phosphate buffer, etc. These materials can be used as needed to aid stability of the formulation, to enhance activity or to produce acceptable mouthfeel and odor in the case of oral administration.
In another aspect the present application provides the use of the aforementioned gene editing system or the aforementioned pharmaceutical composition for the preparation of a product having any one or more of the following functions:
1) Upregulating mRNA expression levels of Atoh 1;
2) Inducing hair cells to regenerate and mature;
3) Treating vestibular dysfunction caused by vestibular hair cell injury.
In the application provided herein, the causes of vestibular hair cell injury include congenital anomalies, ototoxic drugs, viral diseases, degenerative diseases, and the like. In some embodiments, the ototoxic drug comprises one or more combinations of an aminoglycoside antibiotic, a macrolide antibiotic, an anticancer drug, a salicylic antipyretic analgesic, an antimalarial drug, a loop diuretic, an anti-heparinization agent, further, the aminoglycoside antibiotic is selected from the group consisting of streptomycin, kanamycin, neomycin, gentamicin, or 3, 3-iminodipropionitrile; the macrolide antibiotic is selected from erythromycin; the anticancer drug is selected from vincristine, 2-nitroimidazole and cisplatin; the salicylic acid antipyretic analgesic is selected from aspirin; the antimalarial is selected from quinine and chloroquine; the loop diuretic is selected from the group consisting of tachyuria or diuretic acid; the anti-heparinization agent is selected from lanbolin.
The present application is further illustrated by the following examples, which are not intended to limit the scope of the present application.
Unless otherwise indicated, the practice of the present invention employs conventional techniques of molecular biology, microbiology, cell biology, biochemistry, and immunology, which are within the understanding of the skilled artisan. These techniques are widely used and can be fully described in the following documents, such as: "molecular cloning: ALaboratory Manual, fourth edition" (M.R. Green, et al 2014); "Oligonucleotide Synthesis" (M.J. Gait, et al 1984); "Polymerase Chain Reaction: principles, applications and Troubleshooting" (M.E. Babar, et al 2011); "Short Protocols in Molecular Biology, fifth edition" (F.M. Ausubel, et al 2002); "Methods in Molecular Biology" (Humana Press); "Gene Transfer Vectors for Mammalian Cells" (J.H.Miller and M.P.Calos.1987); "Culture of Animal Cell" (R.I. Fresnel, et al 2010); "Methods in Enzymology" (Academic Press, inc.); "Using Antibodies: ALaboratory Manual" (E.Harlow and D.Lane.1999); "Handbook ofExperimental Immunology" (L.A. Herzenberg, et al 1997); "Current Protocols in Immunology" (J.E. Coligan, et al 2002).
Example 1
Screening for gRNA that efficiently up-regulates Atoh1
1) Construction of grna sequence plasmid:
a) The steps are as follows:
the sequence of interest was designed as follows, and was constructed from Shanghai organisms. And (3) performing PCR amplification, performing agar coacervation electrophoresis on the amplified product, cutting a specific electrophoresis band to be thick, melting and purifying.
The vector pcDN3.1 was selected. The amplified products and the vector are respectively subjected to enzyme digestion and then are religated by T4 ligase. And (3) converting the connection product into a receptor bacterium for extraction and amplification to obtain a large number of plasmids.
b) Sequence:
grna1：ACGCGCCAGTGTATCTCCCCCGG(SEQ ID NO：1)
grna2：GGCGCGTGCCGCTTTTAAAGGG(SEQ ID NO：2)
grna3：ACACGCGACTGGCGCAAGGAGGG(SEQ ID NO：3)
grna4：TGGCGCGTGCCGCTTTTTAAAGG(SEQ ID NO：4)
2) Primary culture of P1dCAs9-SPH transgenic mice MEF
a) Mouse MEF culture: taking transgenic mice (information shown in figure 2) of 1 day new year, taking out abdominal skin in 75% alcohol, removing fat in PBS+ double antibody, and cutting with sterile eye for short for 1mm 2 Tissues of size were digested with 0.25% edta-pancreatin at 37 degrees celsius for 60 minutes. Digestion was stopped by adding sufficient MEF medium (DMEM/F12+15% FBS+diabody), repeated pipetting, filtration, centrifugation and removal of supernatant. Resuspension with sufficient MEF medium followed by culture.
3) Electric transfer of grna plasmid and harvesting of cells after 24h
a) Plasmid transfection was performed using a BIO-RAD GENE PULSER XCELL electrotransfer apparatus under conditions of 300V,20ms,1pulse, cell size 1X10 7 Plasmid 5ug. Cells were harvested after 24 hours of post-transfection culture.
4)RT-PCR
a) The expression level of Atoh1 was detected by RT-PCR experiments using SYBR Green dye.
5) Results: see fig. 3.
6) Conclusion: grna1 is most efficient.
Example 2
Screening AAV for efficient transfection of vestibular support cells
1) Construction of AAV1, AAV2, AAV8, adeno-GFP Virus
The plasmid vector information is shown in FIG. 4.
The plasmid vectors were packaged and purified with AAV1, AAV2, AAV8, adeno-GFP: AAV-293 cells (50-70% confluence, no mycoplasma contamination) used for packaging viruses and viral plasmids were co-transfected, cells were collected for 72 hours, supernatant was collected by repeated freeze thawing with liquid nitrogen three times, then digested with Benonase enzyme, the obtained supernatant was purified by Biomiga brand purification column, and the obtained viruses were stored at-80℃after split charging.
2) Round window injection of neonatal wild mice
i. Anesthetizing newborn 1 day mice on ice, sterilizing epidermis, and injecting target virus into cochlea round window of mice with glass electrode of anterior segment opening, total injection dose of about 1×10 11 gc. Mice were resuscitated on a 42 degree heating pad following dosing.
3) Vestibule staining for transfection efficiency after 4 weeks
i. Mice were removed from their vestibular ellipsis 4 weeks after injection, scraped off the otolith, and fixed in 4 degrees 4% pfa for 1 hour. The hair cells were labeled with Myo7a, the transfection was labeled with Gfp, the support cells were labeled with sox2, and after staining, the cells were observed by a laser confocal microscope. Statistics of Gfp +Sox2+ positive cells to total supporting cells.
4) The results are shown in FIG. 5.
5) Conclusion: AAV8 is most efficient in transfection.
Example 3
Construction of mice vestibular injury model
1) 2wk mice were intraperitoneally injected with different doses of 2mg/kg,4mg/kg,6mg/kg IDPN and PBS control
2) Vestibular immunofluorescence was harvested at various times post-dose to observe lesions: all elliptic cyst hair cell number statistics by marking hair cells with Myo7a
3) The results are shown in FIG. 6.
4) Conclusion: as the death rate of 6mg/kg is too high, 4mg/kg is used
Example 4
Constructing a mouse endogenous Atoh1 up-regulation system; assessment of vestibular regeneration
1) Construction of the virus: the method is the same as that
i.AAV-grna-cre
Plasmid information is shown in FIG. 7.
Virus: AAV8
sgnna sequence: ACGCGCCAGTGTATCTCCCCCGG (SEQ ID NO: 1)
2) The neonatal mice were injected with virus at the p1 round window, and were given IDPN, RT-PCR by intraperitoneal injection after two weeks, and were given RT-PCR 4 weeks after administration, suggesting successful upregulation of Atoh1 levels, and the results are shown in FIG. 8.
3) Mice elliptical sacs were harvested 1 month after IDPN, fixed and immunofluorescent, with Myo7 a-labeled hair cells, gfp-labeled transfection, phalloidin-labeled cilia, β -Tubulin (Tuj 1) -labeled nerve fibers, secreted Phosphoprotein (Spp 1) bound to Myo7 a-labeled type I hair cells, annexin A4 (Anx 4) bound to Myo7 a-labeled type II hair cells. The counts were observed with a laser confocal microscope. As shown in fig. 9-10, it can be seen that the number of hair cells increases after treatment, including I, II type hair cells. And a large number of cells have intact cilia and neural connections.
4) Mice were tested for vestibular behavioural function 1.3.6 months after IDPN injection:
i. balance beam experiment: the balance beam facility is a wood rod with the length of 80cm and the width of 1cm, and is suspended in the air and horizontally arranged. After the animals are trained for many times, the animals are placed at the starting end of the balance beam, and the time for the animals to walk to the other end of the balance beam is counted to be more than 120s or the time for the animals to be unable to stand is counted to be 120s.
Open field experiment: the mice were placed in a corner of a 40cm x 40cm x 40cm open field box, and the movement time was recorded for 5 minutes with a camera, and the movement track and the number of rotations were analyzed. The field box was cleaned with 75% alcohol and chlorine-containing sterilizing solution between each experiment and left to air dry.
Stick turning experiment: the speed of the rotor bars was chosen to be fixed at 5rpm, 10rpm, 20rpm for a total of 300s, and accelerated from 5rpm to 44rpm within 60s, respectively. After training the mice, the mice were placed on the rotating rod when the rotating rod reached the rotational speed, and the duration of their stay on the rotating rod was recorded.
Vemp: after anesthesia, the mice were placed on a 37 degree heating pad and the heads were lifted 45 degrees. The neck muscle was elongated, the ground electrode was placed under the skin at the hind limb, the reference electrode was placed under the mid-scalp skin, and the recording electrode was inserted into the test side sternocleidomastoid muscle. The horn was placed 10cm in front of the mouse head and myoelectric potential was recorded after short acoustic stimulation. The stimulation gradually decreased from 120db, with a gradient of 5 db.
vsep: after the mice were anesthetized, the heads of the mice were fixed to a vibration generator on a 37-degree heating pad, a ground electrode was placed under the skin at the hind limb, a reference electrode was placed under the mastoid skin, and a recording electrode was placed under the median skin of the scalp. Cortex potential was recorded after stimulation with vibration. The stimulus was decremented from 2 g/ms.
5) Conclusion: as shown in fig. 11, recovery after vestibular hair cell number and functional impairment can be achieved, lasting for half a year or more.
Example 5
Defining the source of new hair cells
1) Construction of a mouse model supporting lineage follow-up: PLP1CreERT x tdTomato fl/fl xdCAs9-SPH transgene mice
2) The same virus as in example 4 was injected into the round window of the inner ear of P1, and the vestibular immunofluorescence and laser confocal microscopy were performed 1 month after injury at 2 weeks of intraperitoneal IDPN. Myo7a labeled hair cells, gfp suggested the virus transfection status, tdmate labeled support cell-derived cells, and the results are shown in fig. 12.
3) Conclusion: the vast majority of hair cells are derived from support cell transdifferentiation.
Example 6
Adult mouse treatment
1) Adult mice were intraperitoneally injected with IDPN 4 weeks, virus was injected 1 day later into the inner ear, and immunofluorescence and behavioural function were observed 1 month after injury
i. Post-adult mice semi-regulated injection: the mice were anesthetized and then opened to the front of the ear, exposing the posterior semicircular canal. After opening the semicircular canal, the PI tube is used for realizing the followingEXAMPLE 4 the same virus was injected into the inner ear of mice at a dose of 6.6x10 10 gc。
Myo7a labeled hair cells, gfp suggested viral transfection. Phlloidin marks cilia, tuj1 marks nerve fibers, spp1 binds Myo7a marks type I hair cells, and Anx4 binds Myo7a marks type II hair cells. The laser confocal microscope observed the counts, and the results are shown in fig. 13.
The behavioural experiment was the same as in example 4, and the results are shown in fig. 14.
2) Conclusion: can realize the regeneration of vestibular hair cells and the treatment after the vestibular function injury of adult mice, and can last for half a year or longer.
Example 7
7. Double virus mediated gene editing and repairing vestibular function
1) dCAS9-VPR-gRNA double virus system construction
The two vector sequences are shown in FIG. 15, and the construction method is the same.
Virus: AAV8
sgnna sequence: ACGCGCCAGTGTATCTCCCCCGG (SEQ ID NO: 1)
2) Different viral ratios were injected into the inner ear of p1 wild type mice, IDPN was injected intraperitoneally for 2 weeks, and vestibular electrophysiology (VsEP and VEMP) was observed at different times after injection, and the results are shown in fig. 16, suggesting that 1:1 is best
3) Mice oval vesicles were harvested 4 weeks after IDPN, immunofluorescence: myo7 a-labeled hair cells, phlloidin-labeled cilia, tuj 1-labeled nerve fibers, spp 1-labeled type I hair cells bound to Myo7a, and Anx 4-labeled type II hair cells bound to Myo7 a. And (5) observing by a laser confocal microscope. The results are shown in FIG. 17.
4) Vestibular functional recovery was observed in mice at different times 1:1: vestibular behavioural function is the same as example 6 and can last for half a year or more.
To sum up, the application constructs a mouse vestibular injury model by using a wild type C57 mouse, the mice are injected with IDPN in the abdominal cavity after 15 days of birth, and the number of vestibular oval bursa hair cells is calculated by dissecting 1 day, 3 days, 7 days, 14 days and 28 days after administration. Two vestibular electrophysiological indices of mice VsEP and VEMP were recorded. It was found that some mice had a tendency to recover vestibular function after injury, but none was able to recover overall balance function (purple group in fig. 18).
In vitro transfection was performed after dCAS9-SPH mouse MEF cells were cultured in vitro, AAV-sgRNA-Cre-GFP virus was constructed, the level of transfection was observed by GFP fluorescence level, and the level of Atoh1 expression was detected by RT-qPCR, which revealed that the virus was able to successfully up-regulate the level of Atoh1 expression (see FIG. 3).
Detection of the mice vestibule Atoh1 expression level by RT-qPCR suggests a significant increase in overall Atoh1 expression level, and after dissection and immunofluorescence of the oval vesicles, significant transfection of the mice vestibule was found after administration and significant increase in the number of vestibule hair cells in the mice after treatment (see fig. 17).
By using two different dosing ratios, the mice after dosing are subjected to overall vestibular function detection, including vestibular electrophysiology indexes (VsEP and VEMP) and behavioral detection (open field experiment, rod rotation test, balance beam experiment), and the vestibular potential and overall balance function of the mice after treatment are found to be greatly improved, wherein 1: the curative effect of the administration group 1 is better than that of the administration group 2:1, suggesting that CRISPR-mediated activation of Atoh1 transcription can restore function in vestibular injured mice (see figure 18).
The application provides application of utilizing a CRISPR activation system to up-regulate Atoh1 to realize vestibular hair cell regeneration and treat vestibular dysfunction caused by ototoxic drugs. The inner ear gene activation system based on CRISPRa is established, the accurate, controllable and moderate up-regulation of genes is realized, and a new means is provided for the inner ear gene regulation of newborn and adult mammals. The CRISPRa based on the double viruses can up-regulate Atoh1 in vivo to promote the regeneration of vestibular hair cells, rebuild the vestibular function of rodent with vestibular injury, and provide a new strategy for the treatment of vestibular dysfunction.
The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the application. Modifications and variations may be made to the above-described embodiments by those of ordinary skill in the art without departing from the spirit and scope of the present application. Accordingly, it is intended that all equivalent modifications and variations which can be accomplished by persons skilled in the art without departing from the spirit and technical spirit of the present disclosure shall be covered by the claims of this application.
Claims (10)
1. A gene editing system, the gene editing system comprising: DNA editing element and coding gene thereof, CRISPR nuclease and coding gene thereof, gene regulation element and coding gene thereof, and vector, wherein the DNA editing element targets Atoh1, and the gene regulation element is used for activating Atoh1.
2. The gene editing system of claim 1, wherein the DNA editing element encodes a gene selected from the group consisting of seq id NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. or SEQ ID NO: 4.
3. The gene editing system of claim 1, wherein the CRISPR nuclease is selected from Cas9, cas12, cas13 protein family, or variants thereof; preferably, the CRISPR nuclease variant is selected from dCas9, cas9Nickase, spRY; still further, the CRISPR nuclease is selected from dCas9.
4. The gene editing system of claim 1, wherein the vector is selected from an adenovirus vector or an adeno-associated virus vector, further wherein the adeno-associated virus vector is selected from AAV1, AAV2, or AAV8.
5. The gene editing system of claim 1, wherein the DNA editing element encodes a gene selected from the group consisting of seq id NO:1, said CRISPR nuclease is selected from dCas9 and said vector is selected from AAV8.
6. The gene editing system of claim 5, further comprising a tracer Cre enzyme or a gene encoding the same; further, the Cre enzyme encoding gene is located on the same vector as the DNA editing element encoding gene and CRISPR nuclease encoding gene.
7. The gene editing system of claim 5, wherein said dCas9 comprises an N-terminal dCas9N and a C-terminal dCas9C, further wherein said dCas9N and dCas9C are on different vectors; further, the dCas9N and DNA editing element encoding genes are located on the same vector.
8. A pharmaceutical composition comprising the gene editing system of any one of claims 1-7, and a pharmaceutically acceptable carrier.
9. Use of a gene editing system according to any one of claims 1 to 7 or a pharmaceutical composition according to claim 8 for the preparation of a product having any one or more of the following functions:
1) Upregulating mRNA expression levels of Atoh 1;
2) Inducing hair cells to regenerate and mature;
3) Treating vestibular dysfunction caused by vestibular hair cell injury.
10. The use according to claim 9, wherein the cause of vestibular hair cell injury comprises congenital abnormalities, ototoxic drugs, viral diseases, or degenerative diseases; preferably, the ototoxic drug comprises one or more combinations of an aminoglycoside antibiotic, a macrolide antibiotic, an anticancer drug, a salicylic antipyretic analgesic, an antimalarial drug, a loop diuretic, an anti-heparinization agent, further, the aminoglycoside antibiotic is selected from streptomycin, kanamycin, neomycin, gentamicin, or 3, 3-iminodipropionitrile; the macrolide antibiotic is selected from erythromycin; the anticancer drug is selected from vincristine, 2-nitroimidazole and cisplatin; the salicylic acid antipyretic analgesic is selected from aspirin; the antimalarial is selected from quinine and chloroquine; the loop diuretic is selected from the group consisting of tachyuria or diuretic acid; the anti-heparinization agent is selected from lanbolin.
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| US20230181767A1 (en) * | 2020-05-14 | 2023-06-15 | Decibel Therapeutics, Inc. | Compositions and methods for promoting hair cell regeneration |
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| CN106011104A (en) * | 2015-05-21 | 2016-10-12 | 清华大学 | Method for carrying out gene editing and expression regulation by utilizing Cas splitting system |
| WO2021000928A1 (en) * | 2019-07-04 | 2021-01-07 | 中美瑞康核酸技术(南通)研究院有限公司 | Oligomeric nucleic acid molecule activating atoh1 gene and use thereof |
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