WO2021081826A1 - Applications of ptbp1 inhibitor in preventing and/or treating retinal diseases - Google Patents

Abstract

Disclosed are applications of a PTBP1 inhibitor in preventing and/or treating retinal diseases. Specifically, provided are uses of an inhibitor of PTBP1 gene or an encoded protein thereof, for use in preparing a composition or preparation. The composition or preparation is used for preventing and/or treating the retinal diseases. The present invention, by inhibiting the expression or activity of PTBP1 gene or the encoded protein thereof of the MG cells in the retina, effectively induces the differentiation of the MG cells towards the RGC cells, thus preventing and/or treating the retinal diseases.

Description

Application of Ptbp1 inhibitors in the prevention and/or treatment of retinal diseases Technical field

The invention relates to the field of biomedicine. More specifically, the present invention relates to the use of Ptbp1 inhibitors in the prevention and/or treatment of retinal diseases.

Background technique

Retinal ganglion cells (RGCs) are the only output neurons in the retina, which can send visual information from the eyes to the brain. After retinal injury, Müller cells (MG) in zebrafish can re-enter the cell cycle as retinal stem cells and replenish damaged neurons including RGCs. However, in mammals, mature MG loses its neurological performance, and at the same time damaged RGCs cannot regenerate, which will eventually lead to permanent vision loss in the adult retina. This is a very common cause of blindness, so finding a way to repair RGCs has been regarded as a bold goal.

Interestingly, a recent study showed that mouse fibroblasts and N2a cells can be transformed into neurons in vitro by knocking down a single endogenous gene Ptbp1. However, the method of reprogramming neurons in vivo by inhibiting Ptbp1 has not yet been explored.

Therefore, there is an urgent need in the art to develop methods that can use Ptbp1 to reprogram neurons in the body to treat retinal diseases.

Summary of the invention

The purpose of the present invention is to provide an application of a Ptbp1 inhibitor in the prevention and/or treatment of retinal diseases.

In the first aspect of the present invention, there is provided a use of a Ptbp1 gene or its encoded protein inhibitor for preparing a composition or preparation, and the composition or preparation is used to prevent or treat retinal diseases.

In another preferred example, the retinal disease is a retinal disease caused by neurodegeneration.

In another preferred embodiment, the composition or preparation induces the transdifferentiation of MG cells into RGC cells to treat retinal diseases caused by neurodegeneration.

In another preferred embodiment, the MG cells are Müller glial cells.

In another preferred embodiment, the MG cells are derived from the retina.

In another preferred example, the RGC cells are retinal ganglion cells.

In another preferred example, the RGC cells are functional RGCs.

In another preferred example, the RGC cells can be integrated into the visual pathway and improve visual function.

In another preferred example, the RGC cells can realize functional projection to the central visual area and improve visual function.

In another preferred example, the improvement of visual function is to improve the visual function of mammals suffering from retinal diseases caused by neurodegeneration.

In another preferred embodiment, the mammals include human or non-human mammals.

In another preferred embodiment, the non-human mammals include rodents (such as mice, rats, or rabbits) and primates (such as monkeys).

In another preferred embodiment, the MG cells are transdifferentiated into RGC cells while also being differentiated into axonal cells.

In another preferred embodiment, the inhibitor is selected from the group consisting of antibodies, small molecule compounds, microRNA, siRNA, shRNA, gene editors, or a combination thereof.

In another preferred embodiment, the gene editor includes a DNA gene editor and an RNA gene editor.

In another preferred embodiment, the gene editor includes optional gRNA and gene editing protein.

In another preferred example, the gRNA is RNA that guides the gene editing protein to specifically bind to the Ptbp1 gene.

In another preferred embodiment, the gRNA guide gene editing protein specifically binds to the mRNA of the Ptbp1 gene.

In another preferred embodiment, the gene editing protein is selected from the group consisting of CasRx, CRISPR/Cas9, Cpf1, Cas9, Cas13a, Cas13b, Cas13c, or a combination thereof.

In another preferred embodiment, the source of the gene editing protein is selected from the group consisting of Streptococcus pyogenes, Staphylococcus aureus, Acidaminococcus sp, Lachnospiraceae bacterium ), Ruminococcus Flavefaciens, or a combination thereof.

In another preferred embodiment, the Ptbp1 is derived from mammals; preferably, it is derived from humans, mice, rats, or rabbits; more preferably, it is derived from humans.

In another preferred example, the Ptbp1 gene includes a wild-type Ptbp1 gene and a mutant Ptbp1 gene.

In another preferred example, the mutant type includes a mutant form in which the function of the encoded protein is not changed after the mutation (that is, the function is the same or substantially the same as that of the wild-type encoded protein).

In another preferred embodiment, the gene editing protein is CasRx, and the nucleotide sequence of gRNA is selected from the following group: SEQ ID NO.: 1, 2, 3, 4, 5, and 6.

In another preferred example, the region targeted by the ptbp1 gene or the inhibitor of the encoded protein (such as a gene editing protein) is the 4758-4787 and/or 5381-5410 positions of the ptbp1 gene sequence.

In another preferred embodiment, the inhibitor of the ptbp1 gene or its encoded protein inhibits the activity and/or expression of ptbp1.

In another preferred embodiment, the concentration of the inhibitor gene or its encoded protein ptbp1 (virus titer)> 1 × 10 ^13, preferably, is ^{1 × 10 13 -1 × 10 14} .

In another preferred example, the inhibitor of the ptbp1 gene or its encoded protein has an inhibitory rate of more than 90%, preferably 90%-95%, on the activity and/or expression of ptbp1.

In another preferred embodiment, the inhibitor targets MG cells of the retina.

In the second aspect of the present invention, there is provided a composition comprising:

(a) A gene editing protein or an expression vector thereof, the gene editing protein is selected from the group consisting of CasRx, CRISPR/Cas9, Cpf1, Cas9, Cas13a, Cas13b, Cas13c, or a combination thereof; and

(b) gRNA or its expression vector, the gRNA is RNA that guides the gene editing protein to specifically bind to the Ptbp1 gene, and the nucleotide sequence of the gRNA is selected from the following group: SEQ ID NO.: 1, 2, 3, 4, 5, and 6.

In another preferred embodiment, the gRNA guide gene editing protein specifically binds to the mRNA of the Ptbp1 gene.

In another preferred embodiment, the composition includes a pharmaceutical composition.

In another preferred embodiment, the composition further includes:

(c) Other drugs for the prevention and/or treatment of retinal diseases.

In another preferred embodiment, the expression vector of the gene editing protein includes a vector targeting retinal MG cells.

In another preferred embodiment, the expression vector includes a viral vector.

In another preferred example, the viral vector is selected from the group consisting of adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, herpes virus, SV40, poxvirus, or a combination thereof.

In another preferred example, the vector is selected from the following group: lentivirus, adenovirus, adeno-associated virus (AAV), or a combination thereof, preferably, the vector is adeno-associated virus (AAV).

In another preferred embodiment, the dosage form of the composition is selected from the following group: a lyophilized preparation, a liquid preparation, or a combination thereof.

In another preferred embodiment, the dosage form of the composition is a liquid preparation.

In another preferred embodiment, the dosage form of the composition is an injection dosage form.

In another preferred embodiment, the composition is a cell preparation.

In another preferred embodiment, the expression vector of the gene editing protein and the expression vector of gRNA are the same vector or different vectors.

In another preferred example, the weight ratio of the component (a) to the component (b) is 100:1-0.01:1, preferably, 10:1-0.1:1, more preferably, 2: 1-0.5:1.

In another preferred example, the content of the component (a) in the composition is 0.001%-99%, preferably, 0.1%-90%, more preferably, 1%-70%.

In another preferred example, the content of the component (b) in the composition is 0.001%-99%, preferably, 0.1%-90%, more preferably, 1%-70%.

In another preferred example, the content of the component (c) in the composition is 1%-99%, preferably, 10%-90%, more preferably, 30%-70%.

In another preferred example, in the composition, the component (a), component (b) and optional component (c) account for 0.01-99.99 wt% of the total weight of the composition, which is greater than Preferably 0.1-90wt%, more preferably 1-80wt%.

In the third aspect of the present invention, a medicine kit is provided, including:

(a1) The first container, and the gene editing protein or its expression vector in the first container, or a drug containing the gene editing protein or its expression vector, the gene editing protein is selected from the following group: CasRx, CRISPR/ Cas9, Cpf1, Cas9, Cas13a, Cas13b, Cas13c, or a combination thereof;

(b1) A second container, and gRNA or its expression vector, or a drug containing gRNA or its expression vector, in the second container, and the gRNA is RNA that guides the gene editing protein to specifically bind to the Ptbp1 gene.

In another preferred example, the gRNA is mRNA that guides the gene editing protein to specifically bind to the Ptbp1 gene.

In another preferred embodiment, the nucleotide sequence of the gRNA is selected from the group consisting of SEQ ID NO.: 1, 2, 3, 4, 5, and 6.

In another preferred example, the region targeted by the gRNA is positions 4758-4787 and/or positions 5381-5410 of the Ptbp1 gene sequence.

In another preferred embodiment, the kit further includes:

(c1) The third container, and other medicines for preventing and/or treating retinal diseases in the third container.

In another preferred embodiment, the first container, the second container, and the third container are the same or different containers.

In another preferred embodiment, the medicine in the first container is a unilateral preparation containing a gene-edited protein or an expression vector thereof.

In another preferred embodiment, the medicine in the second container is a unilateral preparation containing gRNA or its expression vector.

In another preferred embodiment, the medicine in the third container is a single preparation containing other medicines for preventing and/or treating retinal diseases.

In another preferred embodiment, the dosage form of the drug is selected from the group consisting of a lyophilized preparation, a liquid preparation, or a combination thereof.

In another preferred embodiment, the dosage form of the drug is an oral dosage form or an injection dosage form.

In another preferred embodiment, the kit also contains instructions.

In the fourth aspect of the present invention, there is provided a composition according to the second aspect of the present invention or the use of the kit according to the third aspect of the present invention to prepare a medicine for preventing and/or treating retinal diseases.

In another preferred embodiment, the composition, the concentration of the role of protein or gene expression vector editing (viral titer)> 1 × 10 ^{13, preferably, 1 × 10 13 -1 × 10} 14.

In another preferred embodiment, the composition or expression of the gRNA concentration of the carrier (viral titer)> 1 × 10 ^{13, preferably, 1 × 10 13 -1 × 10} 14.

In another preferred embodiment, in the pharmaceutical composition, the concentration (viral titer) of the other drugs for preventing and/or treating retinal diseases is> 1×10 ¹³ , preferably, 1×10 ¹³ -1 ×10 ¹⁴ .

In another preferred embodiment, the composition or kit includes (a) gene editing protein or its expression vector; and (b) gRNA or its expression vector; and (c) optionally other prevention and/or treatment of retina Drugs for diseases; and (d) pharmaceutically acceptable carriers.

In another preferred embodiment, in the composition or kit, (a) gene editing protein or its expression vector; and (b) gRNA or its expression vector; and (c) optional other prevention and/or treatment The medicine for retinal diseases accounts for 0.01-99.99% by weight of the total weight of the composition or the kit, preferably 0.1-90% by weight, more preferably 1-80% by weight.

In the fifth aspect of the present invention, a method for promoting the differentiation of MG cells into RGC cells is provided, which includes the steps:

In the presence of the Ptbp1 gene or its encoded protein inhibitor or the composition according to the second aspect of the present invention, MG cells are cultured to promote the differentiation of MG cells into RGC cells.

In another preferred embodiment, the effect of the concentration of the gene or its encoded protein Ptbp1 inhibitors (viral titer)> 1 × 10 ^{13, preferably, 1 × 10 13 -1 × 10} 14.

In another preferred embodiment, the effect of concentration of the second aspect of the present invention, the composition (viral titer)> 1 × 10 ^{13, preferably, 1 × 10 13 -1 × 10} 14.

In another preferred embodiment, the method is a non-diagnostic and non-therapeutic method.

In the sixth aspect of the present invention, a method for preventing and/or treating retinal diseases is provided, including:

Ptbp1 gene or its encoded protein inhibitor, or the composition according to the second aspect of the present invention, or the kit according to the third aspect of the present invention is administered to a subject in need.

In another preferred embodiment, the subject includes humans or non-human mammals suffering from retinal diseases.

In another preferred embodiment, the non-human mammals include rodents and primates, preferably mice, rats, rabbits, and monkeys.

In the seventh aspect of the present invention, there is provided a method for screening candidate compounds for the prevention and/or treatment of retinal diseases, the method comprising the steps:

(a) In the test group, add a test compound to the cell culture system, and observe the expression (E1) and/or activity (A1) of Ptbp1 in the cells of the test group; in the control group, in the same cell No test compound is added to the culture system, and the expression (E0) and/or activity (A0) of Ptbp1 in the cells of the control group is observed;

Among them, if the Ptbp1 expression (E1) and/or activity (A1) of the cells in the test group is significantly lower than that of the control group, it indicates that the test compound is a preventive and/or inhibitory effect on the expression and/or activity of Ptbp1. Candidate compounds for the treatment of retinal diseases.

In another preferred example, the expression level of Ptbp1 is obtained by qPCR.

In another preferred embodiment, the method further includes the steps:

(b) For the candidate compound obtained in step (a), further test its promoting effect on the differentiation of MG cells into RGC cells; and/or further test whether it has a down-regulation effect on the Ptbp1 gene.

In another preferred embodiment, the method includes step (c): administering the candidate compound determined in step (a) to a mammalian model, and determining its effect on the mammal.

In another preferred example, the mammal is a mammal suffering from retinal diseases.

In another preferred example, the "significantly lower" means that E1/E0≤1/2, preferably,≤1/3, more preferably≤1/4.

In another preferred example, the "significantly lower" means that A1/A0≤1/2, preferably,≤1/3, more preferably≤1/4.

In another preferred embodiment, the cells include MG cells.

In another preferred embodiment, the cell is a cell cultured in vitro.

In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as the embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, I will not repeat them here.

Description of the drawings

Figure 1 shows that RGCs can be obtained by reprogramming MG in the intact retina.

Figure 1a shows 5 independent replicates of knockdown of Ptbp1 in N2a cells.

Figure 1b shows the expression levels of all detected genes in the CasRx-Ptbp1 RNA-seq library (y-axis) in log2 (FPKM+1) values, compared with the CasRx control (x-axis). The experiment was repeated 4 times independently, with similar results.

Figure 1c shows a schematic diagram of the regeneration of RGCs from MG. Vector 1 (GFAP-GFP-Cre) encodes Cre recombinase and GFP driven by MG-specific promoter GFAP, and vector 2 (GFAP-CasRx-Ptbp1 or GFAP-CasRx) encodes CasRx and guide. In order to regenerate RGCs, GFAP-GFP-Cre and GFAP-CasRx-Ptbp1 or GFAP-CasRx were injected into the retina (5-week-old Ai9 mice). Check for transdifferentiation one month after injection. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner plexiform layer; IPL, inner plexiform layer; GCL, ganglion cell layer.

Figure 1d shows a representative image of the co-localization of tdTomato and Brn3a in GCL. The white arrow indicates the MG terminal endplate that is not co-localized with tdTomato, and the yellow arrow indicates the co-localization of tdTomato and Brn3a in the retina injected with GFAP-GFP-Cre and GFAP-CasRx-Ptbp1. Brn3a is a specific marker of RGCs. Scale bar, 20μm.

Figure 1e shows the number of tdTomato ⁺ or tdTomato ⁺ , Brn3a ⁺ cells in GCL one month after AAV injection. GFAP-GFP-Cre plus GFAP-CasRx, n=6 retinas; GFAP-GFP-Cre plus GFAP-CasRx-Ptbp1, n=7 retinas.

Figure 1f shows a representative image of tdTomato co-localized with another RGC-specific marker Rbpms in GCL. The yellow arrow indicates the co-localization of tdTomato and Rbpms. Scale bar, 20μm.

Figure 1g shows the number of tdTomato ⁺ or tdTomato ⁺ Rbpms ⁺ cells in GCL one month after AAV injection. GFAP-GFP-Cre plus GFAP-CasRx, n=6 retinas; GFAP-GFP-Cre plus GFAP-CasRx-Ptbp1, n=8 retinas. Data are expressed as mean±sem, *p<0.05, **p<0.01, ***p<0.001, unpaired t test.

Figure 2 shows that reprogramming MG can produce RGC in a mouse model of NMDA-induced retinal damage.

Figure 2a shows an outline of the experimental design. Retinal damage was induced by injecting NMDA (200mM, 1.5μl) into Ai9 mice aged 4-8 weeks. 2-3 weeks after NMDA injection, AAV is introduced by subretinal injection. Immunostaining and behavioral experiments were performed one month after AAV injection.

Figure 2b shows that NMDA injection basically depletes the RGCs in the GCL. Scale bar, 50μm.

Figure 2c shows the co-localization of tdTomato and Brn3a. The white arrow indicates the co-localization of Brn3a and tdTomato in GCL. n=6 retinas, scale bar: 20 μm.

Figure 2d shows the number of Brn3a+ or tdTomato+ or tdTomato+Brn3a+ cells in GCL. GFAP-CasRx plus GFAP-GFP-Cre, n=6 retinas; GFAP-CasRx-Ptbp1 plus GFAP-GFP-Cre, n=7 retinas.

Figure 2e shows the co-localization of tdTomato and Rbpms. The white arrow indicates the co-localization of Rbpms and tdTomato in GCL injected with GFAP-CasRx-Ptbp1 plus GFAP-GFP-Cre. Scale bar: 20μm.

Figure 2f shows the number of Rbpms ⁺ or tdTomato ⁺ or tdTomato ⁺ Rbpms ⁺ cells in GCL. Each group n=7 retinas.

Figure 2g shows an image showing representative tdTomato ⁺ RGC-like cells recorded by a two-photon microscope. Scale bar, 20μm.

Figure 2h shows the peak response of representative tdTomato+ON cells to the LED light in the response window relative to the baseline window. A total of 8 cells were recorded, 6 of which showed a response to LED light, 5 of which were ON cells and 1 was OFF cells. Data are expressed as mean ±s.e.m., *p<0.05, **p<0.01, ***p<0.001, unpaired t test.

Figure 3 shows that the regenerated RGC forms the correct connection with its target in the brain and improves the visual function of the damaged retina.

Figure 3a shows a schematic diagram of the visual pathway. RGC projects its axons to dLGN and SC in the brain through the optic nerve, which is responsible for transmitting visual signals outside the retina.

Figure 3b shows the preparation of the retinal spread. Orange arrows indicate MG-derived tdTomato ⁺ RGC axons. Scale bar, 100μm. The experiment was repeated 3 times independently for each group, and the results were similar.

Figure 3c shows a representative image of RGCs tdTomato+ axons regenerated in the optic nerve. Scale bar, 200μm. The experiment was repeated 5 times independently in each group, and the results were similar.

Figure 3d and Figure 3e show representative images of strong signals observed in the target area of RGC axons in the brain, contralateral SC and dLGN. The experiment was repeated 4 times independently in each group, and the results were similar. Please note that the signal on the side part is very weak. Scale bar, 500μm.

Figure 3f shows a schematic diagram of VEP recordings (C57BL/6 mice).

Figure 3g shows the response to the scintillation VEP on the main visual cortex. The graph shows the response of mice from the same group, with each line representing a single retina. The number of retinas in each group is shown in the figure.

Figure 3h shows wild type (WT, C57BL/6 mice, n=8 retinas), NMDA and GFAP-mCherry (n=12 retinas), NMDA, GFAP-mCherry and GFAP-CasRx (n=11 retinas) ) And the response amplitude of NMDA, GFAP-mCherry and -GFAP-CasRx-Ptbp1 (n=8 retinas). Each point represents a single mouse.

Figure 3i shows the percentage of time spent in the dark box. WT (C57BL/6 mice), n=13 mice; NMDA and GFAP-mCherry, n=14 mice; NMDA, GFAP-mCherry and GFAP-CasRx, n=12 mice; NMDA, GFAP- mCherry and GFAP-CasRx-Ptbp1, n=12 mice. All values are expressed as mean; unpaired t-test; *p<0.05, **p<0.01, ***p<0.001.

Figure 4 shows the results of the efficient wizard screening.

Figure 4a shows a schematic diagram of the locations of all the guides.

Figure 4b shows the knockdown efficiency of different combinations of wizards. Guides 5 and 6 showed the highest knock-down efficiency, so they were incorporated into the dual-guide array for the next experiment. The number above the bar graph indicates the number of repetitions for each group. All values are expressed as mean ±s.e.m.

Figure 5 shows the specificity of GFAP-GFP-Cre and confirmation of AAV expression.

Figure 5a shows the GFP expression specifically driven by GFAP-GFP-Cre and the tdTomato expression initiated by GFAP-GFP-Cre in the MG of Ai9 mice. Sox9 is a MG-specific marker. Scale bar: 50μm.

Figure 5b shows the percentage of GFP ⁺ cells expressing tdTomato, and the percentage of tdTomato ⁺ cells expressing Sox9.

Figure 5c shows that qPCR analysis of the infected retina confirmed the expression of GFAP-CasRx and GFAP-CasRx-Ptbp1. By immunostaining the Flag tag (in combination with CasRx) to confirm that the expression of AAV in the brain is successful, but not in the retina. The number above the bar graph indicates the number of repetitions for each group. All values are expressed as mean ±s.e.m.

Figure 6 shows that over time, MG-derived RGC finally stopped GFP expression. One month after injection (Ai9 mice), the white arrow indicates that tdTomato ⁺ Rbpms ⁺ cells express GFP at a low level, and the yellow arrow indicates that MG-derived RGC stops GFP expression. Scale bar, 20μm. The experiment was repeated 6 times independently in each group with similar results.

Figure 7 shows that knocking down Ptbp1 on the intact retina of C57BL/6 mice converts MG to RGCs.

Figure 7a shows a schematic diagram of the regeneration of RGCs from MG. Vector 1 (GFAP-mCherry) encodes mCherry driven by the MG-specific promoter GFAP, and vector 2 (EFS-CasRx-Ptbp1) encodes the guide and CasRx under the universal promoter. In order to regenerate RGCs, GFAP-mCherry plus EFS-CasRx-Ptbp1 was injected into the retina, or only GFAP-mCherry was injected as a control. After 2-3 weeks of injection, it was checked whether transdifferentiation occurred.

Figure 7b shows a representative image of the co-localization of mCherry and MG marker Sox9, which shows that GFAP-mCherry is specifically expressed in MG. Scale bar, 50μm.

Figure 7c and Figure 7d show representative images ^{of mCherry +} Brn3a ⁺ and mCherry ⁺ Rbpms ^{+ cells in GCL.} n = 3 retinas per group. Scale bar, 50μm.

Figure 7e shows that representative RGC-like mCherry ⁺ cells exhibit (4 out of 4 cells, all ON cells) response to LED light.

Figure 8 shows that knocking down Ptbp1 turns MG into axonal cells instead of other types of MG-derived neurons.

^{Figure 8a shows that tdTomato +} Pax6 ⁺ cells were observed in the intact retina of Ai9 mice injected with GFAP-CasRx-Ptbp1. The green arrow indicates that the tdTomato ⁺ cells are not co-localized with Pax6, and the yellow arrow indicates the co-localization of Pax6 and tdTomato. Please note that Pax6 is a marker for amacrine cells. Scale bar, 20μm.

Figure 8b shows cells ^{where tdTomato +} Prox1 ^{+ is not observed.} The arrow indicates that tdTomato cells are not co-localized with the bipolar cell marker Prox1. Scale bar, 20μm.

^{Figure 8c shows that tdTomato+} cells are not observed in the light-sensitive cell layer (ONL). The white arrow indicates the ^{RGC-like cells of tdTomato +} in GCL, the yellow arrow indicates the ^{amacrine cells of tdTomato +} in INL, and the green arrow indicates the dTomato ⁺ projection of MG, scale bar, 20 μm. The experiment was repeated at least three times independently for each group, and the results were similar.

Figure 9 shows that RGCs regenerated in the intact retina form a correct connection with their targets in the brain.

^{Figure 9a shows the tdTomato+} axons regenerating RGCs in the optic nerve of Ai9 mice. Scale bar, 200μm. The experiment was repeated 3 times independently for each group, and the results were similar.

Figure 9b shows a strong signal observed in the target area of the RGC axon in the brain in SC and dLGN. The experiment was repeated 3 times independently for each group, and the results were similar. Scale bar, 500μm.

Detailed ways

After extensive and in-depth research, the inventors unexpectedly discovered for the first time that inhibiting the expression of Ptbp1 in the retina can directly transform MG into functional RGC. More importantly, the regenerated RGC can be integrated into the visual pathway and improve RGC. Impairs the visual function of the mouse model. The present invention is completed on this basis.

Specifically, the degeneration of retinal ganglion cells (RGC) is the main cause of permanent blindness. The transdifferentiation of Müller glial cells (MG) into functional RGC can help restore vision. The inventors found that knocking down Ptbp1 by using the RNA-targeted CRISPR system CasRx in the retina of mature mice can directly convert MG into functional RGC. In addition, in a mouse model of retinal injury induced by NMDA (N-methyl-d-aspartic acid), the RGC transformed from MG achieves a functional projection to the central visual area and improves visual function . Therefore, Ptbp1 knockdown mediated by CasRx will be a promising treatment for retinal diseases caused by neurodegeneration.

This application uses CasRx, a recently characterized RNA targeting CRISPR system, to inhibit Ptbp1. CasRx-mediated regeneration avoids the occurrence of substantial off-target effects induced by shRNA and the risk of permanent gene changes through DNA editing nucleases, and provides an excellent tool that can treat a variety of diseases.

As used herein, Müller glial cells (MG) are the main glial cells in the retinal tissue, and retinal ganglion cells (RGC) are nerve cells located in the innermost layer of the retina. Its dendrites are mainly connected with bipolar cells. Its axons extend to the optic nerve head to form the optic nerve.

Retinal disease is regarded as an eye disease. There are five common types of retinal diseases: ① Diseases of blood vessels and vascular system. Such as retinal vascular occlusion, arteriosclerosis, hypertension, blood disease and diabetic fundus disease. ② Inflammation of the retina. It is closely related to the mutual influence of choroiditis and optic neuritis. ③Retina detachment. Refers to the separation of the retinal nerve layer and the pigment epithelium. ④Retinal degeneration and malnutrition. Has genetic factors. ⑤ Retinal tumors. Among them, retinoblastoma is more common.

In the present invention, a preferred retinal disease is a retinal disease caused by neurodegeneration, and the symptoms are mainly manifested in decreased vision or blindness.

In the present invention, the gene editor includes a DNA gene editor and an RNA gene editor. In a preferred embodiment, the gene editor of the present invention includes a gene editing protein and optionally gRNA.

Gene editing protein

In the present invention, the nucleotides of the gene editing protein can be obtained by genetic engineering techniques, such as genome sequencing, polymerase chain reaction (PCR), etc., and the amino acid sequence can be deduced from the nucleotide sequence. The source of the wild-type gene editing protein includes (but is not limited to): Ruminococcus Flavefaciens, Streptococcus pyogenes, Staphylococcus aureus, and Acidaminococcus sp. , Lachnospiraceae bacterium (Lachnospiraceae bacterium).

In a preferred embodiment of the present invention, the gene editing protein includes, but is not limited to Cas13 (such as CasRx), CRISPR/Cas9, Cpf1, SaCas9, Cas13a, Cas13b, and Cas13c.

ptbp1 protein and polynucleotide

In the present invention, the terms "protein of the present invention", "ptbp1 protein" and "ptbp1 polypeptide" are used interchangeably, and all refer to a protein or polypeptide having an amino acid sequence of ptbp1. They include the ptbp1 protein with or without the starting methionine. In addition, the term also includes full-length ptbp1 and fragments thereof. The ptbp1 protein referred to in the present invention includes its complete amino acid sequence, its secreted protein, its mutant and its functionally active fragments.

The ptbp1 protein is a polypyrimidine domain binding protein 1, which is an RNA binding protein that regulates RNA splicing. At the same time, it also plays a very critical role in other functions of RNA.

In the present invention, the terms "ptbp1 gene" and "ptbp1 polynucleotide" are used interchangeably, and both refer to a nucleic acid sequence having a ptbp1 nucleotide sequence.

The full length of the human ptbp1 gene genome is 14936bp (NCBI GenBank accession number is 5725).

The full length of the mouse ptbp1 gene genome is 10004 bp (NCBI GenBank accession number is 19205).

The similarity of human and mouse ptbp1 at the DNA level is 88%, and the similarity of protein sequence is 84%. It should be understood that when encoding the same amino acid, the substitution of nucleotides in the codon is acceptable. In addition, it should be understood that when conservative amino acid substitutions are produced by nucleotide substitutions, nucleotide changes are also acceptable.

When the amino acid fragment of ptbp1 is obtained, a nucleic acid sequence encoding it can be constructed based on it, and a specific probe can be designed based on the nucleotide sequence. The full-length nucleotide sequence or its fragments can usually be obtained by PCR amplification, recombination or artificial synthesis. For the PCR amplification method, primers can be designed according to the ptbp1 nucleotide sequence disclosed in the present invention, especially the open reading frame sequence, and a commercially available cDNA library or a cDNA prepared by a conventional method known to those skilled in the art can be used. The library is used as a template to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.

Once the relevant sequence is obtained, the recombination method can be used to obtain the relevant sequence in large quantities. This is usually done by cloning it into a vector, then transferring it into a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.

In addition, artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is short. Usually, by first synthesizing multiple small fragments, and then ligating to obtain fragments with very long sequences.

At present, the DNA sequence encoding the protein (or fragment or derivative thereof) of the present invention can be obtained completely through chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (such as vectors) and cells known in the art.

Through conventional recombinant DNA technology, the polynucleotide sequence of the present invention can be used to express or produce recombinant ptbp1 polypeptide. Generally speaking, there are the following steps:

(1) Use the polynucleotide (or variant) encoding human ptbp1 polypeptide of the present invention, or use a recombinant expression vector containing the polynucleotide to transform or transduce a suitable host cell;

(2). Host cells cultured in a suitable medium;

(3). Separate and purify protein from culture medium or cells.

In the present invention, the ptbp1 polynucleotide sequence can be inserted into a recombinant expression vector. In short, any plasmid and vector can be used as long as it can be replicated and stabilized in the host. An important feature of an expression vector is that it usually contains an origin of replication, a promoter, a marker gene, and translation control elements.

Methods well known to those skilled in the art can be used to construct an expression vector containing the ptbp1 coding DNA sequence and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology. The DNA sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selecting transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.

A vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell so that it can express the protein.

The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, bacterial cells of the genus Streptomyces; fungal cells such as yeast; plant cells; insect cells; animal cells, etc.

Transformation of host cells with recombinant DNA can be performed by conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl ₂ method. The steps used are well known in the art. Another method is to use MgCl ₂ . If necessary, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

The obtained transformants can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the medium used in the culture can be selected from various conventional mediums. The culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

The recombinant polypeptide in the above method can be expressed in the cell or on the cell membrane, or secreted out of the cell. If necessary, the physical, chemical, and other characteristics can be used to separate and purify the recombinant protein through various separation methods. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitation agent (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

Adeno-associated virus

Because Adeno-associated virus (AAV) is smaller than other viral vectors, is non-pathogenic, and can transfect dividing and undivided cells, gene therapy methods based on AAV vectors for genetic diseases have been affected. Widespread concern.

Adeno-associated virus (adeno-associated virus, AAV), also known as adeno-associated virus, belongs to the Parvoviridae dependent virus genus. It is the simplest type of single-stranded DNA-deficient virus found so far. Viruses) participate in replication. It encodes the cap and rep genes in the inverted repeat (ITR) at both ends. ITRs play a decisive role in virus replication and packaging. The cap gene encodes the viral capsid protein, and the rep gene is involved in virus replication and integration. AAV can infect a variety of cells.

Recombinant adeno-associated virus vector (rAAV) is derived from non-pathogenic wild-type adeno-associated virus. Due to its good safety, wide range of host cells (dividing and non-dividing cells), and low immunogenicity, it can express foreign genes in vivo. Long and other characteristics, it is regarded as one of the most promising gene transfer vectors and has been widely used in gene therapy and vaccine research worldwide. After more than 10 years of research, the biological characteristics of recombinant adeno-associated virus have been deeply understood, especially in terms of its application effects in various cells, tissues and in vivo experiments. A lot of information has been accumulated. In medical research, rAAV is used in the research of gene therapy for a variety of diseases (including in vivo and in vitro experiments); at the same time, as a characteristic gene transfer vector, it is also widely used in gene function research, disease model construction, and gene preparation. Knockout mice and other aspects.

In a preferred embodiment of the present invention, the vector is a recombinant AAV vector. AAVs are relatively small DNA viruses that can integrate into the genome of the cells they infect in a stable and site-specific manner. They can infect a large range of cells without any impact on cell growth, morphology or differentiation, and they do not seem to be involved in human pathology. The AAV genome has been cloned, sequenced and characterized. AAV contains an inverted terminal repeat (ITR) region of approximately 145 bases at each end, which serves as the origin of replication of the virus. The rest of the genome is divided into two important regions with encapsidation functions: the left part of the genome containing the rep gene involved in viral replication and viral gene expression; and the right part of the genome containing the cap gene encoding the viral capsid protein.

AAV vectors can be prepared using standard methods in the art. Adeno-associated viruses of any serotype are suitable. Methods for purifying vectors can be found in, for example, U.S. Patent Nos. 6,566,118, 6,989,264, and 6,995,006, the disclosures of which are incorporated herein by reference in their entirety. The preparation of hybrid vectors is described in, for example, PCT Application No. PCT/US2005/027091, the disclosure of which is incorporated herein by reference in its entirety. The use of AAV-derived vectors for transferring genes in vitro and in vivo has been described (see, for example, International Patent Application Publication Nos. WO91/18088 and WO93/09239; U.S. Patent Nos. 4,797,368, 6,596,535 and 5,139,941, and European Patent No. 0488528, all of which are incorporated herein by reference in their entirety). These patent publications describe various AAV-derived constructs in which the rep and/or cap genes are deleted and replaced by the gene of interest, and these constructs are in vitro (into cultured cells) or in vivo (into the organism directly). ) The purpose of transporting the gene of interest. Replication-deficient recombinant AAV can be prepared by co-transfecting the following plasmids into a cell line infected with a human helper virus (such as adenovirus): the nucleic acid sequence of interest is flanked by two AAV inverted terminal repeats (ITR) Region plasmids, and plasmids carrying AAV encapsidation genes (rep and cap genes). The resulting AAV recombinants are then purified by standard techniques.

In some embodiments, the recombinant vector is capsidized to viral particles (e.g., including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 And AAV virus particles of AAV16). Therefore, the present disclosure includes recombinant viral particles (recombinant because they contain recombinant polynucleotides) containing any of the vectors described herein. Methods of producing such particles are known in the art and are described in U.S. Patent No. 6,596,535.

ptbp1 inhibitor and pharmaceutical composition

Using the protein of the present invention, through various conventional screening methods, substances that interact with the ptbp1 gene or protein, especially inhibitors, can be screened out.

The ptbp1 inhibitor (or antagonist) that can be used in the present invention includes any substance that can inhibit the expression and/or activity of the ptbp1 gene or its encoded protein.

For example, the inhibitor of ptbp1 includes an antibody of ptbp1, antisense RNA of ptbp1 nucleic acid, siRNA, shRNA, miRNA, gene editor, or an activity inhibitor of ptbp1. A preferred inhibitor of ptbp1 refers to a gene editor capable of inhibiting the expression of ptbp1.

Preferably, the inhibitors of ptbp1 of the present invention include inhibitors targeting positions 4758-4787 and/or positions 5381-5410 of the ptbp1 gene sequence. The targets of the ptbp1 inhibitor of the present invention include MG cells.

In a preferred embodiment, the methods and steps for inhibiting ptbp1 include neutralizing its protein with an antibody of ptbp1, and silencing the ptbp1 gene using shRNA or siRNA or a gene editor carried by a virus (such as adeno-associated virus).

The inhibition rate of ptbp1 is generally at least 50% or more inhibition, preferably 60%, 70%, 80%, 90%, 95% inhibition, which can be based on conventional techniques, such as flow cytometry, fluorescent quantitative PCR or Western Methods such as blot control and detect the inhibition rate of ptbp1.

The inhibitor of the ptbp1 protein of the present invention (including antibodies, antisense nucleic acids, gene editors and other inhibitors), when administered (administered) therapeutically, can inhibit the expression and/or activity of the ptbp1 protein, thereby inducing MG The cells differentiate into RGC cells, thereby preventing and/or treating retinal diseases. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 5-8, preferably about 6-8, although the pH can be The nature of the formulated substance and the condition to be treated vary. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): local, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, topical administration, autologous cell extraction and culture, and infusion Wait.

The present invention also provides a pharmaceutical composition, which contains a safe and effective amount of the inhibitor of the present invention (such as antibodies, gene editors, antisense sequences (such as siRNA), or inhibitors) and a pharmaceutically acceptable carrier or excipient. Shape agent. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of injections, for example, with physiological saline or an aqueous solution containing glucose and other adjuvants for preparation by conventional methods. Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets and capsules should be manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram to 10 mg/kg body weight per day.

The main advantages of the present invention include:

(a) The present invention directly converts MG into functional RGC by inhibiting the expression of Ptbp1 in the retina.

(b) The regenerated RGC can be integrated into the visual pathway and improve the visual function of the mouse model of RGC injury.

(c) The present invention uses the RNA-targeted CRISPR system CasRx to knock down Ptbp1, avoiding the occurrence of substantial off-target effects induced by shRNA and the risk of permanent gene changes, and provides an excellent tool that can treat a variety of diseases.

The present invention will be further explained below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods that do not indicate specific conditions in the following examples usually follow the conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

General materials and methods

All animal experiments were conducted and approved by the Animal Care and Use Committee of the Institute of Neuroscience, Chinese Academy of Sciences.

Guide RNA sequence

Guide 1: 5’-tttgtaccgactgctatgtctgggacgat-3’ (SEQ ID NO:1);

Guide 2: 5’-ggctggctgtctccagagggcaggtcaggt-3’ (SEQ ID NO: 2);

Guide 3: 5’-gtatagtagttaaccatagtgttggcagcc-3’ (SEQ ID NO: 3);

Guide 4: 5’-gctgtcggtcttgagctctttgtggttgga-3’ (SEQ ID NO: 4);

Guide 5: 5’-tgtagatgggctgtccacgaagcactggcg-3’ (SEQ ID NO: 5);

Guide 6: 5’-gcttggagaagtcgatgcgcagcgtgcagc-3’ (SEQ ID NO: 6).

Transient transfection of N2a cells, qPCR and RNA-seq

N2a cells were seeded in 6-well plates. Lipofectamine 3000 (Thermo Fisher Scientific) was used according to standard procedures, and cells were transfected with 7 μg gRNA-CasRx-GFP vector. The control plasmid does not express gRNA. Two days after transfection, about 50,000 GFP-positive cells were collected from each sample by fluorescence activated cell sorting (FACS), and lysed for qPCR analysis. At the same time, the retina was separated to determine the expression of AAV. The RNA was extracted using Trizol (Ambion) and converted into cDNA using a reverse transcription kit (HiScript QRT SuperMix for qPCR, Vazyme, Biotech). Use AceQ qPCR SYBR Green Master Mix (Vazyme, Biotech) to track the amplification process.

Ptbp1qPCR primers are:

Upstream primer, 5’-AGAGGAGGCTGCCAACACTA-3’ (SEQ ID NO: 7);

Downstream primer, 5'-GTCCAGGGTCACTGGGTAGA-3' (SEQ ID NO: 8).

CasRx qPCR primers:

Upstream primer, 5’-CCCTGGTGTCCGGCTCTAA-3’ (SEQ ID NO: 9);

Downstream primer, 5'-GGACTCGCCGAAGTACCTCT-3' (SEQ ID NO: 10).

For RNA-seq, N2a cells were cultured in a 15-cm culture dish and transiently transfected with 70 μg plasmid. Collect ~500,000 GFP-positive (top 20% GFP) N2a cells by FACS, extract RNA, convert it into cDNA, and use it for full-transcriptome RNA-seq.

Intravitreal injection and subretinal injection

As mentioned earlier, NMDA and AAV (AAV-PHP.eB) were introduced by intravitreal and subretinal injections, respectively. ^{For subretinal injection, high titer (>1×10 13} ) AAV was injected into the eye with a Hamilton syringe (32G needle) under an Olympus microscope (Olympus, Tokyo, Japan). To determine reprogramming in the intact retina, a total of 1μl of GFAP-GFP-Cre (0.2μl) and GFAP-CasRx-Ptbp1 (0.8μl) were injected under the retina (Ai9 and C57BL/6 mice, 5 weeks old), Or GFAP-Cre-GFP (0.2μl) and GFAP-CasRx (0.8μl) are delivered to the retina. To determine reprogramming in the damaged retina, dissolve NMDA to a concentration of 200 mM in PBS, and then inject 1.5 μl of NMDA solution into 4-8 week old Ai9 mice or 5-6 week old C57BL/6 mice by intravitreal injection. Mouse eyes (for VEP and black and white scene preference testing). Two to three weeks after NMDA injection, GFAP-GFP-Cre and GFAP-CasRx-Ptbp1 or GFAP-CasRx were co-delivered to the retina by subretinal injection. In order to evaluate the functional rescue of the damaged retina (VEP and light-dark box shuttle test), 5-6 weeks old mice (C57BL/6) were injected with NMDA to induce retinal damage, and GFAP-mCherry( 0.2μl) and GFAP-CasRx-Ptbp1 (0.8μl) or GFAP-CasRx (0.8μl) mixture.

Immunofluorescence staining

One month after AAV injection, the eyes, optic nerve and brain were taken, fixed with 4% paraformaldehyde (PFA) for 2 hours (eyes and optic nerve) or 24 hours (brain), and then stored in 30% sucrose solution 2 (eyes) And optic nerve) or 24 (brain) hours. After embedding and freezing, the eyes and brain were sliced at a thickness of 30 μm. Primary antibodies used for immunofluorescence staining: mouse anti-Brn3a (1:100, MAB1585, Millipore), rabbit anti-RBPMS (1:500, 15187-1-AP, Proteintech), rabbit anti-Sox9 (1:500, AB5535, Millipore), rabbit anti-Pax6 (1:500, 901301, Biolegent), rabbit anti-Prox1 (1:500, AB5475, Millipore), and secondary antibody: Cy ^TM 5 AffiniPure Donkey mouse anti-IgG (H+L) (1: 500, 715-175-150, Jackson ImmunoResearch), Cy™ 5 AffiniPure Donkey rabbit anti-IgG (H+L) (1: 500, 711-175-152, Jackson ImmunoResearch). After applying the antibody, wash and mount the film. Use Olympus FV3000 microscope for imaging.

Electrophysiology

The mice were dark-adapted overnight before euthanasia. Under an infrared microscope at room temperature, the retina was dissected in an oxygenated (95% O2 / 5% CO2) artificial cerebrospinal fluid (ACSF) containing 126mM NaCl, 2.5mM KCl, 1.25mM NaH2PO4, 2mM CaCl2, 2mM NaHCO3 and 10mM glucose. Place the RGC of the retina facing the cell recording groove on the table of the upright microscope. A two-photon (λ=1030nm) microscope was used to identify the tdTomato-positive cells in the ganglion cell layer and record them under infrared light. ASCF and 0.25mM Alexa488 hydration were added to the pipette (4-7MΩ) used for recording. Use Multiclamp 700A amplifier and pClamp10 software package (Molecular Devices) for recording. The signal is low-pass filtered at 1kHz and digitized at 10kHz. A white LED light is used to deliver a full-field light stimulus. After recording, inject current pulses to fill the cells to visualize their morphology.

Visual evoked potential

A mixture of fentanyl (0.05mg/kg), midazolam (5mg/kg) and medetomidine (0.5mg/kg) was injected intraperitoneally in mice. The head of the mouse was fixed in a stereotaxic instrument, and the body temperature was maintained at 37°C through a heating blanket. A craniotomy (approximately 1mm in diameter) was performed on both sides of the main visual cortex (V1) (AP-3.6 to -3.9mm, ML 2.2mm), and the dura mater was removed. The visual stimulus is emitted by a 17-inch LCD display (Dell P170S, maximum brightness 69cd/m2), which is 8 cm away from the eyes on the recording end, and at the same time shields the side of the eye on the same side of the recording end from visual stimuli. We performed 100 repetitive flash stimuli (full field of view, 100% contrast) for 2 seconds with an interval of 2 seconds. Use a multi-point silicon probe (A1×16-5mm-50-177, NeuroNexus Technologies) to record at V1 (AP-3.6 to -3.9mm, ML 2.2mm), and the cortical depth reached by the electrode tip of each recording is about 900μm. Both the reference wire and the ground wire are placed in a small craniotomy at least 3 mm away from the recording point. A Cerebus 32-channel system (Blackrock microsystems) was used to amplify and filter neural responses. A wideband front-end filter (0.3～500Hz) is used to sample the local field potential (LFP) signal at 2kHz or 10kHz. LFP response to stimulation is used to flash the full screen current source density (CSD) analysis to determine the location of the cortical layer ^43. In order to generate the CSD distribution profile, we used the following equation to calculate the second-order spatial derivative of LFP:

among them

Is the LFP, z is the coordinates of the recording end, Δz is the distance between adjacent recording ends, and nΔz is the differentiation grid (n=2). Layer 4 (granular layer) is defined as those recorded positions at the initial current receptor. We used the layer 4 channel showing the maximum average amplitude to analyze the visual evoked response of each mouse.

Black and white box preference test

The equipment used for the light-dark box shuttle experiment includes a box with a door, which is divided into a small (one-third) dark box part and a large (two-thirds) lighting part (550 lumens). The mouse can move freely between the two compartments for 10 minutes. The time the mice spend in each compartment is recorded by the camera and then analyzed using Ethovision XT. After each test, the compartment was cleaned with 70% ethanol to avoid olfactory cues.

Statistical Analysis

All values are shown as mean ±s.e.m. An unpaired t-test was used to determine statistical significance (p<0.05). All experiments were randomized and no statistical method was used to estimate the sample size, but our sample size was similar to that reported in previous publications. It is assumed that the data distribution is normal but has not been formally tested.

Example 1

RGCs can be obtained by reprogramming MG in intact retina

Recently, the RNA of the RNA-targeted CRISPR system has been characterized, and the engineered type VI CRISPR-Cas13d ortholog CasRx is suitable for in vivo applications due to its very small size and high specificity. This example explores the possibility of MG regenerating RGCs by knocking out Ptbp1 using CasRx on the mature retina,

In order to achieve efficient knockdown of Ptbp1, six guides are designed. The position diagram of each guide is shown in Figure 4a, and the sequence is shown in SEQ ID NO: 1-6. Figure 4b shows the knockdown efficiency of different combinations of wizards. Guides 5 and 6 showed the highest knock-down efficiency. The guide RNA 5 and 6 (gRNA5, 6) were packaged into a gRNA array, and the plasmid encoding CasRx and gRNA array transiently transfected thereafter confirmed the specific knockdown of Ptbp1 in N2a cells (Figure 1a, b).

In order to mark MG specifically and permanently, AAV-GFAP-GFP-Cre (hereinafter referred to as GFAP-GFP-Cre) was injected into the eyes of Ai9 mice (Rosa-CAG-LSL-tdTomato-WPRE) to specifically start The expression of tdTomato in MG (Figure 5).

The inventors also constructed AAV-GFAP-CasRx-Ptbp1 (hereinafter referred to as GFAP-CasRx-Ptbp1) expressing CasRx driven by the MG-specific promoter GFAP and two gRNAs targeting Ptbp1, and AAV-GFAP- CasRx (hereinafter referred to as GFAP-CasRx) was used as a control (Figure 1c).

In order to determine the possibility of regenerating RGCs in vivo, GFAP-CasRx-Ptbp1 or GFAP-CasRx and GFAP-Cre-GFP were injected into the eyes of 5-week-old Ai9 mice by subretinal injection. One month after AAV injection, the retina was marked with RGC-specific Brn3a and Rbpms. ^{It was found that basically no tdTomato +} cells were detected on the ganglion cell layer (GCL) of the control eye injected with GFAP-CasRx (Figure 1d-e). ^{In contrast, tdTomato+} cells were observed on the GCL of the experimental eyes injected with GFAP-CasRx-Ptbp1, and most of these cells expressed RGC Brn3a and Rbpms markers (Figure 1d-g). It should be noted that GFAP-driven GFP expression decreased frequently in regenerated RGC cells (Figure 6), which means that the transformed MG loses their characteristics.

Another method of co-injecting GFAP-mCherry and EFS-CasRx-Ptbp1 (CasRx driven by the ubiquitous promoter EFS) into the retina of C57BL/6 mice also confirmed that functional RGCs can be Successfully regenerated (Figure 7).

Previous studies have shown that overexpression of Ascl1 or a mixture of transcription factors will convert MG into different types of retinal neurons. Similarly, the inventors found that in addition to RGCs, MG can also be transformed into amacrine cells (Figure 8).

In summary, the above results indicate that RGCs can be regenerated from MG by knocking down Ptbp1 in the mature retina without damaging the retina.

Example 2

Reprogramming MG in a mouse model of NMDA-induced retinal damage can produce RGC

In order to explore whether MG-derived RGCs can be used to replace damaged RGCs in a mouse model with RGC injury, NMDA injections into the vitreous body of Ai9 mice aged 4-8 weeks will lose nearly all of the RGCs. The thickness of the plexiform layer (IPL) also becomes thinner. Two to three weeks after NMDA injection, GFAP-CasRx-Ptbp1 plus GFAP-GFP-Cre or control virus GFAP-CasRx plus GFAP-GFP-Cre were injected into the eyes (Figure 2a, b).

One month after AAV injection, it was found that the ^{number of Brn3a +} or Rbpms ⁺ cells in the retinal GCL injected with GFAP-CasRx-Ptbp1 increased significantly. Most of these cells are tdTomato ⁺ (Figure 2c-f), and more than half of tdTomato ⁺ cells Both express Brn3a and Rbpms (Figure 2c-f).

In order to determine whether MG-derived RGCs can be integrated into the retinal pathway and have the ability to receive visual information, cell attachment recordings were performed under two photon microscopes to record the action potentials of MG-derived RGCs in response to light stimulation (Figure 2g) , And found that 6 of the 8 cells showed a response to light stimulation, of which 5 were ON cells and 1 was OFF cells (Figure 2h).

The above results indicate. Functional RGCs can be regenerated from MG by knocking down Ptbp1 in the damaged retina.

Example 3

The regenerated RGC forms the correct connection with its target in the brain and improves the visual function of the damaged retina

The neural connections in the nervous system are highly precise. In the visual system, in order to transmit visual information to the cortex, MG-derived RGCs need to project their axons to the lateral geniculate dorsal nucleus (dLGN) and superior colliculus (SC) of the brain (Figure 3a).

^{It is worth noting that a large number of tdTomato +} axons were observed in the retina and optic nerve of the experimental group, but not in the control group (Figure 3b, 3c, Figure 9). Further analysis showed that MG-derived RGCs projected their axons to target neurons in dLGN and SC (Figure 3d, e, Figure 9), which indicates that the regenerated RGCs can form correct projections to the central visual area.

Next, we tested whether the MG-derived RGC in the damaged retina can be stimulated in the brain. One month after injection of AAV, visual evoked potentials (VEPs) were recorded in the primary visual cortex of anesthetized mice (Figure 3f). The experimental group injected with GFAP-mCherry and GFPA-CasRx-Ptbp1 showed an amazing stress response However, in the control group injected with only GFAP-mCherry or GFAP-mCherry plus GFPA-CasRx, only a weaker response was recorded (Figure 3g). In particular, the aroused amplitude of the experimental group was close to that of the healthy group without NMDA-induced injury (Figure 3h), which indicates that the regenerated RGC can efficiently transmit visual information to the primary visual cortex.

Finally, the inventors studied whether Ptbp1-mediated regeneration can repair the vision of mice with retinal damage. It is worth noting that in the light-dark box shuttle experiment, the mice in the experimental group spent a longer time in the dark box than the control mice (Figure 3i), which indicates that the visual function of these behavioral mice has been improved.

All documents mentioned in the present invention are cited as references in this application, as if each document was individually cited as a reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (10)

1. A use of Ptbp1 gene or its coded protein inhibitor is characterized in that it is used to prepare a composition or preparation, and the composition or preparation is used to prevent or treat retinal diseases.
2. The use according to claim 1, wherein the retinal disease is a retinal disease caused by neurodegeneration.
3. The use according to claim 1, wherein the composition or preparation induces the transdifferentiation of MG cells into RGC cells to treat retinal diseases caused by neurodegeneration.
4. The use according to claim 1, wherein the inhibitor is selected from the group consisting of antibodies, small molecule compounds, microRNA, siRNA, shRNA, gene editors, or a combination thereof.
5. The use according to claim 4, wherein the gene editor includes optional gRNA and gene editing protein.
6. The use according to claim 5, wherein the gene editing protein is CasRx, and the nucleotide sequence of gRNA is selected from the following group: SEQ ID NO.: 1, 2, 3, 4, 5, and 6 .
7. A composition characterized by comprising:

   (a) A gene editing protein or an expression vector thereof, the gene editing protein is selected from the group consisting of CasRx, CRISPR/Cas9, Cpf1, Cas9, Cas13a, Cas13b, Cas13c, or a combination thereof; and

   (b) gRNA or its expression vector, the gRNA is RNA that guides the gene editing protein to specifically bind to the Ptbp1 gene, and the nucleotide sequence of the gRNA is selected from the following group: SEQ ID NO.: 1, 2, 3, 4, 5, and 6.
8. A medicine box is characterized in that it comprises:

   (a1) The first container, and the gene editing protein or its expression vector in the first container, or a drug containing the gene editing protein or its expression vector, the gene editing protein is selected from the following group: CasRx, CRISPR/ Cas9, Cpf1, Cas9, Cas13a, Cas13b, Cas13c, or a combination thereof;

   (b1) A second container, and gRNA or its expression vector, or a drug containing gRNA or its expression vector, in the second container, and the gRNA is RNA that guides the gene editing protein to specifically bind to the Ptbp1 gene.
9. A use of the composition according to claim 7 or the kit according to claim 8, characterized in that it is used to prepare a medicine for the prevention and/or treatment of retinal diseases.
10. A method for promoting the differentiation of MG cells into RGC cells is characterized in that it comprises the steps:

    In the presence of the Ptbp1 gene or its encoded protein inhibitor or the composition according to claim 2, MG cells are cultured to promote the differentiation of MG cells into RGC cells.

Images