CN112451669A

CN112451669A - Use of Ptbp1 inhibitors for the prevention and/or treatment of neurological disorders associated with functional neuronal death

Info

Publication number: CN112451669A
Application number: CN202010740568.2A
Authority: CN
Inventors: 杨辉; 周海波
Original assignee: Center for Excellence in Brain Science and Intelligence Technology Chinese Academy of Sciences
Current assignee: Center for Excellence in Brain Science and Intelligence Technology Chinese Academy of Sciences
Priority date: 2019-08-16
Filing date: 2020-07-28
Publication date: 2021-03-09
Also published as: WO2021031810A1; WO2021031565A1

Abstract

The invention relates to application of a Ptbp1 inhibitor in preventing and/or treating a nervous system disease related to functional neuron death. Specifically, the invention provides an application of Ptbp1 gene or RNA or protein inhibitor coded by the gene or RNA in preparing a composition or a preparation, wherein the composition or the preparation is used for preventing and/or treating functional neuron death-related nervous system diseases. The invention can effectively induce the transdifferentiation and differentiation of astrocytes into dopamine neurons by inhibiting the expression or activity of Ptbp1 gene or RNA or coding protein thereof of astrocytes in brain, and can effectively induce the transdifferentiation of Muller glia cells into visual ganglion cells by inhibiting the expression or activity of Ptbp1 gene or RNA or coding protein thereof in Muller glia cells in retina, thereby preventing and/or treating functional neuron death-related nervous system diseases.

Description

Use of Ptbp1 inhibitors for the prevention and/or treatment of neurological disorders associated with functional neuronal death

Technical Field

The invention relates to the field of biomedicine. More specifically, the present invention relates to the use of Ptbp1 inhibitors for the prevention and/or treatment of neurological diseases associated with functional neuronal death.

Background

Parkinson's Disease (PD) is a serious neurodegenerative disease characterized by the loss of mesolimbic nigral dopamine neurons. Previous studies have achieved direct reprogramming of astrocytes into dopamine neurons in vitro and in animal models by simultaneous overexpression of several transcription factors. However, so far, Ptbp 1-mediated in vivo neuronal reprogramming has not been reported.

The current main treatment means for parkinson's disease is a drug represented by levodopa preparations. Meanwhile, the symptoms can be improved to a certain extent by surgical treatment. It should be noted that all these measures can only partially alleviate the disease and do not achieve the effect of stopping the disease.

Therefore, there is an urgent need in the art to develop targets that can effectively treat neurodegenerative diseases.

Disclosure of Invention

The invention aims to provide a target point capable of effectively treating neurodegenerative diseases.

Another objective of the invention is to provide a novel target Ptbp1 for treating Parkinson's disease, which can directly transform astrocytes in striatum into dopamine neurons and restore the Parkinson's disease phenotype by inhibiting the expression of Ptbp 1.

Another objective of the invention is to provide a novel target Ptbp1 for treating vision impairment, which can directly transform Mueller's glia cells in retina into visual ganglion cells and relieve permanent vision impairment phenotype by inhibiting the expression of Ptbp 1.

In a first aspect of the invention, there is provided the use of an inhibitor of the Ptbp1 gene or RNA or the protein encoded thereby for the preparation of a composition or formulation for the treatment of a neurological disease associated with functional neuronal death.

In another preferred embodiment, the composition or formulation is further used for one or more uses selected from the group consisting of:

(a1) inducing the astrocytes to transdifferentiate into dopamine neurons;

(b1) treating dyskinesia in parkinson's disease in mammals;

(c1) inducing glial cell transformation or differentiation into functional neurons;

(d1) treating a neurological disorder associated with degeneration of optic ganglion cells (RGCs);

(e1) preventing or treating retinal diseases;

(f1) preventing and/or treating neurodegenerative diseases;

(g1) transdifferentiation of muller glial cells to optic ganglion cells;

(h1) treating visual impairment caused by death of the optic ganglia of the mammal.

In another preferred embodiment, the neurological disease is selected from the group consisting of: glaucoma, age-related RGC loss, optic nerve injury, retinal ischemia, Leber's hereditary optic neuropathy, alzheimer's disease, huntington's disease, schizophrenia, depression, drug withdrawal, movement disorders (e.g., chorea, cholesteatosis, and movement disorders), motor neuron injury diseases (e.g., amyotrophic lateral sclerosis, spinal cord injury), bipolar disorder, Autism Spectrum Disorder (ASD), dysfunction, parkinson's disease, or combinations thereof.

In another preferred embodiment, the glial cell is selected from the group consisting of: astrocytes, MG cells, oligodendrocytes, ependymal cells, Schwan cells, NG2 cells, satellite cells, or combinations thereof.

In another preferred embodiment, the functional neuron is selected from the group consisting of: RGC neurons, dopamine neurons, or a combination thereof.

In another preferred embodiment, the functional neuron is derived from the striatum.

In another preferred example, the functional neurons are derived from the mature retina.

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

In another preferred embodiment, the composition or formulation treats retinal diseases caused by neurodegeneration by inducing transdifferentiation of MG cells into RGC cells.

In another preferred embodiment, the MG cells are muller glial cells (muller glial cells).

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

In another preferred embodiment, the RGC cell is a retinal ganglion cell.

In another preferred embodiment, the RGC cell is a functional RGC.

In another preferred embodiment, the RGC cells can integrate into the visual pathway and improve visual function.

In another preferred embodiment, the RGC cells can achieve functional projection to the central visual region and improve visual function.

In another preferred embodiment, the improvement of visual function is improvement of visual function of a mammal suffering from a retinal disease caused by neurodegeneration.

In another preferred embodiment, the MG cell transdifferentiates into an RGC cell and also into an axon-free cell.

In another preferred embodiment, RGC (1) expresses Brn3a, Rbpms, Foxp2, Brn3c, and/or mini-albumin; (2) is F-RGC, RGC type 3 or PV-RGC; (3) integrated in the existing retinal pathway in the subject (e.g., central information can be projected to dLGN, and vision can be partially restored by relaying visual information to V1); and/or (4) capable of receiving visual information characterized by the ability to establish action potentials upon light stimulation, synaptic connections (e.g., with existing functional dLGN neurons in the brain), biogenesis of pre-synaptic neurotransmitters, and/or subsequent actions.

In another preferred embodiment, the muller glial cells in the mature retina are reprogrammed and converted to RGCs.

In another preferred embodiment, the dopamine neurons (1) express Tyrosine Hydroxylase (TH), dopamine transporter (DAT), vesicular monoamine transporter 2(VMAT2), hybrid homeobox 1(Enl), FoxA2 and/or LIM homeobox transcription factor 1alpha (lmxla); (2) exhibit biogenesis of presynaptic neurotransmitters; (3) integrated into existing neuronal circuits in the brain of the subject; and/or (4) characterized by its ability to establish action potentials, synaptic connections, biogenesis of presynaptic neurotransmitters and/or postsynaptic responses.

In another preferred embodiment, the plurality of glial cells in the striatum are reprogrammed, and wherein at least 10% or at least 30% of the glial cells are converted to dopamine neurons.

In another preferred embodiment, the mammal includes a mammal suffering from a neurodegenerative disease.

In another preferred embodiment, the mammal comprises a human or non-human mammal.

In another preferred embodiment, the non-human mammal includes a rodent (e.g., a mouse, rat, or rabbit), a primate (e.g., monkey).

In another preferred embodiment, the astrocytes are derived from the striatum, substantia nigra, spinal cord, dorsal mesencephalon or cerebral cortex, preferably the astrocytes are derived from the striatum.

In another preferred embodiment, the astrocytes comprise striatal astrocytes.

In another preferred embodiment, the astrocytes are astrocytes of brain tissue.

In another preferred embodiment, the inhibitor is selected from the group consisting of: antibodies, small molecule compounds, microRNAs, siRNAs, shRNAs, antisense oligonucleotides, aptamers, gene editors, or combinations thereof.

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

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

In another preferred embodiment, the gene editor is driven by a glial cell specific promoter (e.g., the GFAP promoter) to express.

In another preferred embodiment, the gene editor comprises 2 or more gNRA and a gene editing protein.

In another preferred example, the gRNA is an RNA that directs gene editing proteins to specifically bind the Ptbp1 gene.

In another preferred example, the gRNA directs the gene editing protein to specifically bind the mRNA of the Ptbp1 gene.

In another preferred embodiment, the gene-editing protein is selected from the group consisting of: cas13d, CasRx, Cas13e, CRISPR/Cas9, Cpf1, Cas9, Cas13a, Cas13b, Cas13c, Cas13f, RNA-targeted gene-editing proteins, or a combination thereof.

In another preferred embodiment, the gene-editing protein is CasRx, and the nucleotide sequence of the gRNA is selected from the group consisting of: 1, 2,3, 4, 5 and 6.

In another preferred embodiment, the source of the gene-editing protein is selected from the group consisting of: streptococcus pyogenes (Streptococcus pyogenes), Staphylococcus aureus (Staphylococcus aureus), Aminococcus sp, Lachnospiraceae (Lachnospiraceae bacteria), Ruminococcus Flavefaciens (Ruminococcus Flavefaciens), or a combination thereof.

In another preferred embodiment, the Ptbp1 is derived from a mammal; preferably, it is derived from human, mouse, rat, or rabbit; more preferably, it is of human origin.

In another preferred embodiment, the Ptbp1 gene comprises a wild-type Ptbp1 gene and a mutant Ptbp1 gene.

In another preferred embodiment, the mutant form comprises a mutant form in which the function of the encoded protein is not altered after mutation (i.e., the function is the same or substantially the same as the wild-type encoded protein).

In another preferred embodiment, the mutant Ptbp1 gene encodes a polypeptide which is the same or substantially the same as the polypeptide encoded by the wild Ptbp1 gene.

In another preferred example, the mutant Ptbp1 gene comprises polynucleotide with homology more than or equal to 80% (preferably more than or equal to 90%, more preferably more than or equal to 95%, more preferably more than or equal to 98% or 99%) compared with the wild Ptbp1 gene.

In another preferred embodiment, the mutant Ptbp1 gene comprises a polynucleotide which is truncated or added with 1-60 (preferably 1-30, more preferably 1-10) nucleotides at the 5 'end and/or 3' end of the wild-type Ptbp1 gene.

In another preferred embodiment, the Ptbp1 gene comprises a cDNA sequence, a genomic sequence, or a combination thereof.

In another preferred embodiment, the Ptbp1 protein comprises an active fragment of Ptbp1 or a derivative thereof.

In another preferred embodiment, the active fragment or derivative thereof has at least 90%, preferably 95%, more preferably 98%, 99% homology with Ptbp 1.

In another preferred embodiment, the active fragment or derivative thereof has at least 80%, 85%, 90%, 95%, 100% of Ptbp1 activity.

In another preferred embodiment, the amino acid sequence of the Ptbp1 protein is selected from the group consisting of:

(i) a polypeptide having an amino acid sequence as set forth in SEQ ID No. 11;

(ii) (ii) a polypeptide derived from (i) having the function of said protein, which is formed by substituting, deleting or adding one or several (e.g. 1-10) amino acid residues to the amino acid sequence shown in SEQ ID NO. 11; or

(iii) The homology of the amino acid sequence and the amino acid sequence shown in SEQ ID NO. 11 is more than or equal to 90 percent (preferably more than or equal to 95 percent, more preferably more than or equal to 98 percent or 99 percent), and the polypeptide has the function of the protein.

In another preferred embodiment, the nucleotide sequence of the Ptbp1 gene is selected from the group consisting of:

(a) a polynucleotide encoding a polypeptide as set forth in SEQ ID No. 11;

(b) a polynucleotide having a sequence as set forth in SEQ ID No. 12;

(c) polynucleotide having a nucleotide sequence homology of 95% or more (preferably 98% or more, more preferably 99% or more) with the sequence shown in SEQ ID No. 12;

(d) a polynucleotide in which 1 to 60 (preferably 1 to 30, more preferably 1 to 10) nucleotides are truncated or added at the 5 'end and/or the 3' end of the polynucleotide shown in SEQ ID No. 12;

(e) a polynucleotide complementary to any one of the polynucleotides of (a) - (d).

In another preferred example, the ptbp1 protein is shown as SEQ ID No. 11.

In another preferred example, the coding nucleic acid of the ptbp1 protein is shown as SEQ ID No. 12.

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

In another preferred example, the inhibitor of the ptbp1 gene or the protein encoded by the ptbp1 gene inhibits the activity and/or expression level of ptbp 1.

In another preferred embodiment, the concentration of the inhibitor of the ptbp1 gene or its encoded protein (titer of virus) > 1X 10¹³Preferably 1 × 10¹³—1×10¹⁴。

In another preferred embodiment, the inhibition rate of the ptbp1 gene or the protein encoded by the ptbp1 gene on the activity and/or expression level of ptbp1 is more than 90%, preferably 90% -95%.

In another preferred embodiment, the inhibitor targets astrocytes of brain tissue.

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

In another preferred embodiment, the neurodegenerative disease includes parkinson's disease.

In a second aspect, the present invention provides a composition comprising:

(a) a gene-editing protein or an expression vector thereof, said gene-editing protein selected from the group consisting of: CasRx, CRISPR/Cas9, Cpf1, Cas9, Cas13a, Cas13b, Cas13c, RNA-targeted gene editing proteins, or a combination thereof; and

(b) a gRNA, or an expression vector thereof, the gRNA being a DNA or RNA that directs 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: 1, 2,3, 4, 5 and 6.

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

In another preferred embodiment, the composition further comprises:

(c) other drugs for the prevention and/or treatment of neurodegenerative diseases.

In another preferred embodiment, the composition further comprises:

(d) other drugs for treating neurological disorders associated with functional neuronal death.

In another preferred embodiment, the composition further comprises:

(e) other drugs for the prevention and/or treatment of retinal diseases.

In another preferred embodiment, the expression vector for the gene-editing protein comprises a vector targeting glial cells.

In another preferred embodiment, the expression vector for the gene-editing protein comprises a vector targeting brain tissue astrocytes.

In another preferred embodiment, the expression vector for the gene-editing protein comprises a vector targeting retinal MG cells.

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

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

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

In another preferred embodiment, the vector comprises AAV2 or AAV 9.

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

In another preferred embodiment, the composition is in the form of a liquid formulation.

In another preferred embodiment, the composition is in the form of an injectable formulation.

In another preferred embodiment, the other agent for preventing and/or treating neurodegenerative disease is selected from the group consisting of: a dopamine prodrug, a non-ergot dopamine receptor agonist, a monoamine oxidase B inhibitor, or a combination thereof.

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

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

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

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

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

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

In another preferred embodiment, the component (a) and the component (b) and optionally the component (c) in the composition represent 0.01 to 99.99 wt%, preferably 0.1 to 90 wt%, more preferably 1 to 80 wt% of the total weight of the composition.

In a third aspect the invention provides a kit comprising:

(a1) a first container, and a gene-editing protein or an expression vector thereof, or a medicament containing a gene-editing protein or an expression vector thereof, located in the first container, the gene-editing protein being selected from the group consisting of: cas13d, CasRx, Cas13e, CRISPR/Cas9, Cpf1, Cas9, Cas13a, Cas13b, Cas13c, Cas13f, RNA-targeted gene-editing proteins, or a combination thereof;

(b1) a second container, and a gRNA or an expression vector thereof, or a medicament containing a gRNA or an expression vector thereof, located in the second container, the gRNA being a DNA or RNA that directs a gene-editing protein to specifically bind to the Ptbp1 gene.

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

In another preferred embodiment, the kit further comprises:

(c1) a third container and other medicines for preventing and/or treating neurodegenerative diseases and/or other medicines for preventing and/or treating retinal diseases and/or other medicines for treating nervous system diseases related to functional neuron death, which are positioned 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 drug in the first container is a single formulation containing a gene-editing protein or an expression vector thereof.

In another preferred embodiment, the medicament in the second container is a single preparation containing gRNA or an expression vector thereof.

In another preferred embodiment, the drug in the third container is a single formulation containing other drugs that are intended for the treatment of neurological diseases associated with functional neuronal death.

In another preferred embodiment, the dosage form of the drug is selected from the group consisting of: a lyophilized formulation, a liquid formulation, 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 further comprises instructions.

In a fourth aspect, the present invention provides a composition according to the second aspect of the invention or a use of a kit according to the third aspect of the invention for the manufacture of a medicament for the treatment of a neurological condition associated with functional neuronal death.

In another preferred embodiment, the concentration of the gene-editing protein or its expression vector in the composition (viral titer) > 1X 10¹³Preferably, 1 × 10¹³—1×10¹⁴。

In another preferred embodiment, the gRNA or its expression vector has an action concentration (viral titer) >1 × 10 in the composition¹³Preferably, 1 × 10¹³—1×10¹⁴。

In another preferred embodiment, the concentration of the other drug for treating the nervous system disease related to functional neuron death (viral titer) > 1X 10 in the pharmaceutical composition¹³Preferably, 1 × 10¹³—1×10¹⁴。

In another preferred embodiment, the composition or kit comprises (a) a gene-editing protein or an expression vector thereof; and (b) a gRNA or an expression vector thereof; and (c) optionally other agents for treating a neurological disorder associated with functional neuronal death; and (d) a pharmaceutically acceptable carrier.

In another preferred embodiment, in the composition or kit, (a) a gene-editing protein or an expression vector thereof; and (b) a gRNA or an expression vector thereof; and (c) optionally other agents for treating neurological disorders associated with functional neuronal death in an amount of from 0.01 to 99.99 wt%, preferably from 0.1 to 90 wt%, more preferably from 1 to 80 wt% based on the total weight of the composition or kit.

In a fifth aspect, the present invention provides a method for promoting differentiation of glial cells into functional neurons, comprising the steps of:

culturing glial cells in the presence of an inhibitor of the Ptbp1 gene or RNA or protein encoding the same, or a composition according to the second aspect of the invention, to promote differentiation of the glial cells into functional neurons.

In another preferred embodiment, the glial cells are in vitro cells.

In another preferred embodiment, the Ptbp1 gene or its encoded protein inhibitor has an action concentration (viral titer) > 1X 10¹³Preferably, 1 × 10¹³—1×10¹⁴。

In another preferred embodiment, the composition of claim 2 has an action concentration (viral titer) > 1X 10¹³Preferably, 1 × 10¹³—1×10¹⁴。

In a sixth aspect, the present invention provides a method for preventing and/or treating a neurological disease associated with functional neuron death, comprising:

administering to a subject in need thereof an inhibitor of the Ptbp1 gene or RNA or a protein encoding the same, or a composition according to the second aspect of the invention, or a kit according to the third aspect of the invention.

In another preferred embodiment, the subject comprises a human or non-human mammal suffering from a neurological condition associated with functional neuronal death.

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

In a seventh aspect, the present invention provides a method of screening for a candidate compound for the prevention and/or treatment of a neurological disease associated with functional neuronal death, the method comprising the steps of:

(a) in the test group, a test compound was added to a culture system of cells, and the expression amount (E1) and/or activity (a1) of Ptbp1 in the cells of the test group was observed; in the control group, no test compound was added to the culture system of the same cells, and the expression amount (E0) and/or activity (a0) of Ptbp1 in the cells of the control group were observed;

wherein, if the expression level (E1) and/or activity (A1) of Ptbp1 of the cells in the test group is significantly lower than that of the control group, it indicates that the test compound is a candidate compound for preventing and/or treating a functional neuron death-related neurological disease having an inhibitory effect on the expression and/or activity of Ptbp 1.

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

In another preferred example, the method further comprises the steps of:

(b) further testing the candidate compound obtained in step (a) for its promoting effect on the differentiation of glial cells into functional neurons; and/or further tested for its effect on Ptbp1 gene downregulation.

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

In another preferred embodiment, the mammal is a mammal suffering from a neurological disease associated with functional neuronal death.

In another preferred embodiment, the phrase "substantially less than" means E1/E0 ≦ 1/2, preferably ≦ 1/3, more preferably ≦ 1/4.

In another preferred embodiment, the phrase "substantially less than" means A1/A0 ≦ 1/2, preferably ≦ 1/3, more preferably ≦ 1/4.

In another preferred embodiment, the cells comprise glial cells.

In another preferred embodiment, the cells are cultured in vitro.

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

The eighth aspect of the present invention provides a method for promoting differentiation of astrocytes into dopaminergic neurons, comprising the steps of:

culturing astrocytes in the presence of an inhibitor of Ptbp1 gene or RNA or its encoded protein or a composition according to the second aspect of the present invention, thereby promoting differentiation of astrocytes into dopamine neurons.

In another preferred embodiment, the astrocytes comprise striatal astrocytes.

In another preferred embodiment, the astrocytes are astrocytes of brain tissue.

In another preferred embodiment, the astrocytes are in vitro cells.

In another preferred embodiment, the concentration of action (viral titer) of the composition is > 1X 10¹³Preferably, 1 × 10¹³—1×10¹⁴。

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

The ninth aspect of the present invention provides a method for promoting differentiation of muller glia cells into optic ganglion cells, comprising the steps of: in the presence of an inhibitor of the Ptbp1 gene or RNA or protein encoding the same or a composition according to the second aspect of the invention, thereby promoting differentiation of retinal muller glia cells into retinal ganglion cells.

In another preferred embodiment, the concentration of the inhibitor of the Ptbp1 gene or RNA or protein encoded thereby (viral titer) > 1X 10¹³Preferably, 1 × 10¹³—1×10¹⁴。

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows that CasRx can specifically knock down Ptbp1 mRNA1 in vitro. (A) Schematic representation of CasRx mediated knockdown of Ptbp1 mRNA. (B) The schematic shows the target sites of six gRNAs in the Ptbp1 gene. (C and D) knockdown efficiency of different combinations of gRNAs. grnas 5 and 6 showed the highest knockout efficiency in both N2a cells (C) and astrocytes (D). The upper numbers indicate the number of repetitions of each group. All values are expressed as mean ± SEM. (E and F) CasRx-Ptbp1 knockdown Ptbp1 specifically. N2a cells (E), N ═ 4 independent repeats; astrocytes (F), n ═ 2 independent repeats.

FIG. 2 shows AAV specificity and expression, GFP expression turned off over time, and the subgenotypes of RGCs transformed, in relation to FIG. 1. (A) Comparison with CasRx (x-axis)₎In contrast, log of all detected genes in the RNA-seq pool of CasRx-Ptbp1 (y-axis)₂Expression level of (FPKM +1) value. N2a cells, N ═ 4 independent repeats; astrocytes, n ═ 2 independent repeats. Typical neuronal derived genes are marked as black dots. (B) Specificity of AAV-GFAP-GFP-Cre. AAV-GFAP-GFP-Cre drives GFP expression in MG of Ai9 mice and unlocks tdTomato expression. Sox9 is a MG-specific marker. Scale bar, 50 microns. (C) Determination of AAV expression. Percentage of GFP + cells expressing tdTomato and percentage of tdTomato + cells expressing Sox 9. All values are expressed as mean ± SEM. (D) qPCR analysis confirmed that the infected retinas expressed AAV-GFAP-CasRx and AAV-GFAP-CasRx-Ptbp 1. The upper numbers indicate the number of repetitions of each group. All values are expressed as mean ± SEM. (E) MG-induced RGCs eventually shut off GFP expression over time. And (4) a scale. The experiment was repeated 6 times independently for each 20 micron group with similar results. (F-H) staining of Foxp2, Brn3c and PV. Note that Foxp2, Brn3c, and PV are markers for RGC subtypes F-RGC, RGC type 3, and PV-RGC, respectively. Yellow arrows indicate co-localization of tdTomato + cells with different markers, white arrows indicate co-localization of tdTomato + cells with different markers. Scale bar, 20 microns. The experiment was repeated 3 times per group with similar results.

FIG. 3 shows that Ptbp1 combination converts MG to RGC in intact mature retina. (A) Schematic of MG to RGC conversion. Vector I (AAV-GFAP-GFP-Cre) encodes Cre recombinase and GFP driven by the MG-specific promoter GFAP, and vector II (AAV-GFAP-CasRx-Ptbp1) encodes CasRx and gRNA. To induce RGC, retinas (5 week old Ai9 mice) were injected with AAV-GFAP-CasRx-Ptbp1 or control vector AAV-GFAPCasRx and AAV-GFAP-GFP-Cre. The occurrence of transformation was checked about one month after injection. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner core layer; IPL, inner plexiform layer; GCL, ganglion cell layer. (B) Representative images show the co-localization of tdTomato and Brn3a in GCL (dashed lines). White arrows indicate the ends of MG not co-localized with Brn3a, yellow arrows indicate co-localization of tdTomato and Brn3a in retinas injected with AAV-GFAP-GFP-Cre and AAV-GFAP-CasRx-Ptbp 1. Brn3a is a specific marker for RGCs. Scale bar, 20 microns. (C) 1 month after AAV injection, tdTomato in GCL⁺Or tdTomato⁺Brn3a⁺The number of cells. AAV-GFAP-GFP-Cre plus AAV-GFAP-CasRx, n 6 retinas; AAV-GFAP-GFP-Cre plus AAV-GFAP-CasRx-Ptbp1, n ═ 7 retinas. Data are expressed as mean ± SEM,. about.p<0.05，**p<0.01，**p<0.001, unpaired t-test. (D) Representative images show the co-localization of tdTomato with another specific marker for RGC Rbpms in GCL. The yellow arrow indicates co-localization of tdTomato and Rbpms. Scale bar, 20 microns. (E) Number of tdTomato + or tdTomato + Rbpms + cells in GCL 1 month after AAV injection. AAV-GFAP-GFP-Cre plus AAV-GFAP-CasRx, n 6 retinas; AAV-GFAP-GFP-Cre plus AAV-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 4 shows that knockdown of Ptbp1 converts MG to RGC in the intact retina of C57BL mice, which correlates with figure 3.

(A) Schematic of RGC induction from MG. Vector 1(GFAP-mCherry) expresses mCherry driven by the MG-specific promoter GFAP, and vector 2(AAV-EFS-CasRx-Ptbp1) expresses gRNA and CasRx under the promoters of the spectrum. To induce RGC, the retina was injected with AAV-GFAP-mCherry, simultaneously with AAV-EFS-CasRx-Ptbp1 or control virus AAV-GFAP-mCherry. The occurrence of transformation was checked 2-3 weeks after injection. (B) Representative images show the co-localization of mCherry and MG marker Sox9, indicating that AAV-GFAP-mChery is specifically expressed in MG. Scale bar, 50 microns. Representative images (C, D) show mCherry + Brn3a + and mCherry + Rbpms + cells in GCL. Each group of n is 3 retinas. Scale bar, 50 microns. (E) mCherry + cells like RGCs respond electrophysiologically to the production of LED light.

FIG. 5 shows that knocking down Ptbp1 converts MG into amacrine cells, which correlates with FIG. 3. (A) tdTomato + Pax6+ cells were observed in intact retinas of Ai9 mice injected with AAV-GFAP-CasRx-Ptbp 1. Green arrows indicate tdTomato + cells do not co-localize with Pax6, yellow arrows indicate Pax6 and tdTomato co-localization. Note that Pax6 is a marker for amacrine cells. Scale bar, 20 microns. (B) no tdTomato + Prox1+ cells were observed. The arrows indicate that tdTomato cells do not co-localize with Prox 1(a marker for bipolar cells). Scale bar, 20 microns. (C) tdTomato + cells were not observed in the photoreceptor layer (ONL). White arrows indicate tdTomato positive RGC-like cells in GCL, yellow arrows indicate tdTomato + protamine-like cells in INL, green arrows indicate tdTomato + projection of MG. Scale bar, 20 microns. Each group independently repeated the experiment at least 3 times, and the results were similar.

Figure 6 shows the induction of MG to RGC conversion in a mouse model of NMDA-induced retinal damage. (A) experimental design. Retinal damage was caused by intravitreal injection of NMDA (200mM, 1.5mL) into Ai9 mice of 4-8 weeks of age. Two to three weeks after NMDA injection, AAV was injected subretinally. Immunostaining and behavioral testing were performed 1 month after AAV injection. (B) NMDA injection essentially kills most of the RGCs in GCL. Scale bar, 50 microns. (C) Co-localization of tdTomato and Brn3 a. Yellow arrows indicate co-localization of Brn3a and tdTomato in the GCL. n-6 retinas, scale bar, 20 μm. (D) Number of Brn3a + or tdTomato + Brn3a + cells in GCL. Intact retina, n ═ 6 retinas; GFAP-CasRx plus GFAP-GFP-Cre, n ═ 6 retinas; GFAP-CasRx-Ptbp1 plus GFAP-GFP-Cre, n ═ 7 retinas. Data are presented as mean ± SEM, # p <0.05, # p <0.01, # p <0.001, unpaired t-test. (E) Co-location of tdTomato and Rbpms. Yellow arrows indicate co-localization of Rbpms and tdTomato in GCL injected with GFAP-CasRx-Ptbp1 plus GFAP-GFP-Cre. Scale bar, 20 microns. (F) Number of Rbpms + or tdTomato + Rbpms + cells in GCL. (ii) uninjured retinas, n ═ 6 retinas; GFAP-CasRx plus GFAP-GFP-Cre, n ═ 7 retinas; GFAP-CasRx-Ptbp1 plus GFAP-GFP-Cre, n ═ 7 retinas. Data are presented as mean ± SEM, # p <0.05, # p <0.01, # p <0.001, non-paired t-test. (G) Images show representative RGC-like tdTomato + cells recorded under a two-photon microscope. Scale bar, 20 microns. (H) Induced response of the optic ganglion cells to light. A total of 8 cells were recorded, 6 of which showed a response to LED light. Of these cells, 5 were ON cells and 1 was OFF cells.

Fig. 7 shows the progress of MG to RGC conversion, in relation to fig. 6. (A) Representative images show the conversion of MG to RGC in the intact retina at five different time points (no NMDA injection). Arrows indicate induced RGCs. Scale bar, 20 microns. The experiment is independently repeated for more than or equal to 3 times, and the results are similar. Note that representative pictures in "2 months" are also shown in fig. 2B and 2D. (B) Absolute numbers of tdTomato + Brn3a + and tdTomato + Rbpms + cells in GCL. Note that the values in "1 month" are also shown in fig. 1C and 3E. All values are expressed as mean ± SEM. (C) Representative images show the intermediate stage of induction of cells at 1.5 weeks post AAV injection (white arrows). Each group of n > -3 mice. Note that the intermediate unit typically loses its projection in the ONL. Scale bar, 10 microns. The experiment is independently repeated for more than or equal to 3 times, and the results are similar. (D) Representative images show the process of MG conversion to RGC in NMDA-damaged retinas at four different time points. Arrows indicate induced RGCs. Scale bar, 20 microns. Note that representative images in "3 months" are also shown in fig. 3C and 3E. The experiment is independently repeated for more than or equal to 2 times, and the results are similar.

Figure 8 shows that the transformed RGCs can project to the brain and partially restore visual function. (A) A schematic view of a visual pathway. RGCs send axons through the optic nerve, carrying optic nerve signals to the dorsal geniculate nucleus and superior colliculus outside the retina. (B) Retinal planopies. Yellow arrows indicate MG-derived tdTomato-positive RGC axons. Scale bar, 100 microns. The experiment was repeated 3 times per group with similar results. (C) Representative images show tdTomato + axons of RGCs induced in the optic nerve. Scale bar, 200 microns. Each group was independently repeated 5 times with similar results. Representative images (D and E) show that strong signals are observed in the contralateral dorsal geniculate nucleus (D) and superior colliculus (E) (the target area of RGC projection in the brain). Each group was independently repeated 4 times with similar results. Note that the ipsilateral dorsal geniculate nucleus and SC showed a weak tdTomato + signal. Scale bar, 500 microns (left), 50 microns (right). (F) Schematic of VEP recording. (G) The VEP gives up in the primary visual cortex. Responses from the same group of mice are shown, with each line representing one retina. The number of retinas per group is represented above the histogram. (H) Wild type (WT, n ═ 8 retinas), NMDA and AAV-GFAP-mCherry (n ═ 12 retinas), NMDA, AAV-GFAP-mCherry and AAV-GFAP-CasRx (n ═ 11 retinas), and NMDA, AAV-GFAP-mCherry and AAV-GFAP-CasRx-Ptbp1(n ═ 8 retinas). Each dot represents one mouse. Data are presented as mean ± SEM, # p <0.05, # p <0.01, # p <0.001, unpaired t-test. (I) Design of dark/light preference test. Note that both eyes received NMDA treatment and were injected with the same AAV2 weeks after NMDA injection. (J) Percentage of time spent in dark room. WT, n ═ 13 mice; NMDA and GFAP-mCherry, n ═ 14 mice; NMDA, AAV-GFAP-mCherry and AAV-GFAP-CasRx, n 12 mice; NMDA, AAV-GFAP-mCherry and AAV-GFAP-CasRx-Ptbp1, n ═ 12 mice. All values are expressed as mean ± SEM; unpaired t-test; p <0.05, p <0.01, p < 0.001.

FIG. 9 shows the projection of induced RGCs onto the optic nerve and brain. (A) Representative images are shown at five different time points (1 week, 1.5 weeks, 2 weeks, 3 weeks and 1 month). tdTomato + axons were first seen 1.5 weeks after AAV injection (yellow arrows). Scale bar, 50 microns. The experiment was independently repeated 3 times with similar results. (B) The density of tdTomato + axons (yellow arrows) in the dorsal geniculate nucleus gradually increased. Note that at 1.5 weeks post-AAV injection, tdTomato + axons were first observed in the contralateral dorsal geniculate nucleus. Scale bar, 500 microns (top), 50 microns (bottom). The experiment was independently repeated 3 times with similar results. (C) Projection of tdTomato + axons (yellow arrows) onto SCs. Note that tdTomato + axons were first observed in contralateral SCs 2 weeks after AAV injection. Scale bar, 500 microns (upper), 50 microns (lower). The experiment was independently repeated 3 times with similar results. (D) The schematic shows the step-wise projection process of the induced RGC.

Figure 10 shows the progression of RGC projection induced in the damaged retina, relative to figure 9. (A) Representative images show a gradual increase in tdTomato + axons in NMDA-induced optic nerves at three different time points (1 week, 2 weeks, 3 weeks). Scale bar, 50 microns. The experiment was independently repeated 2 times with similar results. (B) The density of tdTomato + axons (yellow arrows) in the dorsal geniculate nucleus gradually increased. Scale bar, 500 microns (top), 50 microns (bottom). The experiment was independently repeated 2 times with similar results. (C) Projection progression of tdTomato + axons (yellow arrows) in superior colliculus. Scale bar, 500 microns (top), 50 microns (bottom). The experiment was independently repeated 2 times with similar results.

FIG. 11 shows CasRx mediated glial cell to neuronal transformation. (A, B) schematic representation of the injection strategy. The efficiency of transformation was evaluated about one month after injection. ST, striatum. (C) Co-localization of mCherry and GFAP. Percentage of mCherry + cells expressing GFAP, n-3 mice. Scale bar, 20 microns. (D) One week after AAV injection, Flag (fused to CasRx) and GFAP co-localized. Scale bar, 20 microns. (E) One week after AAV injection, Ptbp1 expression (detected by the Ptbp1 antibody) was down-regulated in the striatum. The expression of CasRx (fused to Flag) was detected by an antibody against the Flag tag. Each group of n-4 mice. Scale bar, 10 microns. (F) The fluorescence intensity of Ptbp1 was quantified using ImageJ. a.u. represents arbitrary units. (G) Representative image of striatum. NeuN is a specific marker for mature neurons. White arrows indicate that NeuN expression is not co-localized with mCherry, yellow arrows indicate NeuN and mCherry co-localized. Scale bar, 50 microns. (H) Percentage of mCherry + NeuN + cells in mCherry + cells (n ═ 6 mice per group; t ═ 4.7, p < 0.001). (I) The image shows that mCherry + NeuN + glutaminase positive cells (pink arrow) are adjacent to mCherry + NeuN + glutaminase negative cells (yellow arrow). Scale bar, 10 microns. (J) Percentage of mCherry + glutaminase + NeuN + cells in mCherry + NeuN + cells. N-5 mice. (K) Representative images show that mCherry + NeuN + cells rarely co-localize with somastatin. Yellow arrows indicate mCherry + NeuN + somastatin cells, pink arrows indicate mCherry + NeuN + somastatin cells. Control AAV showed no presence of mCherry + SST + cells, indicating that SST + cells could not be infected by AAV-GFAP-mChery. The experiment was repeated 5 times independently with similar results. Scale bar, 20 microns. (L) representative images show that mCherry + NeuN + cells do not co-localize with Palvabumin. The experiment was independently repeated 4 times with similar results. Scale bar, 20 microns. (M) mCherry + TH + cells were observed in the intact striatum (white arrows). Scale bar, 20 microns. (N) percentage of mCherry + TH + cells in mCherry + cells, N-5 mice per group. (O) generally, mCherry + TH + cells show low (upper, white arrow) or undetectable levels of NeuN expression (lower, yellow arrow). Scale bar, 5 microns. The experiment was independently repeated 5 times with similar results. All values are expressed as mean ± SEM; unpaired t-test; p <0.05, p <0.01, p < 0.001.

FIG. 12 shows the conversion of astrocytes into dopamine neurons in Parkinson's model mice. (A and B) summary of the experiments. 6-OHDA was injected unilaterally into substantia nigra. After 3 weeks, AAV-GFAP-CasRx-Ptbp1 plus AAV-GFAP-mCherry, AAV-GFAP-CasRx plus AAV-GFAP-mCherry or saline was injected into the striatum of rats ipsilateral (relative to the 6-OHDA-injected side) for immunostaining at about 1 or 3 months after AAV injection. (C) Confocal images of transformed mCherry + TH + (yellow arrow) and mCherry + DAT + (yellow arrow)

cells

1 or 3 months after AAV injection. Orange arrows indicate the neurosynaptic of transformed mCherry + TH + DAT + cells. Note that TH and DAT are dopamine neuron specific markers. Scale bar, 50 microns or 10 microns. (D) percentage of mCherry + TH + cells in mCherry + cells. AAV-GFAP-CasRx, 1 month: n-5 mice; AAV-GFAP-CasRx-Ptbp1, 1 month: n-5 mice; AAV-GFAP-CasRx-Ptbp1, 3 months: n-3 mice. (E) Percentage of mCherry + TH + cells in TH + cells, n-5 mice per group. Scale bar, 50 microns. (F) Percentage of mCherry + DAT + cells in mCherry + cells. AAV-GFAP-CasRx, 1 month: n-5 mice; AAV-GFAP-CasRx-Ptbp1, 1 month: n-5 mice; AAV-GFAP-CasRx-Ptbp1, 3 months: n-3 mice. (G) Percentage of mCherry + DAT + TH + cells of mCherry + TH + cells. AAV-GFAP-CasRx, 1 month: n-5 mice; AAV-GFAP-CasRx-Ptbp1, 1 month: n-5 mice; AAV-GFAP-CasRx-Ptbp1, 3 months: n-3 mice. (H) Representative images show that ALDH1a1 and GIRK2 expression co-localized with mCherry + TH + (yellow arrow), while Calbindin expression (orange arrow) did not co-localized with mCherry + DAT +. ALDH1A1 and GIRK2 are dopamine neuron specific markers in the SNc A9 region, while calbindin is a VTA dopamine neuron specific marker. Each group of n-3 mice. Scale bar, 50 microns. (I) Percentage of mCherry + ALDH1a1+, mCherry + GIRK2+ and mCherry + calabin + cells in mCherry + cells. Each group of n-3 mice. (J) The percentage of mCherry + TH + ALDH1a1+ and mCherry + TH + GIRK2+ in mCherry + TH + cells; and mCherry + DAT + calabindin + cells among mCherry + DAT + cells. Each group of n-3 mice. (K) Whole cell recordings were performed in striatal sections (n-22 cells). The figure shows the ability of neuron-like mCherry + cells to produce repetitive action potentials (20 out of 22 cells) and rectification (4 out of 10 cells, green). (L) images show the induction of spontaneous synaptic currents in neurons. (M) hyperpolarized voltage-gated current. Representative images (N) show the percentage of mCherry + VMAT2+ TH + cells among mCherry + TH + cells expressed VMAT2 (yellow arrow), and mCherry + TH + cells, N3 mice. Scale bar, 20 microns. (o) images show that mCherry + cells can take up FFN206 (blue), a fluorescent dopamin derivative, 1 month after AAV injection. n > -10 slices per group. Scale bar, 30 microns (top), 20 microns (bottom). (P) FFN206 can be released at mCherry + cells (yellow arrow). The images show high KCl-induced FFN206 release. Scale bar, 10 microns. (Q) statistical analysis of KCl-induced FFN206 release in sections injected with AAV-GFAP-mCherry plus AAV-GFAP-CasRx-Ptbp1, n-25 mCherry + blue + cells. From 5 sections (n-3 mice) 25 cells were analyzed. All values are expressed as mean ± SEM. Unpaired t-test; p <0.05, p <0.01, p < 0.001.

FIG. 13 shows that knocking down Ptbp1 converts astrocytes into dopamine neurons in a mouse model of 6-OHDA-induced Parkinson's disease. (A) Schematic experimental diagram. (B) Staining showed ipsilateral substantia nigra (relative to the 6-OHDA injection side) TH + neuronal (green) death. Scale bar, 100 microns. The experiment was independently repeated 12 times with similar results. (C) DAT staining showed disappearance of dopamine neuron fibers (green) in the ipsilateral striatum. Scale bar, 500 microns. The experiment was independently repeated >10 times with similar results. (D, E) representative confocal images showing quantification of mCherry + TH + and mCherry + DAT + cells, and mCherry + TH + and mCherry + DAT + cells at different time points after AAV injection. Each group of n > -3 mice. Scale bar, 50 microns. Note that data "GFAP-CasRx, 1 month", "GFAP-CasRx-

Ptbp

1, 1 month" and "GFAP-CasRx-

Ptbp

1, 3 months" are also shown in FIGS. 6C, 6D and 6. Scale bar, 50 microns. (F) Absolute number of TH + cells in parkinson disease model mice. (G) Confocal images of mCherry + TH + cells and the percentage of mCherry + TH + cells in TH + cells, n-5 mice per group. Scale bar, 50 microns. (H) Percentage of mCherry + TH + cells in mCherry + cells. (I) Absolute number of DAT + cells in PD model mice. (J, K) confocal images of mCherry + DDC + cells and the percentage of mCherry + DDC + cells in mCherry + cells, n-5 mice per group. DDC is a dopamine neuron marker. Scale bar, 50 microns. The (L, M) representative images show the co-localization of FOXA2 and mCherry (yellow arrow), and the percentage of mCherry + FOXA2+ cells in the cells. Each group of n-5 mice. FOXA2 is a dopamine neuron marker. Scale bar, 30 microns. (N) representative images show that mCherry + TH + cells are not co-localized with representative striatal interneuron markers PV, SST and CR. Yellow arrows indicate mCherry + TH + cells, blue arrows indicate PV +, SST + or CR + cells. Note that SST is co-localized with mCherry, but not with TH, consistent with the results in fig. S1H, suggesting that knock-out Ptbp1 may sparsely induce SST + neurons. The experiment was independently repeated 3 times with similar results. Scale bar, 20 microns. (O) comparison of net revolutions before and 1 month after AAV injection. The numbers above the dots indicate the number of mice per group. And (5) pairing t test. Data for "1 month" is also shown in figure 7. (P) Net rotation (rpm) caused by apomorphine injection. Each mouse was evaluated for behavior at 1 and 3 months, with n ═ 3 mice per group. Two-way ANOVA and bonferroni tests. The data for "1 month" is also shown in figure 7. (Q) net spin (rpm) induced by amphetamine injection, n-3 mice per group. Two-way ANOVA and bonferroni tests. The data for "1 month" is also shown in figure 7. All values are expressed as mean ± SEM; unpaired t-test; p <0.05, p <0.01, p < 0.001.

Fig. 14 shows that induced neurons reduced motor dysfunction in parkinson disease model mice (a) experimental design. (B) Net rotation (contralateral-ipsilateral) caused by apomorphine injection. (C) Net rotation induced by amphetamine injection (ipsilateral-contralateral). (D) Percentage of ipsilateral versus total rotations (ipsilateral/total rotations) following systemic amphetamine injection. (E) Percentage of spontaneous ipsilateral touches relative to total number of touches. (F) And (5) testing by a bar rotating instrument. Results are expressed as the time (seconds) that the mouse stayed on the accelerated rotating bar before dropping. The numbers above the bars indicate the number of mice per group. All values are expressed as mean ± SEM. And (4) carrying out one-way analysis of variance and then carrying out Tukey test. P <0.05, p <0.01, p < 0.001.

Detailed Description

The present inventors have conducted extensive and intensive studies and have surprisingly found for the first time that inhibition of expression or activity of ptbp1 gene or RNA of glial cells or protein encoded thereby can effectively induce differentiation of glial cells into functional neurons, thereby treating neurological diseases associated with functional neuron death. On this basis, the present inventors have completed the present invention.

In the present invention, Retinal Ganglion Cell (RGC) degeneration is the primary cause of permanent blindness. Whereas the transdifferentiation of Muller glial cells (MG) into functional RGCs can help restore vision. The inventors found that MG can be directly converted to functional RGC by knocking down Ptbp1 in mature mouse retina using the RNA-targeted CRISPR system CasRx. In addition, RGC converted from MG achieves a functional projection to the central visual region and results in improved visual function in a mouse model of NMDA (N-methyl-d-aspartate) -induced retinal damage. Therefore, the CasRx-mediated knock-down of Ptbp1 would be a promising therapy for the treatment of retinal diseases caused by neurodegeneration.

The present application uses the recently characterized RNA-targeted CRISPR system CasRx to inhibit Ptbp 1. CasRx-mediated regeneration avoids the emergence of shRNA-induced substantial off-target effects and the risk of permanently altering genes by DNA editing nucleases, providing an excellent tool for treating a variety of diseases.

As used herein, muller glial cells (MG) are the major glial cells in retinal tissue, Retinal Ganglion Cells (RGCs) are nerve cells located in the innermost layers of the retina, whose dendrites are primarily in communication with bipolar cells, whose axons extend to the optic nerve head, forming the optic nerve.

Retinal disease is considered to be an ocular disease. The following 5 types of retinal diseases are common: lesions of the blood vessels and the vascular system. Such as retinal vascular occlusion, arteriosclerotic, hypertensive, hematologic, and diabetic ocular fundus disease. ② inflammation of retina. Is closely related to the interplay of choroiditis and optic neuritis. And thirdly, retinal detachment. Refers to the separation of the retinal nerve layer from the pigment epithelium layer. Retinal degeneration and poor nutrition. Has a genetic factor. Retinal tumor. Among them, retinoblastoma is the most common.

In the present invention, the retinal disease is preferably a retinal disease caused by neurodegeneration, and the symptoms are mainly manifested by visual deterioration 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 invention comprises a gene editing protein and optionally a gRNA.

The term "reprogramming" or "transdifferentiation" may refer to the process of generating cells of a particular lineage (e.g., neuronal cells) from different types of cells (e.g., fibroblasts) without intermediate differentiation.

Neurological disorders associated with functional neuronal death

In the present invention, the functional neuron death-related neurological diseases mainly include Parkinson's disease and visual impairment due to optic ganglion death.

In a preferred embodiment, the neuronal system disorder associated with functional neuronal degeneration includes, but is not limited to, glaucoma, age-related RGC loss, optic nerve injury, retinal ischemia, Leber hereditary optic neuropathy, alzheimer's disease, huntington's disease, schizophrenia, depression, drug abuse, movement disorders (e.g., chorea, cholesteatosis and movement disorders), motor neuron injury disorders (e.g., amyotrophic lateral sclerosis, spinal cord injury), manic depression, Autism Spectrum Disorders (ASD), dysfunction, parkinson's disease.

Astrocytes

Astrocytes are the largest group of cells in the brain of mammals. They perform a number of functions, including biochemical support (e.g., forming the blood-brain barrier), providing nutrition to neurons, maintaining extracellular ionic balance, and participating in repair and scarring following brain and spinal cord injury. Astrocytes can be classified into two types according to the content of glial filaments and the shape of the apophysis: fibrous astrocytes (fibro astrocytes) are mostly distributed in the white matter of the brain and spinal cord, have slender processes and fewer branches, and contain a large amount of glial filaments in the cytoplasm; protoplasmic astrocytes (protoplasmic astrocytes) are mostly distributed in gray matter, and have coarse and short cell processes and many branches.

Astrocytes useful in the present invention are not particularly limited, and include various astrocytes of mammalian central nervous system origin, for example, derived from the striatum, spinal cord, dorsal mesencephalon or cerebral cortex, preferably, from the striatum.

Functional neuron

In the present invention, a functional neuron may refer to a neuron capable of transmitting or receiving information by a chemical or electrical signal. In some embodiments, functional neurons exhibit one or more functional properties of mature neurons found in the normal nervous system, including but not limited to: excitability (e.g., the ability to exhibit an action potential, such as a rapid rise and subsequent fall) (voltage across the cell membrane or membrane potential), formation of synaptic connections with other neurons, presynaptic neurotransmitter release, and postsynaptic responses (e.g., excitatory postsynaptic current or inhibitory postsynaptic current).

In some embodiments, the functional neuron is characterized in that it expresses one or more markers of the functional neuron, including, but not limited to, synaptoprotein, synaptophysin, glutamate decarboxylase 67(GAD67), glutamate decarboxylase 67(GAD65), microalbumin, dopamine-and cAMP-regulated neuronal phosphoprotein 32(DARPP32), vesicular glutamate transporter 1 (vgut 1), vesicular glutamate transporter 2 (vgut 2), acetylcholine, Tyrosine Hydroxylase (TH), dopamine, vesicular GABA transporter (VGAT), and gamma-aminobutyric acid (GABA).

Dopamine neurons

Dopaminergic neurons (dopaminergic neurons) contain and release Dopamine (DA) as the neurotransmitter neurons. Dopamine belongs to catecholamine neurotransmitters and plays an important biological role in the central nervous system, and dopaminergic neurons in the brain are mainly concentrated in the substantia nigra compact area (SNc) of the midbrain, the Ventral Tegmental Area (VTA), the hypothalamus, and around the ventricles. Many experiments have demonstrated that dopaminergic neurons are critically associated with a variety of diseases in the human body, most typically parkinson's disease.

Neurodegenerative diseases

Neurodegenerative diseases are diseases caused by the loss of neurons in the brain and spinal cord. Neurons are the most important component of the nervous system, and their death ultimately leads to dysfunction of the nervous system. After suffering from neurodegenerative diseases, patients suffer from mobility or cognitive impairment, and the development of the disease often causes a plurality of complications, which cause serious harm to the lives of the patients. Clinically, neurodegenerative diseases mainly include alzheimer's disease, parkinson's disease, huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis and the like. At present, the neurodegenerative diseases can only be relieved or the progress of the diseases is delayed, and the complete cure can not be achieved. Parkinson's Disease (PD) is a serious neurodegenerative disease characterized by the loss of mesolimbic dopamine neurons.

Gene editor

Gene editing proteins

In the present invention, the nucleotide of the gene-editing protein can be obtained by genetic engineering techniques such as genomic sequencing, Polymerase Chain Reaction (PCR), etc., and the amino acid sequence thereof can be deduced from the nucleotide sequence. Sources of the wild-type gene-editing protein include (but are not limited to): examples of the microorganisms include Ruminococcus Flavefaciens, Streptococcus pyogenes, Staphylococcus aureus, Acidococcus sp, and Lachnospiraceae bacteria.

In a preferred embodiment of the present invention, the gene-editing proteins include, but are not limited to, Cas13d, CasRx, Cas13e, CRISPR/Cas9, Cpf1, Cas9, Cas13a, Cas13b, Cas13c, Cas13f, RNA-targeted gene-editing proteins.

ptbp1 protein and polynucleotide

In the present invention, the terms "protein of the invention", "ptbp 1 protein", "ptbp 1 polypeptide" and "PTB" are used interchangeably and refer to a protein or polypeptide having the amino acid sequence ptbp 1. They include ptbp1 protein with or without an initiating 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 mutants and functionally active fragments thereof.

The ptbp1 protein is a polypyrimidine domain binding protein 1, is an RNA binding protein and regulates the regulation of RNA splicing. Meanwhile, the method plays a very critical role in other functions of RNA.

In the present invention, the terms "ptbp 1 gene", "ptbp 1 polynucleotide" and "PTB gene" are used interchangeably and all refer to a nucleic acid sequence having a ptbp1 nucleotide sequence.

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

The genome of the murine ptbp1 gene has a total length of 10004bp (NCBI GenBank accession 19205).

Human and murine ptbp1 showed 88% similarity at the DNA level and 84% protein sequence similarity. It is understood that nucleotide substitutions in codons are acceptable when encoding the same amino acid. It is also understood that nucleotide changes are also acceptable when conservative amino acid substitutions are made by nucleotide substitutions.

In the case where an amino acid fragment of ptbp1 is obtained, a nucleic acid sequence encoding it can be constructed therefrom, and a specific probe can be designed based on the nucleotide sequence. The full-length nucleotide sequence or a fragment thereof can be obtained by PCR amplification, recombination, or artificial synthesis. For the PCR amplification method, primers can be designed based on the ptbp1 nucleotide sequence disclosed in the present invention, especially open reading frame sequence, and the relevant sequence can be amplified using a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art as a template. When the sequence is long, two or more PCR amplifications are often required, and then the amplified fragments are spliced together in the correct order.

Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the expanded host cell by conventional methods.

In addition, the sequence can be synthesized by artificial synthesis, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.

At present, DNA sequences encoding the proteins of the present invention (or fragments, derivatives thereof) have been obtained completely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (e.g., vectors) and cells known in the art.

The polynucleotide sequences of the present invention may be used to express or produce a recombinant ptbp1 polypeptide by conventional recombinant DNA techniques. Generally, the following steps are performed:

(1) transforming or transducing a suitable host cell with a polynucleotide (or variant) of the invention encoding a human ptbp1 polypeptide, or with a recombinant expression vector comprising the polynucleotide;

(2) a host cell cultured in a suitable medium;

(3) isolating and purifying the protein from the culture medium or the cells.

In the present invention, the ptbp1 polynucleotide sequence may be inserted into a recombinant expression vector. In general, any plasmid or vector can be used as long as it can replicate and is stable in the host. An important feature of expression vectors is that they generally contain 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 ptbp 1-encoding DNA sequence and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to a suitable promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

Furthermore, the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and Green Fluorescent Protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for E.coli.

Vectors comprising the appropriate DNA sequences described above, together with appropriate promoter or control sequences, may be used to transform appropriate host cells to enable expression of the protein.

The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: coli, bacterial cells of the genus streptomyces; fungal cells such as yeast; a plant cell; an insect cell; animal cells, and the like.

Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, e.g., E.coli, competent cells capable of DNA uptake can be harvested after exponential growth phase using CaCl₂Methods, the steps used are well known in the art. Another method is to use MgCl₂. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, and the like.

The obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.

Adeno-associated virus

Since Adeno-associated virus (AAV) is smaller than other viral vectors, it is not pathogenic, and can transfect dividing and non-dividing cells, and gene therapy methods for genetic diseases based on AAV vectors are receiving wide attention.

Adeno-associated virus (AAV), also known as adeno-associated virus, belongs to the genus dependovirus of the family parvoviridae, is the single-stranded DNA-deficient virus with the simplest structure that is currently found, and requires a helper virus (usually adenovirus) to participate in replication. It encodes the cap and rep genes in inverted repeats (ITRs) at both ends. ITRs are crucial for replication and packaging of viruses. The cap gene encodes the viral capsid protein, and the rep gene is involved in viral replication and integration. AAV can infect a variety of cells.

The recombinant adeno-associated virus (rAAV) is derived from non-pathogenic wild adeno-associated virus, is considered to be one of the most promising gene transfer vectors due to the characteristics of good safety, wide host cell range (divided and non-divided cells), low immunogenicity, long time for expressing foreign genes in vivo and the like, and is widely applied to gene therapy and vaccine research worldwide. Over 10 years of research, the biological properties of recombinant adeno-associated viruses have been well understood, and many data have been accumulated, especially in terms of their utility in various cells, tissues and in vivo experiments. In medical research, rAAV is used in the study of gene therapy for a variety of diseases (including in vivo, in vitro experiments); meanwhile, the gene transfer vector is used as a characteristic gene transfer vector and is widely applied to the aspects of gene function research, disease model construction, gene knock-out mouse preparation and the like.

In a preferred embodiment of the invention, the vector is a recombinant AAV vector. AAV is a relatively small DNA virus that can integrate into the genome of cells that they infect in a stable and site-specific manner. They are able to infect a large series of cells without any effect on cell growth, morphology or differentiation and they do not appear to be involved in human pathology. AAV genomes have been cloned, sequenced and characterized. AAV contains an Inverted Terminal Repeat (ITR) region of about 145 bases at each end, which serves as the viral origin of replication. The remainder of the genome is divided into two important regions with encapsidation functions: the left part of the genome comprising the rep gene involved in viral replication and viral gene expression; and the right part of the genome comprising the cap gene encoding the viral capsid protein.

AAV vectors can be prepared using standard methods in the art. Any serotype of adeno-associated virus is suitable. Methods for purifying vectors can be found, for example, in U.S. Pat. Nos. 6566118, 6989264, and 6995006, the disclosures of which are incorporated herein by reference in their entireties. The preparation of hybrid vectors is described, for example, in PCT application No. PCT/US2005/027091, the disclosure of which is incorporated herein by reference in its entirety. The use of vectors derived from AAV for in vitro and in vivo gene transfer has been described (see, e.g., International patent application publication Nos. WO91/18088 and WO 93/09239; U.S. Pat. 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 a gene of interest, and the use of these constructs to transport the gene of interest in vitro (into cultured cells) or in vivo (directly into the organism). Replication-defective recombinant AAV can be prepared by co-transfecting the following plasmids into a cell line infected with a human helper virus (e.g., adenovirus): plasmids containing the nucleic acid sequence of interest flanked by two AAV Inverted Terminal Repeat (ITR) regions, and plasmids carrying AAV encapsidation genes (rep and cap genes). The AAV recombinants produced are then purified by standard techniques.

In some embodiments, the recombinant vector is encapsidated into a virion (e.g., an AAV virion including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV 16). Accordingly, the present disclosure includes recombinant viral particles (recombinant as they comprise recombinant polynucleotides) comprising 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 inhibitors and pharmaceutical compositions

By utilizing the protein of the invention, substances, particularly inhibitors and the like, which interact with the ptbp1 gene or protein can be screened out by various conventional screening methods.

Ptbp1 inhibitors (or antagonists) useful in the present invention include any substance that inhibits the expression and/or activity of the ptbp1 gene or protein encoded thereby.

For example, the inhibitor of ptbp1 includes an antibody to ptbp1, an antisense RNA to a ptbp1 nucleic acid, an siRNA, an shRNA, an miRNA, a gene editor, or an inhibitor of the activity of ptbp 1. A preferred inhibitor of ptbp1 refers to a gene editor capable of inhibiting expression of ptbp 1.

Preferably, the inhibitors of ptbp1 of the present invention include inhibitors targeted to the 4758-4787 and/or 5381-5410 positions of the ptbp1 gene sequence. The subjects on which the ptbp1 inhibitor of the present invention acts include astrocytes or MG cells.

In a preferred embodiment, the method and steps of inhibiting ptbp1 comprise neutralizing the protein thereof with antibodies to ptbp1 and silencing of the ptbp1 gene using shRNA or siRNA or gene editors carried by viruses such as adeno-associated virus.

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

The inhibitor of ptbp1 protein (including antibody, antisense nucleic acid, gene editor and other inhibitors) of the present invention, when administered (dosed) therapeutically, can inhibit expression and/or activity of ptbp1 protein, thereby inducing glial cell differentiation into functional neurons, thereby treating nervous system diseases associated with functional neuronal degeneration. Generally, these materials will be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally from about 5 to about 8, preferably from about 6 to about 8, although the pH will vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: topical, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, topical administration, autologous cell extraction culture followed by reinfusion, etc.

The invention also provides a pharmaceutical composition comprising a safe and effective amount of an inhibitor of the invention (e.g., an antibody, gene editor, antisense sequence (e.g., siRNA), or inhibitor) and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions, such as tablets and capsules, can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example, from about 1 microgram to about 10 milligrams per kilogram of body weight per day.

The main advantages of the invention include:

(1) the invention firstly discovers that the expression or activity of the Ptbp1 gene or the protein coded by the gene in the astrocyte is reduced, and the differentiation of the astrocyte into dopamine neurons can be induced, so that neurodegenerative diseases (such as Parkinson's disease) can be prevented and/or treated.

(2) The invention discovers for the first time that the expression of ptbp1 in astrocytes is inhibited by using a gene editor (comprising gene editing protein and gRNA), so that the astrocytes can be transdifferentiated into dopamine neurons, and a potential approach is provided for Parkinson treatment.

(3) The invention discovers for the first time that the induction of dopamine neurons relieves the motor dysfunction of a Parkinson mouse model.

(4) The invention discovers for the first time that the RNA-targeted CRISPR system CasRx can avoid the risk of permanent DNA change caused by traditional CRISPR-Cas9 editing. Therefore, CasRx-mediated RNA editing provides an effective means for treating various diseases.

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

(6) The regenerated RGCs can be integrated into the visual pathway and improve the visual function of RGCs-injured mouse models.

(7) The invention uses the RNA-targeted CRISPR system CasRx to knock down Ptbp1, avoids the occurrence of shRNA-induced substantial off-target effect and the risk of permanently changing genes, and provides an excellent tool capable of treating various diseases.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. The experimental procedures, without specific conditions being noted in the following examples, are generally performed according to conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

Unless otherwise specified, materials and reagents used in examples of the present invention are commercially available products.

General procedure

Ethics of animals: the use and feeding of animals were in accordance with the guidelines of the ethical committee on biomedical research at the neuroscience research institute of the 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 astrocytes and qPCR: isolation and culture of astrocytes as described above¹. Briefly, astrocytes were seeded in 6-well plates. Lipo was used according to standard proceduresfectamine 3000(Thermo Fisher Scientific) was transiently transfected with 3. mu.g of a vector expressing gRNA-CasRx-GFP. Control plasmids express non-targeted guidance. 1-2 days after transient transfection, GFP positive cells were collected by flow fluorescent cell sorting (FACS) and lysed for qPCR analysis: RNA was first extracted using trizol (ambion) and then reverse transcribed into cDNA using a reverse transcription kit (HiScript Q RT SuperMix for qPCR, Vazyme, Biotech). Amplification was followed by AceQ qPCR SYBR Green Master Mix (Vazyme, Biotech). The Ptbp1 qPCR primers were: forward direction, 5'-AGAGGAGGCTGCCAACACTA-3' (SEQ ID NO. 13); reverse, 5'-GTCCAGGGTCACTGGGTAGA-3' (SEQ ID NO.: 14).

Stereotactic injection: AAV8 (fig. 1) and AAV-php. eb (fig. 2 and 3) were used in this study. Stereotactic injection (C57BL/6,1-3 months old) method as described previously². The AAV-CasRx-Ptbp1 titers in FIGS. 1 and 2,3 were approximately 5X 10e12 (2. mu.l per injection) and 1.6X 10e13 (2-3. mu.l per injection), respectively. AAV was injected into the striatum (AP +0.8mm, ML. + -. 1.6mm and DV-2.8 mm).

And (3) immunofluorescence staining: immunofluorescent staining was performed 5-6 weeks (FIG. 1) or 3-4 weeks (FIGS. 2 and 3) after injection. After perfusion, the brains were removed and fixed overnight with 4% Paraformaldehyde (PFA) and maintained in 30% sucrose for at least 12 hours. Sections were frozen after embedding and had a thickness of 35 μm. Brain sections were thoroughly washed with 0.1M Phosphate Buffer (PB) prior to immunofluorescent staining. A first antibody: rabbit polyclonal NeuN antibody (Brain, 1: 500, # ABN78, Millipore), rabbit polyclonal Tbr1 antibody (# NG1854874, Millipore), mouse TH antibody (1: 300, MAB318, Millipore), and rabbit DAT antibody (1: 100, MAB369, Millipore). Secondary antibody: donkey anti-mice (# 715-545-152, Jackson ImmunoResearch), donkey anti-rabbits (#711-545-152, Jackson ImmunoResearch) were used in this study. After antibody incubation, sections were washed and covered with a blocking tablet (Life Technology).

Electrophysiological recording: electrophysiological recordings were made 5-6 weeks after AAV injection, and the procedure was as described above³. Briefly, mice were anesthetized and heart perfused before brain was placed into carbon dioxide infused NMDG artificial cerebrospinal fluid (acss) [ NMDG acss (mM): NMDG 92, potassium chloride 2.5,sodium dihydrogen phosphate 1.25, sodium bicarbonate 30, HEPES 20, glucose 25, thiourea 2, sodium ascorbate 5) at room temperature, sodium pyruvate 3, calcium chloride 0.5, magnesium sulfate 10]. After perfusion, brains were extracted and placed in ice-cold NMDG aCSF solution for 30 seconds. The brains were trimmed and sectioned at a thickness of 250-350 μm at a speed of 0.04-0.05 mm/s. Brain sections were transferred to a dish filled with carbon dioxide NMDG aCSF and held at 32-34 ℃ for ≤ 12 minutes. Sections were transferred to new dishes of carbonated HEPES aCSF at room temperature [ HEPES, containing aCSF (mm): 92 parts of sodium chloride, 2.5 parts of potassium chloride, 1.25 parts of sodium dihydrogen phosphate, 30 parts of sodium bicarbonate, 20 parts of HEPES, 25 parts of glucose, 2 parts of thiourea, 5 parts of sodium ascorbate, 3 parts of sodium pyruvate, 2 parts of calcium chloride and 2 parts of magnesium sulfate]. After 1 hour the sections were transferred to a recording dish containing recording buffer [ record acsf (mm): 119 parts of sodium chloride, 2.5 parts of potassium chloride, 1.25 parts of sodium dihydrogen phosphate, 24 parts of sodium bicarbonate, 12.5 parts of glucose, 2 parts of calcium chloride and 2 parts of magnesium sulfate]. Neuronal-like mCherry positive cells were recorded under a microscope (Olympus BX51WI) and data were obtained using campex 10.

6-OHDA PD mouse model

The study procedure was based on previous studies⁴. Adult C57BL/6 mice were injected intraperitoneally (7-10 weeks). Desipramine hydrochloride (D3900, Sigma-Aldrich) 25mg/kg was injected half an hour prior to anesthesia. After anaesthesia, mice were injected with 3 μ g of 6-OHDA (H116, Sigma-Aldrich) or saline solution into the right medial forebrain tract according to the following coordinates: the anterior and posterior (A/P) positions are-1.2 mm, the medial and lateral (M/L) positions are-1.1 mm, and the dorsoventral (D/V) positions are-5 mm. All mice were injected subcutaneously with 1ml of 4% glucose-saline solution 1 hour after surgery. Mice received the soaked food particles 3 weeks later.

Apomorphine-induced spin test

Mice were intraperitoneally injected with 0.5mg/kg apomorphine (A4393, Sigma-Aldrich) 10 minutes prior to testing. Thereafter, they were each placed in an opaque cylinder (diameter 30cm) and recorded by a camera for 20 minutes above it. Rotation is defined as full body steering with one hind paw as the center and no switching of head orientation. The number of injected side and contralateral rotations was counted. Data were quantified as contralateral derotation over 20 minutes.

Cylinder test

Each mouse was gently placed into a glass beaker (1000ml) and recorded with a camera for 10 minutes in front of it. The number of wall touches by the injecting side and the contralateral paw were calculated separately and the data were quantified as the ratio of the number of wall touches by the same side to the total number of wall touches.

Rotating rod test

All mice were trained for 2 days and tested on day 3. On day 1, mice were trained 4 times on a rotating rod for 300 seconds each time at a fixed speed of 4 cycles/min. Mice were trained or tested 4 times on

days

2 and 3 at an accelerated rate of 4 to 40 cycles/min. The time that the mice stayed on the rod before shedding was recorded as the dwell period and the average of the 3 longest dwell periods was used for the analysis.

Statistical analysis: statistical significance (p) was calculated from s.e.m. error bars set by unpaired two-tailed t-test or one-way anova<0.05). All experiments were randomized, and the sample size was not predetermined using statistical methods, but our sample size was referenced in previous literature reports⁵. Data distribution was assumed to be normal but not formally verified. Data collection and analysis were not performed under blind experimental conditions.

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. mu.g of a vector expressing gRNA-CasRx-GFP. Control plasmids did not express grnas. Two days after transfection, approximately 50000 GFP positive cells were collected for each sample by Fluorescence Activated Cell Sorting (FACS) and lysed for qPCR analysis. Retinas were also isolated to determine AAV expression. RNA was extracted using trizol (ambion) and converted to cDNA using a reverse transcription kit (HiScript Q RT SuperMix for qPCR, Vazyme, Biotech). The amplification process was followed using AceQ qPCR SYBR Green Master Mix (Vazyme, Biotech).

The Ptbp1 qPCR primers were: 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 15-cm dishes and transiently transfected with 70. mu.g of plasmid. 500000GFP positive (first 20% GFP) N2a cells were collected by FACS, RNA extracted, converted to cDNA, and used for whole transcriptome RNA-seq.

Intravitreal and subretinal injections

NMDA and AAV (AAV-php. eb) were introduced by intravitreal and subretinal injections, respectively, as described previously. For subretinal injection, high titer(s) were injected into the eye under an Olympus microscope (Olympus, tokyo, japan) using a Hamilton syringe (32G needle)>1×10¹³) AAV. To determine reprogramming in intact retina, a total of 1 μ l of GFAP-GFP-Cre (0.2 μ l) and GFAP-CasRx-Ptbp1(0.8 μ l), or GFAP-Cre-GFP (0.2 μ l) and GFAP-CasRx (0.8 μ l) were delivered to the retina by subretinal injection (Ai9 and C57BL/6 mice, 5 weeks old). To determine reprogramming in damaged retinas, NMDA was dissolved in PBS to a concentration of 200mM, and then 1.5. mu.l of NMDA solution was injected by intravitreal injection into the eyes of either 4-8 week old Ai9 mice or 5-6 week old C57BL/6 mice (for VEP and black and white scenario preference tests). 2-3 weeks after NMDA injection, GFAP-GFP-Cre was co-delivered to the retina by subretinal injection with GFAP-CasRx-Ptbp1 or GFAP-CasRx. To assess functional rescue of damaged retinas (VEP and light and dark box shuttle experiments), mice 5-6 weeks old (C57BL/6) were injected with NMDA-induced retinal damage and 2-3 weeks after injection delivered either GFAP-mCherry (0.2. mu.l) and GFAP-CasRx-Ptbp1 (0.8. mu.l) or GFAP-CasRx (0.8. mu.l) mixtures.

Immunofluorescence staining

After 1 month of AAV injection, eyes, optic nerves and brain were taken, fixed with 4% Paraformaldehyde (PFA) for 2 hours (eyes and optic nerves) or 24 hours (brain), and then stored in 30% sucrose solution for 2 (eyes and optic nerves) or 24 (brain) hours. After embedding and freezing, the eyes and brain were sectioned at a thickness of 30 μm. For immunofluorescence staining oneResisting: mouse anti-Brn 3a (1: 100, MAB1585, Millipore), rabbit anti-RBPMS (1: 500,15187-1-AP, Proteintech), rabbit anti-Sox 9 (1: 500, AB5535, Millipore), rabbit anti-Pax 6 (1: 500,901301, Biolegent), rabbit anti-Prox 1 (1: 500, AB5475, Millipore), and secondary antibodies: cy is a Cy-^TM5 AffiniPicture Donkey mouse anti-IgG (H + L) (1: 500, 715-175; Jackson ImmunoResearch), Cy TM 5 AffiniPicture Donkey rabbit anti-IgG (H + L) (1: 500, 711-175; Jackson ImmunoResearch). After antibody application, the film is washed and mounted. Imaging was performed using an Olympus FV3000 microscope.

Electrophysiology

Mice were dark-adapted overnight before euthanasia. Retinal dissection was performed in 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 under infrared microscopy at room temperature. The RGCs of the retina were placed facing the cell-track on the stage of the upright microscope. tdTomato positive cells in the ganglion cell layer were identified using two-photon (λ -1030 nm) microscopy and recorded for cell attachment under infrared light. The pipettes used for recording (4-7 M.OMEGA.) were hydrated with ASCF and 0.25mM Alexa 488. Recordings were performed using multiclad 700A amplifier and pClamp10 software suite (Molecular Devices). The signal was low pass filtered at 1kHz and digitized at 10 kHz. Full field light stimulation was delivered with white LED light. After the recording was completed, a current pulse was injected to fill the cells to visualize their morphology.

Visual evoked potential

Mice were injected intraperitoneally with a mixture of fentanyl (0.05mg/kg), midazolam (5mg/kg) and medetomidine (0.5 mg/kg). The mouse head was mounted in a brain stereotaxic apparatus and its body temperature was maintained at 37 ℃ by a heating blanket. Craniotomies (about 1mm in diameter) were performed over both sides of the main visual cortex (V1) (AP-3.6 to-3.9 mm, ML 2.2mm) and the dura was removed. The visual stimulus was emitted from a 17 inch liquid crystal display (Dell P170S, maximum luminance 69cd/m2) that was 8 cm from the recording end eye while shielding the lateral side of the eye on the same side as the recording end from the visual stimulus. We performed 100 repeated flash stimuli for 2 seconds (all over)Field of view, 100% contrast), at intervals of 2 seconds. Recordings were made at V1(AP-3.6 to-3.9 mm, ML 2.2mm) using a multi-site silicon probe (A1X 16-5mm-50-177, neuroNexus Technologies) with each recording reaching a cortical depth of approximately 900 μm at the electrode tip. Both the reference and ground lines were placed in a small craniotomy at least 3 mm from the recording point. Neural responses were amplified and filtered using a Cerebus 32 channel system (Black microsystems). Local Field Potential (LFP) signals are sampled at 2kHz or 10kHz using a wideband front-end filter (0.3-500 Hz). The LFP response to full-screen flash stimulus is used in a Current Source Density (CSD) analysis to determine the cortical layer 4³The position of (a). To generate the CSD profile, we calculated the second spatial derivative of the LFP using the following equation:

wherein

Is LFP, z is the coordinate of the recording end, Δ z is the distance between adjacent recording ends, and n Δ z is the differentiation grid (n ═ 2). Layer 4 (granular layer) is defined as those recording positions at the initial current receptor. We used the layer 4 channel showing the largest mean amplitude to analyze the visually evoked response of each mouse.

Black and white box preference test

The apparatus for the light and dark box shuttle experiment included a box with a door that was divided into a small (one-third) dark box portion and a large (two-thirds) illumination portion (550 lumens). The mouse was free to move between the two compartments for 10 minutes. The time spent by the mouse in each compartment was recorded by the camera and then analyzed using Ethovision XT. After each test, the compartment was washed with 70% ethanol to avoid olfactory cues.

Statistical analysis

All values are shown as mean ± s.e.m. Unpaired t-test was used to determine statistical significance (p < 0.05). All experiments were randomized and the sample size was not predicted using statistical methods, but our sample sizes were similar to those reported in previous publications. Data distribution was assumed to be normal but not formally tested.

Example 1 specific knock-down of Ptbp1 in vitro Using CasRx

To determine the efficiency of CasRx-mediated Ptbp1 knockdown, six grnas targeting Ptbp1 were first designed and their inhibitory efficiency was compared in cultured N2a cells and astrocytes (fig. 1A and 1B). Co-transfection of two grnas targeting Ptbp1 exon IV and VII regions (5 and 6 combinations) was found to achieve 87% ± 0.4% and 76% ± 4% reductions in N2a and astrocytes, respectively (fig. 1C and 1D). Data from RNA whole transcriptome sequencing further showed that this knockdown was very specific (fig. 1E, 1F and 2A).

Example 2 conversion of Mueller glia cells to Optic ganglion cells in mature retina

Previous studies found that cultured mouse fibroblasts and N2a cells could be transformed into functional neurons by shRNA knockdown of Ptbp1, followed by studies of whether Ptbp1 knockdown would convert muller glia into optic ganglion cells in vivo in mature retinas. To specifically and permanently label retinal mueller glial cells, we injected AAV-GFAP-GFP-Cre into eye Ai9 mice (Rosa-CAG-LSL-tdTomato-WPRE) specifically induced the expression of tdTomato in mueller glial cells (fig. 2B and 2C). We also constructed AAV-GFAP-CasRx-Ptbp1(gRNA 5+6) driven by the muller glial cell-specific promoter GFAP, hopefully specifically knock down Ptbp1 in muller glial cells and we simultaneously constructed control virus AAV-GFAP-CasRx that did not target Ptbp1 (fig. 3A and 2D). We first co-injected AAV-GFAP-CasRx-Ptbp1 and AAV-GFAP-Cre-GFP subretinally in Ai9 mice aged approximately 5 weeks, and after 1 month we found that many tdTomato-positive cells co-labeled with the antibody Brn3a or Rbpms specific for optic ganglion cells in the retinal Ganglion Cell Layer (GCL), but we did not find such cells in retinas injected with control AAV vectors (fig. 3B-3E). These results indicate that knocking down Ptbp1 in the mature retina can transform muller glia cells into visual ganglion cells. Notably, the transformed visual ganglion cell cells were no longer muller glial cells, so expression of GFP driven by GFAP was also reduced in the induced visual ganglion cells (fig. 2E). We also demonstrated this presence by other methods (fig. 4). In addition to the optic ganglion cells, we also found amacrine nerve cells (FIG. 5).

Example 3 conversion of MG to RGC in mouse model of retinal damage

To investigate whether MG-induced RGCs could replenish lost RGCs in a mouse model of retinal damage, we intravitreally injected NMDA (200mM) into Ai9 mice (NMDA, 200mM)4 to 8 weeks old, resulting in death of most RGCs and a reduction in Internal Plexiform Layer (IPL) thickness. Two to three weeks after NMDA injection, eyes were injected with AAV-GFAP-CasRx-Ptbp1 plus AAV-GFAP-GFP-Cre or control AAV virus (FIGS. 6A and 6B). One month after AAV injection, we found that the number of Brn3a or Rbpms positive cells in the ganglion cell layer was significantly increased in retinas injected with AAV-GFAP-CasRx-Ptbp 1. Interestingly, most of these cells were tdTomato positive. While we did not find an increase in the ganglion cell layer of the optic nerve injected with control AAV (FIGS. 6C-6F). To determine whether MG-induced RGCs integrate into the retinal circuit and have the ability to receive visual information, we performed extracellular recordings of MG-induced RGCs under a two-photon microscope to monitor the photostimulation-induced responses (fig. 6G). We found that of the 8 cells examined, 6 cells showed light-stimulus-induced action potentials (fig. 6H). Of these cells, five were ON cells and one was OFF cells (fig. 6H). These results indicate that functional RGCs can be transformed by MG by reducing expression of Ptbp1 in muller cells in damaged retinas.

Example 4 partial restoration of visual function by induced optic ganglion cells

In the visual system, visual information is projected through RGCs to the dorsolateral geniculate nucleus and superior colliculus of the brain (fig. 8A). In permanently injured retinas, we observed a large number of tdTomato-positive axons in the retinas and optic nerves by treatment, but no such axons were seen in the control group (fig. 8B and 8C). Notably, we also found a large number of tdTomato positive axons in the dorsal geniculate nucleus and superior colliculus and distributed much more abundantly in the contralateral side of the brain than in the ipsilateral side (fig. 8D and 8E), consistent with the correct projection of RGCs on normal mouse granules. To investigate whether impaired visual function could be improved by induced RGCs. After 1 month of AAV virus injection, we recorded Visual Evoked Potentials (VEPs) in the primary visual cortex (V1) of anesthetized mice (fig. 8F). In mice with retinal damage treated with AAV-GFPA-CasRx-Ptbp1 and AAV-GFAP-mCherry, we found very significant evoked potentials with amplitudes similar to those found in uninjured mice. In the control group, we observed only very weak responses (fig. 8G and 8H). These results support the induction of optic nerve cells, presumably by establishing synaptic connections with the functional dorsal geniculate nuclear neurons present in the brain, thereby partially restoring the transmission of visual information to the primary visual cortex. From a behavioral perspective, we investigated whether CasRx-mediated conversion of MG to RGC could restore visual performance lost due to retinal damage (fig. 8I). We injected NMDA in both eyes of mice first, followed by AAV virus. In the light/dark preference test, untreated mice had no apparent tendency to remain in the dark, consistent with vision loss due to retinal damage. In contrast, the duration of time the treated mice were left in the dark room was significantly increased, even close to the level of healthy control mice (fig. 8J), suggesting that the induced optic nerve cells improved the visual behavior of the vision impaired mice.

Example 5 projection of optic ganglion cells into the brain

To explore at what time points the muller glial-induced visual ganglion cells could begin to be seen, we set five different time points (1 week, 1.5 weeks, 2 weeks, 3 weeks, and 1 month). We found that tdTomato + Rbpms + and tdTomato + Brn3a + cells were first found in the retina at 1.5 weeks after AAV injection, and the number of these cells gradually increased with time (fig. 7A and 7B). Our results also indicate that there is an intermediate stage of induced RGC migration from INL to the optic nerve cell layer at 1.5 weeks after AAV injection (figure 7C). These findings were also confirmed in NMDA-induced permanently damaged retinas (fig. 7D). For when to start projection to the correct brain region, we first found that tdTomato positive axons in the optic nerve increased significantly over time (FIG. 9A). In the brain, we did not find any projections in the brain at 1 week, while we began to find tdTomato positive axons in the contralateral dorsal geniculate nucleus at 1.5 weeks of AAV injection, which did not occur ipsilaterally (fig. 9B). At2 weeks of AAV injection, we began to find projections of induced visual ganglion cells in the contralateral hypothalamus (fig. 5C), and at 3 weeks of AAV injection we found projections of induced visual ganglion cells in both the contralateral and ipsilateral dorsal geniculate nucleus and superior colliculus, and the density of such projections increased further at 1 month (fig. 9B-9D). Similar results were also observed in injured retinas injected with NMDA (fig. 10A-10C).

Example 6 transformation of astrocytes into neurons

To further explore the potential use of CasRx-induced glial-neuronal transformation in other systems, we further investigated whether knocking down Ptbp1 expression in astrocytes in the striatum could convert astrocytes locally into dopamine neurons, a cell type closely associated with the development of Parkinson's Disease (PD). We first injected AAV-GFAP-mCherry into the striatum of wild type mice to label astrocytes, along with AAV-GFAP-CasRx-Ptbp1 (gRNA: 5+6) to downregulate the expression of Ptbp1 in astrocytes (FIGS. 11A and 11B). As a control, we used AAV-GFAP-CasRx that did not contain Ptbp1 gRNA. We found that both mCherry and CasRx are abundantly expressed in astrocytes and show higher coinfection efficiency in the striatum, with 99% + -1% of mChery + cells expressing CasRx (82% + -2% of GFAP)⁺Cells expressed mCherry, while 95% ± 1% of mCherry + cells expressed GFAP) (fig. 11C and 11D). After 1 week of AAV injection into the striatum, we found down-regulation of Ptbp1 expression in astrocytes (fig. 11E and 11F). After one month injection of AAV virus, we found a high proportionmCherry + cells expressed the mature neuronal marker NeuN (48% ± 10%, SEM, n ═ 6 mice), but such expression was not found in control streaks injected with AAV-GFAP-mCherry and AAV-GFAP-CasRx (0.97% ± 0.45%, SEM, n ═ 6) (fig. 11G and 11H). To further explore the specific types of these transformed neurons, we immunostained using antibodies specific for neurons. We found that approximately 50% of the induced neurons expressed glutaminase (fig. 11I and 11J), a marker for excitatory glutamatergic neurons, while we found that a very small fraction of the induced neurons expressed one interneuron subtype marker SST and no cells expressed another interneuron subtype marker PV (fig. 11K and 11L). We also used co-staining of dopamine neuron labeled Tyrosine Hydroxylase (TH) with NeuN to determine if there were dopamine neurons in transformed neurons, and we found that a fraction (7.5% ± 3%, SEM) of mCherry + cells expressed TH (fig. 11M-11O). Furthermore, we found that mCherry expression in transformed neurons persisted after at least 1 month of infection (fig. 11G), a phenomenon consistent with previous reports.

Example 7 Induction of dopamine neurons

To explore the potential clinical application of CasRx-mediated neuronal transformation in the striatum, we induced the parkinsonian mouse model by unilateral infusion of 6-hydroxydopamine (6-OHDA) into the substantia nigra. This infusion resulted in the death of dopamine neurons in the substantia nigra and the degeneration of dopaminergic projections in the ipsilateral striatum (fig. 13A-13C). Three weeks after injection of 6-OHDA, AAV-GFAP-CasRx-Ptbp1 (or AAV-GFAP-CasRx as a control) was co-injected with AAV-GFAP-mCherry into the ipsilateral striatum. Striatal cell transformation was analyzed at different time points simultaneously (fig. 12A and 12B). One month after virus injection, we found that a high proportion of mCherry cells expressing the marker TH of dopamine neurons appeared in the striatum of AAV-GFAP-CasRx-Ptbp1 and AAV-GFAP-mCherry in 6-OHDA induced injured mice, and that this percentage increased at 3 months of AAV injection (19% ± 0.4%, SEM, n ═ 5 mice, one month; 32% ± 7%, SEM, n ═ 3 mice3 months) (fig. 12C, 12D, 13D and 13E). In contrast, in the control group, such cells were rarely observed (FIGS. 12C, 12D and 13D-13F). In addition, about 80% of TH in the virus injection region⁺The cells were mCherry + (fig. 12E and 13G), indicating that these dopamine cells were transformed mainly from astrocytes. We note that the percentage of mCherry + TH + cells in wild type (no 6-OHDA lesion) mice was lower than in 6-OHDA lesion mice (fig. 13H), suggesting that repair mechanisms following injury may promote induction of TH + neurons. We also found that induced dopamine neurons express the mature dopamine neuron marker dopamine transporter Slc6a3(DAT), which is not present in striatal neurons transiently expressing TH. We found that a high proportion of mCherry positive cells expressed DAT (10% ± 3%, SEM, n ═ 5,1 month; 31% ± 7% of mCherry + DAT + cells in the striatum injected with AAV-GFAP-CasRx-Ptbp1, n ═ 3 mice at 3 months), whereas the presence of such cells was barely found in the striatum injected with control group virus (fig. 12C, 12F, 13D and 13I). Co-immunostaining for TH and DAT further showed that most mCherry + TH + cells expressed DAT (fig. 12C, 12G and 13D), suggesting that most induced dopamine neurons expressed mature dopaminergic markers. In addition, mCherry + cells expressed two additional midbrain dopamine neuron markers, Dopa Decarboxylase (DDC) and forkhead box protein a2 (FOXA2) (fig. 13J-13M), further validating transformed dopaminergic neurons at neuronal sites. Previous studies have indicated that TH is expressed in some interneurons transiently in the striatum of mice following 6-OHDA injury. Here we evaluated this, and we co-stained the markers PV +, SST + and calretinin + (CR +) of the interneurons 3 months after AAV injection. We found that none of these interneuron markers co-localized with mCherry + TH + cells, suggesting that transformed TH + neurons were not transiently induced TH + neurons (fig. 13N). To explore dopamine neuron-inducing subtypes, we co-stained TH with two substantia nigra A9 domain-specific dopaminergic neuron markers ALDH1A1 and GIRK2, and calcium binding protein (Calbinin), a Ventral Tegmental Area (VTA) dopamine neuron-specific marker, respectivelyDAT. Our results show that almost all induced neurons expressed ALDH1a1 and GIRK2, but not calbindin (fig. 12H-12J), suggesting that induced dopamine neurons are very similar to those of the substantia nigra. Next two we made whole cell recordings of electrophysiology on brain slices. We found that most neuron-like mCherry positive cells (20 out of 22) were able to induce action potentials in response to depolarizing currents in current clamp mode (fig. 12K). We also observed spontaneous post-synaptic currents in voltage-clamp mode (Vc ═ 70mV), indicating that transformed neurons were able to accept functional synaptic inputs (fig. 12L and 12M). Furthermore, in 4 out of 10 neurons examined, we observed a delayed voltage rectification induced by hyperpolarizing activation current (Ih) (fig. 12K), which is also one of the hallmarks of mesencephalic dopamine neurons. Next to determine whether induced dopamine neurons could release dopamine, we performed immunostaining and found that most induced dopamine cells expressed vesicular monoamine transporter 2(VMAT2) (fig. 12N), a protein that regulates dopamine packaging, storage and release. We also found that many cells in the striatum region of virus injection absorbed fluorescent dopamine derivative (FFN206), a substrate of VMAT2, and could determine whether the expressed VMAT2 was functional. Under high potassium chloride treatment, we found a reduction in the fraction of fluorescence, indicating that transformed cells have dopamine releasing function (FIG. 12O-12Q). Our results indicate that CasRx-mediated Ptbp1 knockdown can efficiently induce dopaminergic neurons in the striatum of PD model mice.

Example 8 improvement of motor function in Parkinson's disease model mice

We further examined whether dopamine neurons induced in the striatum could alleviate motor symptoms in a 6-OHDA-induced parkinson disease mouse model (fig. 14A). We performed simultaneous drug and drug-free induction assessment of motor function. For drug-induced activity, we first studied apomorphine-induced contralateral rotation behavior, which is widely used to test behavioral changes resulting from unilateral dopamine neuron loss. We found that the apomorphine-induced net spin numbers (calculated as ipsilateral-contralateral shear spin numbers) were significantly reduced in Ptbp1 knock-down mice (AAV-GFAP-CasRx-Ptbp1 and AAV-GFAP-mCherry injected) compared to control mice (AAV-GFAP-CasRx and AAV-GFAP-mChery or saline injected) at levels comparable to uninjured healthy mice (FIGS. 14B, 13O and 13P). In another ipsilateral rotational behavior test induced by systemic amphetamine administration, intercellular dopamine concentrations were increased by inhibiting dopamine uptake by DAT in the striatum. The net spin (as ipsilateral-contralateral spin) and ipsilateral spin ratio (as ipsilateral/total spin) were significantly reduced in mice injected with AAV-GFAP-CasRx-Ptbp1 compared to control mice (fig. 14C, 14D and 13Q). These results indicate that neurons induced in the striatum can release enough dopamine to alleviate drug-induced motor dysfunction in PD model mice. In addition, we tested whether the two drug-free motor functions improved separately, namely forelimb use asymmetry and motor coordination. We found that the percentage of ipsilateral touches was significantly reduced in mice injected with AAV-GFAP-CasRx-Ptbp1 and for longer duration on the rotating horse compared to control mice (FIGS. 14E and 14F). Taken together, these results indicate that astrocyte Ptbp1 knockdown-mediated dopamine neuron conversion in the striatum reduces motor dysfunction in parkinson disease model mice.

Discussion of the related Art

In this work, our studies indicate that glial cells can be efficiently converted to neurons through CasRx-mediated down-regulation of Ptbp 1. In the NMDA-induced retinal injury model, Ptbp1 knockdown can induce conversion of MG to RGC in injured retinas, partially restoring visual response and vision-dependent behavior. In a mouse model of 6-OHDA-induced Parkinson's disease, it can induce astrocytes in the striatum into dopamine neurons and alleviate motor dysfunction associated with loss of dopamine neurons in the substantia nigra. These results indicate that down-regulation of the expression of the single RNA binding protein Ptbp1 is sufficient to transform glial cells into specific types of neurons that are lost in different nervous systems, providing a new approach for future therapeutic applications. Short hairpin RNA (shRNA) technology is capable of cleaving or inhibiting the desired transcript, but it has a very severe off-target effect. Cas 13-mediated knockdown is not only more efficient than RNAi, but also can reduce off-target effects to a large extent, with greater potential in therapeutic applications. Furthermore, CasRx is the smallest one in the Cas13 protein family, and can be packaged with CRISPR arrays (encoding multiple guide RNAs) in one AAV. In the field of gene editing, the RNA-targeting CRISPR system cassx can avoid the risk of permanent DNA changes caused by the DNA-targeting CRISPR-Cas9 editing system, making it more safe in clinical applications.

Reference documents:

1.Mccarthy,K.D.&Devellis,J.Preparation Of Separate Astroglial And Oligodendroglial Cell-Cultures From Rat Cerebral Tissue.Journal Of Cell Biology 85,890-902(1980).

2.Zhou,H.et al.Cerebellar modules operate at different frequencies. Elife 3,e02536(2014).

3.Xu,H.T.et al.Distinct lineage-dependent structural and functional organization of the hippocampus.Cell 157,1552-1564(2014).

4.Su,J.et al.Reduction of HIP2 expression causes motor function impairment and increased vulnerability to dopaminergic degeneration in Parkinson's disease models.Cell Death Dis 9,1020(2018).

5.Chavez,A.et al.Comparison of Cas9 activators in multiple species. Nat Methods 13,563-567(2016).

all documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it will be appreciated that various changes or modifications may be made by those skilled in the art after reading the above teachings of the invention, and such equivalents will fall within the scope of the invention as defined in the appended claims.

Sequence listing

<110> China academy of sciences brain science and intelligent technology prominent innovation center

Application of <120> Ptbp1 inhibitor in preventing and/or treating nervous system diseases related to functional neuron death

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<151> 2019-08-16

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<151> 2019-10-30

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<151> 2020-03-26

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Claims

1. Use of an inhibitor of the Ptbp1 gene or RNA or the protein encoded thereby for the preparation of a composition or a formulation for the prevention and/or treatment of a neurological disease associated with functional neuronal death.

2. The use of claim 1, wherein the composition or formulation is further for one or more uses selected from the group consisting of:

(a1) inducing the astrocytes to transdifferentiate into dopamine neurons;

(b1) treating dyskinesia in parkinson's disease in mammals;

(e1) preventing or treating retinal diseases;

(f1) preventing and/or treating neurodegenerative diseases;

(g1) transdifferentiation of muller glial cells to optic ganglion cells;

3. Use according to claim 2, wherein the astrocytes are derived from the striatum, substantia nigra, spinal cord, dorsal mesencephalon or cerebral cortex, preferably the astrocytes are derived from the striatum.

4. The use of claim 1, wherein the functional neuron is selected from the group consisting of: RGC neurons, dopamine neurons, or a combination thereof.

5. The use of claim 1, wherein the inhibitor is selected from the group consisting of: antibodies, small molecule compounds, microRNAs, siRNAs, shRNAs, antisense oligonucleotides, aptamers, gene editors, or combinations thereof.

6. A composition, comprising:

(a) a gene-editing protein or an expression vector thereof, said gene-editing protein selected from the group consisting of: CasRx, CRISPR/Cas9, Cpf1, Cas9, Cas13a, Cas13b, Cas13c, RNA-targeted gene-editing proteins, or a combination thereof; and

(b) a gRNA, or an expression vector thereof, which is a DNA or RNA that directs a gene editing protein to specifically bind to the Ptbp1 gene.

7. A kit, comprising:

(a1) a first container, and a gene-editing protein or an expression vector thereof, or a medicament containing a gene-editing protein or an expression vector thereof, located in the first container, the gene-editing protein being selected from the group consisting of: CasRx, CRISPR/Cas9, Cpf1, Cas9, Cas13a, Cas13b, Cas13c, RNA-targeted gene-editing proteins, or a combination thereof;

8. Use of a composition according to claim 6 or a kit according to claim 7 for the preparation of a medicament for the prevention and/or treatment of a neurological disease associated with functional neuronal death.

9. A method of promoting the differentiation of glial cells into functional neurons comprising the steps of:

culturing glial cells in the presence of an inhibitor of Ptbp1 gene or RNA or its encoded protein, or a composition according to claim 6, thereby promoting differentiation of glial cells into functional neurons.

10. A method of screening for a candidate compound for the prevention and/or treatment of a neurological condition associated with functional neuronal death, said method comprising the steps of:

(a) in the test group, a test compound was added to a culture system of cells, and the expression amount (E1) and/or activity (a1) of Ptbp1 in the cells of the test group was observed; in a control group, a test compound not targeting Ptbp1 is added to a culture system of the same cells, and the expression amount (E0) and/or activity (A0) of Ptbp1 in the cells of the control group is observed;