WO2021031810A1 - Application d'un inhibiteur de ptbp1 dans la prévention et/ou le traitement d'une maladie du système nerveux liée à la mort neuronale fonctionnelle - Google Patents

Application d'un inhibiteur de ptbp1 dans la prévention et/ou le traitement d'une maladie du système nerveux liée à la mort neuronale fonctionnelle Download PDF

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WO2021031810A1
WO2021031810A1 PCT/CN2020/105285 CN2020105285W WO2021031810A1 WO 2021031810 A1 WO2021031810 A1 WO 2021031810A1 CN 2020105285 W CN2020105285 W CN 2020105285W WO 2021031810 A1 WO2021031810 A1 WO 2021031810A1
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cells
ptbp1
gene
aav
another preferred
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杨辉
周海波
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中国科学院脑科学与智能技术卓越创新中心
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    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the invention relates to the field of biomedicine. More specifically, the present invention relates to the use of Ptbp1 inhibitors in the prevention and/or treatment of neurological diseases related to functional neuronal death.
  • Parkinson's disease is a serious neurodegenerative disease characterized by the loss of dopamine neurons in the substantia nigra of the midbrain.
  • Previous studies have achieved the direct reprogramming of astrocytes into dopamine neurons in vitro and in animal models by simultaneously overexpressing several transcription factors.
  • Ptbp1 mediated neuronal reprogramming in vivo has not been reported yet.
  • the main treatment for Parkinson's disease is drugs represented by levodopa preparations.
  • surgical treatment can also improve symptoms to a certain extent. It should be pointed out that all these methods can only partially alleviate the disease, but cannot achieve the effect of preventing the development of the disease.
  • the purpose of the present invention is to provide a target that can effectively treat neurodegenerative diseases.
  • Another purpose of the present invention is to provide a new target Ptbp1 for the treatment of Parkinson's disease.
  • Ptbp1 By inhibiting the expression of Ptbp1, astrocytes in the striatum can be directly converted into dopamine neurons and the phenotype of Parkinson's disease can be restored. .
  • Another objective of the present invention is to provide a new target Ptbp1 for the treatment of visual impairment.
  • Ptbp1 By inhibiting the expression of Ptbp1, the Muller glial cells in the retina can be directly transformed into optic ganglion cells and the phenotype of permanent visual impairment can be alleviated. .
  • an inhibitor of Ptbp1 gene or RNA or its encoded protein for the preparation of a composition or preparation for the treatment of functional neuronal death-related nerves System diseases.
  • composition or preparation is also used for one or more purposes selected from the following group:
  • the neurological disease is selected from the group consisting of glaucoma, age-related RGC loss, optic nerve damage, retinal ischemia, Leber hereditary optic neuropathy, Alzheimer's disease, Huntington's disease, mental Schizophrenia, depression, drug use, movement disorders (such as chorea, hypercholesterolemia and movement disorders), motor neuron injury diseases (such as amyotrophic lateral sclerosis, spinal cord injury), bipolar disorder, autism spectrum disorder (ASD), dysfunction, Parkinson's disease, or a combination thereof.
  • the glial cells are selected from the group consisting of astrocytes, MG cells, oligodendrocytes, ependymal cells, Schwan cells, NG2 cells, satellite cells, or combinations thereof.
  • the functional neuron is selected from the following group: RGC neuron, dopamine neuron, or a combination thereof.
  • the functional neurons are derived from the striatum.
  • the functional neurons are derived from mature retina.
  • the retinal disease is a retinal disease caused by neurodegeneration.
  • composition or preparation can treat retinal diseases caused by neurodegeneration by inducing MG cells to transdifferentiate into RGC cells.
  • the MG cells are Müller glial cells (Muller glial cells).
  • the MG cells are derived from the retina.
  • the RGC cells are retinal ganglion cells.
  • the RGC cells are functional RGCs.
  • the RGC cells can be integrated into the visual pathway and improve visual function.
  • the RGC cells can realize functional projection to the central visual area and improve visual function.
  • the improvement of visual function is to improve the visual function of mammals suffering from retinal diseases caused by neurodegeneration.
  • the MG cells are transdifferentiated into RGC cells while also being differentiated into axonal cells.
  • RGC (1) expresses Brn3a, Rbpms, Foxp2, Brn3c and/or paralbumin; (2) is F-RGC, type 3 RGC or PV-RGC; (3) is integrated in the Existing retinal pathways in the subject (for example, the central information can be projected to dLGN, and the vision can be partially restored by relaying the visual information to V1); and/or (4) the visual information can be received. It is characterized by the ability to establish action potentials in light stimulation, synaptic connections (for example with existing functional dLGN neurons in the brain), pre-synaptic neurotransmitter biogenesis and/or subsequent action.
  • the Muller glial cells in the mature retina are reprogrammed and converted into RGC.
  • dopamine neurons (1) express tyrosine hydroxylase (TH), dopamine transporter (DAT), vesicular monoamine transporter 2 (VMAT2), hybrid homology box 1 (Enl) , FoxA2 and/or LIM homeobox transcription factor 1 alpha (Lmxla); (2) exhibiting the biogenesis of presynaptic neurotransmitters; (3) being integrated into the existing nerves in the subject’s brain And/or (4) is characterized by its ability to establish action potentials, synaptic connections, pre-synaptic neurotransmitter biogenesis and/or post-synaptic reactions.
  • TH tyrosine hydroxylase
  • DAT dopamine transporter
  • VMAT2 vesicular monoamine transporter 2
  • Enl hybrid homology box 1
  • FoxA2 FoxA2
  • LIM homeobox transcription factor 1 alpha Lmxla
  • multiple glial cells in the striatum are reprogrammed, and at least 10% or at least 30% of the glial cells are converted into dopamine neurons.
  • the mammal includes a mammal suffering from a neurodegenerative disease.
  • the mammal includes a human or non-human mammal.
  • the non-human mammal includes rodents (such as mice, rats, or rabbits) and primates (such as monkeys).
  • the astrocytes are derived from the striatum, substantia nigra, spinal cord, dorsal midbrain or cerebral cortex, preferably, the astrocytes are derived from the striatum .
  • the astrocytes include striatal astrocytes.
  • the astrocytes are astrocytes of brain tissue.
  • the inhibitor is selected from the group consisting of antibodies, small molecule compounds, microRNA, siRNA, shRNA, antisense oligonucleotides, nucleic acid aptamers, gene editors, or combinations thereof.
  • the gene editor includes a DNA gene editor and an RNA gene editor.
  • the gene editor includes optional gRNA and gene editing protein.
  • the gene editor is expressed by a glial cell-specific promoter (for example, GFAP promoter).
  • a glial cell-specific promoter for example, GFAP promoter
  • the gene editor includes two or more gNRA and gene editing proteins.
  • the gRNA is RNA that guides the gene editing protein to specifically bind to the Ptbp1 gene.
  • the gRNA guide gene editing protein specifically binds to the mRNA of the Ptbp1 gene.
  • 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 protein, or a combination thereof.
  • the gene editing protein is CasRx
  • the nucleotide sequence of gRNA is selected from the group consisting of SEQ ID NO.: 1, 2, 3, 4, 5, and 6.
  • the source of the gene editing protein is selected from the group consisting of Streptococcus pyogenes, Staphylococcus aureus, Acidaminococcus sp, Lachnospiraceae bacterium ), Ruminococcus Flavefaciens, or a combination thereof.
  • the Ptbp1 is derived from mammals; preferably, it is derived from humans, mice, rats, or rabbits; more preferably, it is derived from humans.
  • the Ptbp1 gene includes wild-type Ptbp1 gene and mutant Ptbp1 gene.
  • the mutant type includes a mutant form in which the function of the encoded protein is not changed after mutation (that is, the function is the same or substantially the same as that of the wild-type encoded protein).
  • polypeptide encoded by the mutant Ptbp1 gene is the same or substantially the same as the polypeptide encoded by the wild Ptbp1 gene.
  • the mutant Ptbp1 gene includes homology of ⁇ 80% (preferably ⁇ 90%, more preferably ⁇ 95%, more preferably ⁇ 98% compared with the wild Ptbp1 gene) Or 99%) polynucleotides.
  • mutant Ptbp1 gene is included in the 5'end and/or 3'end of the wild-type Ptbp1 gene, truncated or added 1-60 (preferably 1-30, more preferably 1 -10) nucleotide polynucleotides.
  • the Ptbp1 gene includes a cDNA sequence, a genome sequence, or a combination thereof.
  • the Ptbp1 protein includes active fragments of Ptbp1 or derivatives thereof.
  • the homology of the active fragment or its derivative with Ptbp1 is at least 90%, preferably 95%, more preferably 98%, 99%.
  • the active fragment or derivative thereof has at least 80%, 85%, 90%, 95%, 100% of Ptbp1 activity.
  • amino acid sequence of the Ptbp1 protein is selected from the following group:
  • amino acid sequence shown in SEQ ID NO.: 11 is formed by the substitution, deletion or addition of one or a few (such as 1-10) amino acid residues, and those with the protein function are formed by ( i) Derived polypeptide; or
  • the homology between the amino acid sequence and the amino acid sequence shown in SEQ ID NO.: 11 is ⁇ 90% (preferably ⁇ 95%, more preferably ⁇ 98% or 99%), and a polypeptide having the protein function.
  • nucleotide sequence of the Ptbp1 gene is selected from the following group:
  • the ptbp1 protein is shown in SEQ ID NO.: 11.
  • nucleic acid encoding the ptbp1 protein is shown in SEQ ID NO.: 12.
  • the region targeted by the ptbp1 gene or the inhibitor of the encoded protein is the 4758-4787 and/or 5381-5410 positions of the ptbp1 gene sequence.
  • the inhibitor of the ptbp1 gene or its encoded protein inhibits the activity and/or expression of ptbp1.
  • the inhibitory rate of the ptbp1 gene or its encoded protein inhibitor on the activity and/or expression of ptbp1 is greater than 90%, preferably, 90%-95%.
  • the inhibitor targets astrocytes in brain tissue.
  • the inhibitor targets MG cells of the retina.
  • the neurodegenerative disease includes Parkinson's disease.
  • the second aspect of the present invention provides a composition comprising:
  • a gene editing protein or an expression vector thereof is selected from the group consisting of CasRx, CRISPR/Cas9, Cpf1, Cas9, Cas13a, Cas13b, Cas13c, RNA targeted gene editing protein, or a combination thereof;
  • the gRNA is DNA or RNA that guides the gene editing protein to specifically bind to the Ptbp1 gene.
  • the gRNA guide gene editing protein specifically binds to the mRNA of the Ptbp1 gene.
  • nucleotide sequence of the gRNA is selected from the following group: SEQ ID NO.: 1, 2, 3, 4, 5, and 6.
  • the composition includes a pharmaceutical composition.
  • composition further includes:
  • composition further includes:
  • composition further includes:
  • the expression vector of the gene editing protein includes a vector targeting glial cells.
  • the expression vector of the gene editing protein includes a vector targeting astrocytes of brain tissue.
  • the expression vector of the gene editing protein includes a vector targeting retinal MG cells.
  • the expression vector includes a viral vector.
  • the viral vector is selected from the following group: adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, herpes virus, SV40, poxvirus, or a combination thereof.
  • AAV adeno-associated virus
  • adenovirus adenovirus
  • lentivirus lentivirus
  • retrovirus lentivirus
  • herpes virus SV40
  • poxvirus poxvirus
  • the vector is selected from the following group: lentivirus, adenovirus, adeno-associated virus (AAV), or a combination thereof, preferably, the vector is adeno-associated virus (AAV).
  • the carrier includes AAV2 or AAV9.
  • the dosage form of the composition is selected from the group consisting of a lyophilized preparation, a liquid preparation, or a combination thereof.
  • the dosage form of the composition is a liquid preparation.
  • the dosage form of the composition is an injection dosage form.
  • other drugs for preventing and/or treating neurodegenerative diseases are selected from the following group: dopamine prodrugs, non-ergot dopamine receptor agonists, monoamine oxidase B inhibitors, or combinations thereof.
  • the composition is a cell preparation.
  • the expression vector of the gene editing protein and the expression vector of gRNA are the same vector or different vectors.
  • 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: 1-0.5:1.
  • the content of the component (a) in the composition is 0.001%-99%, preferably, 0.1%-90%, more preferably, 1%-70%.
  • the content of the component (b) is 0.001%-99%, preferably, 0.1%-90%, more preferably, 1%-70%.
  • the content of the component (c) in the composition is 1%-99%, preferably, 10%-90%, more preferably, 30%-70%.
  • the component (a), component (b) and optional component (c) account for 0.01-99.99 wt% of the total weight of the composition, which is greater than Preferably 0.1-90wt%, more preferably 1-80wt%.
  • the third aspect of the present invention provides a medicine kit including:
  • the second container, and the gRNA or its expression vector, or a drug containing the gRNA or its expression vector, in the second container, the gRNA is DNA or RNA that guides the gene editing protein to specifically bind to the Ptbp1 gene.
  • the gRNA guide gene editing protein specifically binds to the mRNA of the Ptbp1 gene.
  • nucleotide sequence of the gRNA is selected from the following group: SEQ ID NO.: 1, 2, 3, 4, 5, and 6.
  • the region targeted by the gRNA is positions 4758-4787 and/or positions 5381-5410 of the Ptbp1 gene sequence.
  • the kit further includes:
  • the third container, and other drugs for preventing and/or treating neurodegenerative diseases, and/or containing other drugs for preventing and/or treating retinal diseases, and/or containing other drugs in the third container Drugs for neurological diseases related to functional neuronal death.
  • first container, the second container, and the third container are the same or different containers.
  • the medicine in the first container is a unilateral preparation containing gene editing protein or its expression vector.
  • the medicine in the second container is a unilateral preparation containing gRNA or its expression vector.
  • the medicine in the third container is a unilateral preparation containing other medicines intended to treat neurological diseases related to functional neuron death.
  • the dosage form of the drug is selected from the group consisting of a lyophilized preparation, a liquid preparation, or a combination thereof.
  • the dosage form of the drug is an oral dosage form or an injection dosage form.
  • the kit also contains instructions.
  • the fourth aspect of the present invention provides a composition according to the second aspect of the present invention or the use of the kit according to the third aspect of the present invention to prepare a medicine for the treatment of neurological diseases related to functional neuron death .
  • the concentration (viral titer) of the other drugs for treating neurological diseases related to functional neuron death is> 1 ⁇ 10 13 , preferably, 1 ⁇ 10 13 —1 ⁇ 10 14 .
  • the concentration (viral titer) of the other drugs for treating neurological diseases related to functional neuron death is> 1 ⁇ 10 13 , preferably, 1 ⁇ 10 13 —1 ⁇ 10 14 .
  • composition or kit includes (a) gene editing protein or its expression vector; and (b) gRNA or its expression vector; and (c) optionally other functional nerves Drugs for neurological diseases related to death; and (d) pharmaceutically acceptable carriers.
  • composition or kit in another preferred embodiment, (a) gene editing protein or its expression vector; and (b) gRNA or its expression vector; and (c) optionally other functionalities for treatment
  • the drugs for neuron death-related neurological diseases account for 0.01-99.99 wt% of the total weight of the composition or kit, preferably 0.1-90 wt%, more preferably 1-80 wt%.
  • the fifth aspect of the present invention provides a method for promoting the differentiation of glial cells into functional neurons, including the steps:
  • Glial cells are cultured in the presence of inhibitors of Ptbp1 gene or RNA or its encoded protein or the composition of the second aspect of the present invention, thereby promoting the differentiation of glial cells into functional neurons.
  • the glial cells are selected from the group consisting of astrocytes, MG cells, oligodendrocytes, ependymal cells, Schwan cells, NG2 cells, satellite cells, or combinations thereof.
  • the functional neuron is selected from the following group: RGC neuron, dopamine neuron, or a combination thereof.
  • the glial cells are cells in vitro.
  • the effect of the concentration of the gene or its encoded protein Ptbp1 inhibitors > 1 ⁇ 10 13, preferably, 1 ⁇ 10 13 -1 ⁇ 10 14.
  • the sixth aspect of the present invention provides a method for preventing and/or treating neurological diseases related to functional neuron death, including:
  • An inhibitor of Ptbp1 gene or RNA or its encoded protein, or the composition according to the second aspect of the present invention, or the kit according to the third aspect of the present invention is administered to a subject in need.
  • the subject includes a human or non-human mammal suffering from a neurological disease related to functional neuron death.
  • the non-human mammals include rodents and primates, preferably mice, rats, rabbits, and monkeys.
  • the seventh aspect of the present invention provides a method for screening candidate compounds for the prevention and/or treatment of neurological diseases related to functional neuron death, the method comprising the steps:
  • test group In the test group, add the test compound to the cell culture system, and observe the expression (E1) and/or activity (A1) of Ptbp1 in the cells of the test group; in the control group, in the same cell No test compound is added to the culture system, and the expression (E0) and/or activity (A0) of Ptbp1 in the cells of the control group is observed;
  • the expression level of Ptbp1 is obtained by qPCR.
  • the method further includes the steps:
  • step (b) For the candidate compound obtained in step (a), further test its promoting effect on the differentiation of glial cells into functional neurons; and/or further test whether it has a down-regulation effect on the Ptbp1 gene.
  • the method includes step (c): applying the candidate compound determined in step (a) to a mammalian model, and determining its effect on the mammal.
  • the mammal is a mammal suffering from a neurological disease related to functional neuronal death.
  • the "significantly lower” means E1/E0 ⁇ 1/2, preferably, ⁇ 1/3, more preferably ⁇ 1/4.
  • the "significantly lower” means that A1/A0 ⁇ 1/2, preferably, ⁇ 1/3, more preferably ⁇ 1/4.
  • the cells include glial cells.
  • the cell is a cell cultured in vitro.
  • the method is non-diagnostic and non-therapeutic.
  • the eighth aspect of the present invention provides a method for promoting the differentiation of astrocytes into dopamine neurons, including the steps:
  • astrocytes are cultured to promote the differentiation of astrocytes into dopamine neurons.
  • the astrocytes include striatal astrocytes.
  • the astrocytes are astrocytes of brain tissue.
  • the astrocytes are cells in vitro.
  • the effect of the concentration of the gene or its encoded protein Ptbp1 inhibitors > 1 ⁇ 10 13, preferably, 1 ⁇ 10 13 -1 ⁇ 10 14.
  • the method is a non-diagnostic and non-therapeutic method.
  • the ninth aspect of the present invention provides a method for promoting the differentiation of Müller glial cells into optic ganglion cells, which comprises the steps of: in the presence of an inhibitor of Ptbp1 gene or RNA or its encoded protein or the composition of the second aspect of the present invention This promotes the differentiation of retinal Muller glial cells into optic ganglion cells.
  • the method is a non-diagnostic and non-therapeutic method.
  • FIG. 1 shows that CasRx can specifically knock down Ptbp1 mRNA1 in vitro.
  • A Schematic diagram of CasRx-mediated Ptbp1 mRNA knockdown.
  • B The schematic diagram shows the target sites of six gRNAs in the Ptbp1 gene.
  • C and D Knockdown efficiency of different combinations of gRNA. gRNA 5 and 6 showed the highest knockout efficiency in N2a cells (C) and astrocytes (D). The number above indicates the number of repetitions for each group. All values are expressed as mean ⁇ SEM.
  • E and F CasRx-Ptbp1 can specifically knock down Ptbp1.
  • N2a cells E
  • n 4 independent repeats
  • Figure 2 shows the specificity and expression of AAV, the closure of GFP expression over time, and the subtype of transformed RGC, which is related to Figure 1.
  • B Specificity of AAV-GFAP-GFP-Cre. AAV-GFAP-GFP-Cre drives GFP expression and unlocks tdTomato expression in the MG of Ai9 mice.
  • Sox9 is MG specific mark. Scale bar, 50 microns.
  • C Determination of AAV expression. The percentage of GFP+ cells expressing tdTomato and the percentage of tdTomato+ cells expressing Sox9. All values are expressed as mean ⁇ SEM.
  • D qPCR analysis confirmed that the infected retina expressed AAV-GFAP-CasRx and AAV-GFAP-CasRx-Ptbp1. The number above indicates the number of repetitions for each group. All values are expressed as mean ⁇ SEM.
  • E MG-induced RGC will eventually turn off GFP expression over time. scale. The experiment was repeated 6 times independently for each group of 20 microns, and the results were similar.
  • Foxp2, Brn3c and PV are the markers of RGC subtype F-RGC, type 3 RGC and PV-RGC, respectively.
  • the yellow arrow indicates the co-localization of tdTomato+ cells with different markers, and the white arrow indicates the co-localization of tdTomato+ cells with different markers.
  • Scale bar 20 microns. The experiment was repeated 3 times in each group, and the results were similar.
  • Figure 3 shows the combination of Ptbp1 transforming MG into RGC in the intact mature retina.
  • A Schematic diagram of MG to RGC conversion.
  • Vector I AAV-GFAP-GFP-Cre
  • Vector II AAV-GFAP-CasRx-Ptbp1
  • CasRx and gRNA AAV-GFAP-CasRx-Ptbp1
  • AAV-GFAP-CasRx-Ptbp1 or the control vector AAV-GFAP-CasRx and AAV-GFAP-GFP-Cre were injected into the retina (5-week-old Ai9 mice). Check for the occurrence of conversion about one month after injection.
  • (C) The number of tdTomato + or tdTomato + Brn3a + cells in GCL at 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 7 retinas.
  • Data are expressed as mean ⁇ SEM, *p ⁇ 0.05, **p ⁇ 0.01, **p ⁇ 0.001, unpaired t test.
  • D Representative image showing the co-localization of tdTomato with another specific marker of RGC in GCL, Rbpms. The yellow arrow indicates the co-localization of tdTomato and Rbpms.
  • Figure 4 shows that knockdown of Ptbp1 converts MG to RGC in the intact retina of C57BL mice, which is related to Figure 3.
  • FIG. 1 (GFAP-mCherry) expresses mCherry driven by MG-specific promoter GFAP, and vector 2 (AAV-EFS-CasRx-Ptbp1) expresses gRNA and CasRx under the promoter of the spectrum.
  • AAV-GFAP-mCherry was injected into the retina, and AAV-EFS-CasRx-Ptbp1 or the control virus AAV-GFAP-mCherry was injected at the same time. Check the occurrence of conversion 2-3 weeks after injection.
  • (C, D) Representative images showing mCherry+Brn3a+ and mCherry+Rbpms+ cells in GCL. Each group n 3 retinas. Scale bar, 50 microns.
  • Figure 5 shows that knocking down Ptbp1 converts MG into amacrine cells, which is related to Figure 3.
  • A tdTomato+Pax6+ cells were observed in the intact retina of Ai9 mice injected with AAV-GFAP-CasRx-Ptbp1. The green arrow indicates that the tdTomato+ cells are not co-localized with Pax6, and the yellow arrow indicates that Pax6 and tdTomato are co-localized. Please note that Pax6 is a marker for amacrine cells. Scale bar, 20 microns.
  • B No tdTomato+Prox1+ cells were observed.
  • the arrow indicates that tdTomato cells will not co-localize with Prox1 (a marker for bipolar cells). Scale bar, 20 microns.
  • C No tdTomato+ cells were observed in the photoreceptor cell layer (ONL).
  • White arrows indicate tdTomato-positive RGC-like cells in GCL, yellow arrows indicate tdTomato+protamine-like cells in INL, and green arrows indicate tdTomato+ projections of MG.
  • Scale bar 20 microns. Each group independently repeated the experiment at least 3 times, with similar results.
  • Figure 6 shows the induction of MG to RGC conversion in a mouse model of NMDA-induced retinal damage.
  • A Experimental design. Intravitreal injection of NMDA (200mM, 1.5mL) into Ai9 mice aged 4-8 weeks caused retinal damage. Two to three weeks after NMDA injection, AAV is injected under the retina. Immunostaining and behavioral tests were performed one month after AAV injection.
  • B NMDA injection basically killed most of the RGCs in GCL. Scale bar, 50 microns.
  • the yellow arrow indicates the co-localization of Rbpms and tdTomato in GCL injected with GFAP-CasRx-Ptbp1 plus GFAP-GFP-Cre. Scale bar, 20 microns.
  • Figure 7 shows the progress of the MG to RGC conversion, which is related to Figure 6.
  • A Representative images showing the conversion of MG to RGC at five different time points (without NMDA injection) in the intact retina. The arrow indicates induced RGC. Scale bar, 20 microns. The experiment was repeated independently ⁇ 3 times with similar results. Please note that representative pictures in "2 months” are also shown in Figures 2B and 2D.
  • B The absolute number of tdTomato+Brn3a+ and tdTomato+Rbpms+ cells in GCL. Note that the values in "1 month” are also shown in Figures 1C and 3E. All values are expressed as mean ⁇ SEM.
  • (D) Representative images showing the conversion of MG to RGC in the retina damaged by NMDA at four different time points. The arrow indicates induced RGC. Scale bar, 20 microns. Please note that the representative images in "3 months" are also shown in Figures 3C and 3E. The experiment was repeated independently ⁇ 2 times, with similar results.
  • Figure 8 shows that the converted RGC can be projected to the brain and partially restored visual function.
  • A Schematic diagram of the visual pathway. RGC sends axons through the optic nerve and transmits optic nerve signals to the dorsal geniculate nucleus and superior colliculus outside the retina.
  • B Retina tile. Yellow arrows indicate MG-derived tdTomato-positive RGC axons. Scale bar, 100 microns. The experiment was repeated 3 times in each group, and the results were similar.
  • C Representative image showing tdTomato+ axons of RGC induced in the optic nerve. Scale bar, 200 microns. Each group was repeated 5 times independently, with similar results.
  • Figure 9 shows the projection process of induced RGCs to 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). The tdTomato+ axon (yellow arrow) was first seen 1.5 weeks after AAV injection. Scale bar, 50 microns. The experiment was repeated 3 times independently, with similar results.
  • B The density of tdTomato+ axons (yellow arrows) in the dorsal geniculate nucleus gradually increases. Note that 1.5 weeks after AAV injection, tdTomato+ axons were first observed in the contralateral dorsal geniculate nucleus. Scale bars, 500 microns (top), 50 microns (bottom).
  • Figure 10 shows the progression of RGC projection induced in the damaged retina, related to Figure 9.
  • A Representative images show that at three different time points (1 week, 2 weeks, 3 weeks), NMDA-induced tdTomato+ axons in the optic nerve gradually increased. Scale bar, 50 microns. The experiment was repeated 2 times independently, with similar results.
  • B The density of tdTomato+ axons (yellow arrows) in the dorsal geniculate nucleus gradually increases. Scale bars, 500 microns (top), 50 microns (bottom). The experiment was repeated 2 times independently, with similar results.
  • C Projection progress of tdTomato+axons (yellow arrows) in the superior colliculus. Scale bars, 500 microns (top), 50 microns (bottom). The experiment was repeated 2 times independently, with similar results.
  • Figure 11 shows the conversion of glial cells to neurons mediated by CasRx.
  • A, B Schematic diagram of injection strategy. The efficiency of transformation was evaluated about one month after injection. ST, striatum.
  • D One week after AAV injection, Flag (fused with CasRx) and GFAP co-localized. Scale bar, 20 microns.
  • E One week after injection of AAV, the expression of Ptbp1 in the striatum (detected by the Ptbp1 antibody) was down-regulated.
  • the control AAV showed the absence of mCherry+SST+ cells, indicating that SST+ cells could not be infected by AAV-GFAP-mCherry.
  • the experiment was repeated 5 times independently, with similar results.
  • Scale bar 20 microns.
  • L Representative images show that mCherry+NeuN+ cells are not co-localized with Palvabumin. The experiment was repeated 4 times independently, with similar results. Scale bar, 20 microns.
  • M mCherry+TH+ cells (white arrow) are observed in the intact striatum. Scale bar, 20 microns.
  • Figure 12 shows the conversion of astrocytes into dopamine neurons in Parkinson's model mice.
  • a and B Summary of the experiment. Inject 6-OHDA unilaterally into the substantia nigra. After 3 weeks, AAV-GFAP-CasRx-Ptbp1 plus AAV-GFAP-mCherry, AAV-GFAP-CasRx plus AAV-GFAP-mCherry or normal saline were injected into the striae of the rat on the same side (relative to the 6-OHDA injection side).
  • B Immunostaining was performed about 1 month or 3 months after AAV injection in the morphology.
  • F Percentage of mCherry+DAT+ cells in mCherry+ cells.
  • G Percentage of mCherry+DAT+TH+ cells in mCherry+TH+ cells.
  • Figure 13 shows that knockdown of Ptbp1 converts astrocytes into dopamine neurons in a mouse model of 6-OHDA-induced Parkinson's disease.
  • A Experimental schematic diagram.
  • B Staining shows the death of TH+ neurons (green) on the ipsilateral substantia nigra (relative to the 6-OHDA injection side). Scale bar, 100 microns. The experiment was repeated 12 times independently, with similar results.
  • C DAT staining shows the disappearance of dopamine neuron fibers (green) in the ipsilateral striatum. Scale bar, 500 microns. The experiment was repeated independently >10 times with similar results.
  • Figure 14 shows that the induced neurons alleviated the motor dysfunction in Parkinson's disease model mice.
  • A Experimental design.
  • B Net rotation caused by apomorphine injection (contralateral-ipsilateral).
  • C Net rotation caused by amphetamine injection (ipsilateral-contralateral).
  • D After systemic injection of amphetamine, the percentage of ipsilateral rotation relative to the total number of rotations (ipsilateral/total rotation).
  • E The percentage of spontaneous touches on the same side relative to the total number of touches.
  • R Rotary rod instrument test. The results are expressed as the time (seconds) the mouse stays on the accelerated rotating rod before falling. The numbers above the bars indicate the number of mice in each group. All values are expressed as mean ⁇ SEM. One-way analysis of variance followed by Tukey test. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001.
  • the present inventors unexpectedly discovered for the first time that inhibiting the expression or activity of glial cells ptbp1 gene or RNA or its encoded protein can effectively induce glial cells to differentiate into functional neurons, thereby achieving therapeutic functions Neurological diseases related to the death of sexual neurons.
  • the inventor completed the present invention.
  • the degeneration of retinal ganglion cells is the main cause of permanent blindness.
  • the transdifferentiation of Müller glial cells (MG) into functional RGC can help restore vision.
  • the inventors found that knocking down Ptbp1 by using the RNA-targeted CRISPR system CasRx in the retina of mature mice can directly convert MG into functional RGC.
  • the RGC transformed from MG achieves a functional projection to the central visual area and improves visual function . Therefore, Ptbp1 knockdown mediated by CasRx will be a promising treatment for retinal diseases caused by neurodegeneration.
  • CasRx a recently characterized RNA targeting CRISPR system, to inhibit Ptbp1.
  • CasRx-mediated regeneration avoids the appearance of substantial off-target effects induced by shRNA and the risk of permanent gene changes through DNA editing nucleases, and provides an excellent tool that can treat a variety of diseases.
  • Müller glial cells are the main glial cells in the retinal tissue
  • retinal ganglion cells are nerve cells located in the innermost layer of the retina. Its dendrites are mainly connected with bipolar cells. Its axons extend to the optic nerve head to form the optic nerve.
  • Retinal disease is regarded as an eye disease.
  • 3Retina detachment Refers to the separation of the retinal nerve layer and the pigment epithelium.
  • a preferred retinal disease is a retinal disease caused by neurodegeneration, and the symptoms are mainly manifested in decreased vision or blindness.
  • the gene editor includes a DNA gene editor and an RNA gene editor.
  • the gene editor of the present invention includes a gene editing protein and optionally gRNA.
  • reprogramming or “transdifferentiation” may refer to the process of producing cells of a specific lineage (for example, neuronal cells) from different types of cells (for example, fibroblasts) without intermediate differentiation.
  • a specific lineage for example, neuronal cells
  • fibroblasts for example, fibroblasts
  • neurological diseases related to functional neuron death mainly include Parkinson's disease and visual disturbance caused by the death of optic ganglia.
  • neurological diseases related to functional neuronal degeneration include, but are not limited to, glaucoma, age-related RGC loss, optic nerve damage, retinal ischemia, Leber hereditary optic neuropathy, Alzheimer’s Disease, Huntington’s disease, schizophrenia, depression, drug use, movement disorders (such as chorea, hypercholesterolemia and movement disorders), motor neuron injury diseases (such as amyotrophic lateral sclerosis, spinal cord injury), bipolar disorder Disease, autism spectrum disorder (ASD), dysfunction, Parkinson’s disease.
  • Astrocytes are the most abundant type of cells in the mammalian brain. They perform many functions, including biochemical support (such as forming a blood-brain barrier), providing nutrients for neurons, maintaining extracellular ion balance, and participating in repair and scar formation after brain and spinal cord injury. According to the content of glial filaments and the shape of cell processes, astrocytes can be divided into two types: fibrous astrocytes (fibrous astrocytes) are mostly distributed in the white matter of the brain and spinal cord, with slender protrusions and fewer branches , The cytoplasm contains a lot of glial filaments; protoplasmic astrocytes (protoplasmic astrocytes) are mostly distributed in the gray matter, with stubby cell processes and many branches.
  • biochemical support such as forming a blood-brain barrier
  • astrocytes can be divided into two types: fibrous astrocytes (fibrous astrocytes) are mostly distributed in the white matter of the brain and spinal cord, with slender protru
  • the astrocytes that can be used in the present invention are not particularly limited, and include various astrocytes derived from the central nervous system of mammals, such as from the striatum, spinal cord, dorsal midbrain or cerebral cortex, preferably , From the striatum.
  • a functional neuron may refer to a neuron capable of sending or receiving information through chemical or electrical signals.
  • functional neurons exhibit one or more functional properties of mature neurons present in the normal nervous system, including but not limited to: excitability (e.g., the ability to exhibit action potentials, such as rapid Rise and subsequent fall) (voltage or membrane potential across the cell membrane), form synaptic connections with other neurons, release of presynaptic neurotransmitters, and post-synaptic responses (e.g., excitatory postsynaptic current or inhibitory synaptic current Aftertouch current).
  • excitability e.g., the ability to exhibit action potentials, such as rapid Rise and subsequent fall
  • post-synaptic responses e.g., excitatory postsynaptic current or inhibitory synaptic current Aftertouch current.
  • the functional neuron is characterized by the expression of one or more markers of the functional neuron, including but not limited to synapsin, synaptophysin, glutamate decarboxylase 67 (GAD67), glutamine Acid decarboxylase 67 (GAD65), paralbumin, dopamine- and cAMP-regulated neuronal phosphoprotein 32 (DARPP32), vesicle glutamate transporter 1 (vGLUT1), vesicle glutamate transporter 2 (vGLUT2) , Acetylcholine, tyrosine hydroxylase (TH), dopamine, vesicle GABA transporter (VGAT) and ⁇ -aminobutyric acid (GABA).
  • markers of the functional neuron including but not limited to synapsin, synaptophysin, glutamate decarboxylase 67 (GAD67), glutamine Acid decarboxylase 67 (GAD65), paralbumin, dopamine- and cAMP-regulated neuronal
  • Dopaminergic neuron contains and releases dopamine (dopamine, DA) as a neurotransmitter.
  • Dopamine is a catecholamine neurotransmitter and plays an important biological role in the central nervous system.
  • Dopaminergic neurons in the brain are mainly concentrated in the substantria nigra pars compacta (SNc) of the midbrain and the ventral cover Area (ventral tegmental area, VTA), hypothalamus and periventricular. Many experiments have confirmed that dopaminergic neurons are closely related to many diseases of the human body, the most typical being Parkinson's disease.
  • Neurodegenerative diseases are diseases caused by the loss of neurons in the brain and spinal cord. Neurons are the most important part of the nervous system, and his death will eventually lead to dysfunction of the nervous system. After a patient suffers from a neurodegenerative disease, there will be mobility or cognitive impairment, and the development of the disease often leads to many complications, causing serious damage to the patient's life.
  • neurodegenerative diseases mainly include Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and multiple sclerosis. At present, the neurodegenerative diseases can only be relieved or delayed, and cannot be completely cured.
  • Parkinson's disease (PD) is a serious neurodegenerative disease characterized by the loss of dopamine neurons in the substantia nigra of the midbrain.
  • the gene editor includes a DNA gene editor and an RNA gene editor.
  • the gene editor of the present invention includes a gene editing protein and optionally gRNA.
  • the nucleotides of the gene editing protein can be obtained by genetic engineering techniques, such as genome sequencing, polymerase chain reaction (PCR), etc., and the amino acid sequence can be derived from the nucleotide sequence.
  • the source of the wild-type gene editing protein includes (but is not limited to): Ruminococcus Flavefaciens, Streptococcus pyogenes, Staphylococcus aureus, and Acidaminococcus sp. , Lachnospiraceae bacterium (Lachnospiraceae bacterium).
  • the gene editing protein includes, but is not limited to Cas13d, CasRx, Cas13e, CRISPR/Cas9, Cpf1, Cas9, Cas13a, Cas13b, Cas13c, Cas13f, RNA targeted gene editing protein.
  • protein of the present invention refers to a protein or polypeptide having an amino acid sequence of ptbp1. They include ptbp1 protein with or without starting methionine. In addition, the term also includes full-length ptbp1 and fragments thereof.
  • the ptbp1 protein referred to in the present invention includes its complete amino acid sequence, its secreted protein, its mutant and its functionally active fragments.
  • the ptbp1 protein is a polypyrimidine domain binding protein 1, which is an RNA binding protein that regulates RNA splicing. At the same time, it also plays a very critical role in other functions of RNA.
  • ptbp1 gene ptbp1 polynucleotide
  • PTB gene a nucleic acid sequence having a ptbp1 nucleotide sequence
  • the genome of the human ptbp1 gene is 14936bp in length (NCBI GenBank accession number is 5725).
  • the genome of the mouse ptbp1 gene is 10004bp in length (NCBI GenBank accession number is 19205).
  • nucleic acid sequence encoding it can be constructed based on it, and specific probes can be designed based on the nucleotide sequence.
  • the full-length nucleotide sequence or its fragments can usually be obtained by PCR amplification, recombination, or artificial synthesis.
  • primers can be designed according to the ptbp1 nucleotide sequence disclosed in the present invention, especially the open reading frame sequence, and a commercially available cDNA library or a cDNA prepared by a conventional method known to those skilled in the art can be used.
  • the library is used as a template to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.
  • the recombination method can be used to obtain the relevant sequence in large quantities. This usually involves cloning it into a vector, then transferring it into a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is short. Usually, by first synthesizing multiple small fragments, and then ligating to obtain a very long fragment.
  • the DNA sequence encoding the protein (or fragment or derivative thereof) of the present invention can be obtained completely through chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules (such as vectors) and cells known in the art.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant ptbp1 polypeptide. Generally speaking, there are the following steps:
  • the ptbp1 polynucleotide sequence can be inserted into a recombinant expression vector.
  • any plasmid and vector can be used as long as it can replicate and stabilize in the host.
  • An important feature of an expression vector is that it usually contains an origin of replication, a promoter, a marker gene, and translation control elements.
  • the methods well known to those skilled in the art can be used to construct an expression vector containing the ptbp1 coding DNA sequence and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology.
  • the DNA sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selecting transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • selectable marker genes to provide phenotypic traits for selecting transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • a vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell so that it can express the protein.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples include: Escherichia coli, bacterial cells of the genus Streptomyces; fungal cells such as yeast; plant cells; insect cells; animal cells, etc.
  • Transformation of host cells with recombinant DNA can be performed by conventional techniques well known to those skilled in the art.
  • the host is a prokaryotic organism such as Escherichia coli
  • competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl 2 method. The steps used are well known in the art. Another method is to use MgCl 2 . If necessary, transformation can also be performed by electroporation.
  • the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the obtained transformants can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention.
  • the medium used in the culture can be selected from various conventional mediums.
  • the culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • the recombinant polypeptide in the above method can be expressed in the cell or on the cell membrane, or secreted out of the cell. If necessary, the physical, chemical, and other characteristics can be used to separate and purify the recombinant protein through various separation methods. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitation agent (salting out method), centrifugation, osmotic cleavage, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.
  • Adeno-associated virus is smaller than other viral vectors, is non-pathogenic, and can transfect dividing and undivided cells, gene therapy methods for genetic diseases based on AAV vectors have been affected. Widespread concern.
  • Adeno-associated virus also known as adeno-associated virus, belongs to the Parvoviridae dependent virus genus. It is the simplest type of single-stranded DNA-deficient virus found so far and requires a helper virus (usually Viruses) participate in replication. It encodes the cap and rep genes in the inverted repeat (ITR) at both ends. ITRs play a decisive role in virus replication and packaging. The cap gene encodes the viral capsid protein, and the rep gene is involved in virus replication and integration. AAV can infect a variety of cells.
  • Recombinant adeno-associated virus vector is derived from non-pathogenic wild-type adeno-associated virus. Due to its good safety, wide range of host cells (dividing and non-dividing cells), and low immunogenicity, it can express foreign genes in vivo. Long and other characteristics, it is regarded as one of the most promising gene transfer vectors and has been widely used in gene therapy and vaccine research worldwide. After more than 10 years of research, the biological characteristics of recombinant adeno-associated virus have been deeply understood, especially its application effects in various cells, tissues and in vivo experiments have accumulated a lot of data.
  • rAAV is used in the research of gene therapy for various diseases (including in vivo and in vitro experiments); at the same time, as a characteristic gene transfer vector, it is also widely used in gene function research, disease model construction, and gene preparation. Knockout mice and other aspects.
  • the vector is a recombinant AAV vector.
  • AAVs are relatively small DNA viruses that can integrate into the genome of the cells they infect in a stable and site-specific manner. They can infect a large range of cells without any effect on cell growth, morphology or differentiation, and they do not seem to be involved in human pathology.
  • the AAV genome has been cloned, sequenced and characterized.
  • AAV contains an inverted terminal repeat (ITR) region of approximately 145 bases at each end, which serves as the origin of replication of the virus. The rest of the genome is divided into two important regions with encapsidation functions: the left part of the genome containing the rep gene involved in viral replication and viral gene expression; and the right part of the genome containing the cap gene encoding the viral capsid protein.
  • ITR inverted terminal repeat
  • AAV vectors can be prepared using standard methods in the art. Adeno-associated viruses of any serotype are suitable. Methods for purifying vectors can be found in, for example, U.S. Patent Nos. 6,566,118, 6,989,264, and 6,995,006, the disclosures of which are incorporated herein by reference in their entirety. The preparation of hybrid vectors is described in, for example, PCT Application No. PCT/US2005/027091, the disclosure of which is incorporated herein by reference in its entirety. The use of AAV-derived vectors for in vitro and in vivo gene transfer has been described (see, for example, International Patent Application Publication Nos. WO91/18088 and WO93/09239; U.S. Patent Nos.
  • Replication-deficient recombinant AAV can be prepared by co-transfecting the following plasmids into a cell line infected with a human helper virus (such as adenovirus): the nucleic acid sequence of interest is flanked by two AAV inverted terminal repeats (ITR) Region plasmids, and plasmids carrying AAV encapsidation genes (rep and cap genes).
  • a human helper virus such as adenovirus
  • the recombinant vector is capsidized to viral particles (e.g., including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 And AAV virus particles of AAV16). Therefore, the present disclosure includes recombinant virus particles (recombinant because they contain recombinant polynucleotides) containing any of the vectors described herein. Methods of producing such particles are known in the art and are described in US Patent No. 6,596,535.
  • the ptbp1 inhibitor (or antagonist) that can be used in the present invention includes any substance that can inhibit the expression and/or activity of the ptbp1 gene or its encoded protein.
  • the inhibitor of ptbp1 includes an antibody of ptbp1, antisense RNA of ptbp1 nucleic acid, siRNA, shRNA, miRNA, gene editor, or an activity inhibitor of ptbp1.
  • a preferred inhibitor of ptbp1 refers to a gene editor capable of inhibiting the expression of ptbp1.
  • the inhibitors of ptbp1 of the present invention include inhibitors that target positions 4758-4787 and/or positions 5381-5410 of the ptbp1 gene sequence.
  • the ptbp1 inhibitor of the present invention acts on astrocytes or MG cells.
  • the methods and steps for inhibiting ptbp1 include using an antibody of ptbp1 to neutralize its protein, and using shRNA or siRNA or a gene editor carried by a virus (such as adeno-associated virus) to silence the ptbp1 gene.
  • a virus such as adeno-associated virus
  • the inhibition rate of ptbp1 is generally at least 50% or more inhibition, preferably 60%, 70%, 80%, 90%, 95% inhibition, which can be based on conventional techniques, such as flow cytometry, fluorescent quantitative PCR or Western Methods such as blot control and detect the inhibition rate of ptbp1.
  • the inhibitors of the ptbp1 protein of the present invention when administered (administered) therapeutically, can inhibit the expression and/or activity of the ptbp1 protein, thereby inducing glue Plasma cells differentiate into functional neurons to treat neurological diseases related to functional neuronal degeneration.
  • these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 5-8, preferably about 6-8, although the pH can be The nature of the formulated substance and the condition to be treated vary.
  • the formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): local, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, topical administration, autologous cell extraction and culture and reinfusion Wait.
  • the present invention also provides a pharmaceutical composition, which contains a safe and effective amount of the inhibitor of the present invention (such as antibody, gene editor, antisense sequence (such as siRNA), or inhibitor) and a pharmaceutically acceptable carrier or excipient Shape agent.
  • a pharmaceutical composition which contains a safe and effective amount of the inhibitor of the present invention (such as antibody, gene editor, antisense sequence (such as siRNA), or inhibitor) and a pharmaceutically acceptable carrier or excipient Shape agent.
  • Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of injection, for example, prepared by conventional methods with 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 should be manufactured under sterile
  • the present invention found for the first time that reducing the expression or activity of the Ptbp1 gene or its encoded protein in astrocytes can induce the differentiation of astrocytes into dopamine neurons, thereby preventing and/or treating neurodegeneration Diseases (such as Parkinson's disease).
  • the present invention finds for the first time that using a gene editor (including gene editing protein and gRNA) to inhibit the expression of ptbp1 in astrocytes can make astrocytes transdifferentiate into dopamine neurons, which in turn is Parkinson’s Treatment provides a potential way.
  • a gene editor including gene editing protein and gRNA
  • the present invention found for the first time that the induction of dopamine neurons alleviated the motor dysfunction in the Parkinsonian mouse model.
  • the present invention finds for the first time that the RNA-targeted CRISPR system CasRx can avoid the risk of permanent DNA changes caused by traditional CRISPR-Cas9 editing. Therefore, CasRx-mediated RNA editing provides an effective means for the treatment of various diseases.
  • the present invention directly converts MG into functional RGC by inhibiting the expression of Ptbp1 in the retina.
  • the regenerated RGC can be integrated into the visual pathway and improve the visual function of the mouse model of RGC injury.
  • the present invention uses the RNA-targeted CRISPR system CasRx to knock down Ptbp1, avoiding the occurrence of substantive off-target effects induced by shRNA and the risk of permanently changing genes, and provides an excellent tool that can treat a variety of diseases.
  • Transient transfection of astrocytes and qPCR isolated and cultured as previously described 1 astrocytes. In short, astrocytes were seeded in 6-well plates. Using Lipofectamine 3000 (Thermo Fisher Scientific) according to standard procedures, 3 ⁇ g of gRNA-CasRx-GFP expressing vector was used for transient transfection. The control plasmid expresses non-targeted guidance.
  • GFP positive cells were collected by Flow Fluorescence Cell Sorting (FACS) and lysed for qPCR analysis: first use Trizol (Ambion) to extract RNA, and then use reverse transcription kit (HiScript for qPCR) Q RT SuperMix, Vazyme, Biotech) reverse transcription of RNA into cDNA. The amplification was followed by AceQ qPCR SYBR Green Master Mix (Vazyme, Biotech).
  • the Ptbp1 qPCR primers are: forward, 5'-AGAGGAGGCTGCCAACACTA-3' (SEQ ID NO.: 13); reverse, 5'-GTCCAGGGTCACTGGGTAGA-3' (SEQ ID NO. 14).
  • Stereotactic injection AAV8 ( Figure 1) and AAV-PhP.eb ( Figures 2 and 3) were used in this study. Stereotactic injection (C57BL / 6,1-3 months) 2 method as described above.
  • the titers of AAV-CasRx-Ptbp1 in Figure 1 and Figures 2, 3 are about 5 ⁇ 10e12 (2 ⁇ l per injection) and 1.6 ⁇ 10e13 (2-3 ⁇ l per injection). Inject AAV into the striatum (AP+0.8mm, ML ⁇ 1.6mm and DV-2.8mm).
  • Immunofluorescence staining was performed 5-6 weeks ( Figure 1) or 3-4 weeks ( Figures 2 and 3) after injection. After the mice were perfused, their brains were taken and fixed with 4% paraformaldehyde (PFA) overnight, and kept in 30% sucrose for at least 12 hours. The sections were frozen after embedding, and the section thickness was 35 ⁇ m. Before immunofluorescence staining, the brain sections were washed thoroughly with 0.1M phosphate buffer (PB).
  • PB 0.1M phosphate buffer
  • Electrophysiological recordings after AAV injection electrophysiological recordings 5-6 weeks, 3 as previously described.
  • the mice were anesthetized and perfused into the heart, and their brains were put into carbon dioxide-filled NMDG artificial cerebrospinal fluid (aCSF) [NMDG aCSF(mM): NMDG 92, potassium chloride 2.5, sodium dihydrogen phosphate 1.25, bicarbonate Sodium 30, HEPES 20, glucose 25, thiourea 2, sodium ascorbate 5) at room temperature, sodium pyruvate 3, calcium chloride 0.5, magnesium sulfate 10].
  • aCSF NMDG artificial cerebrospinal fluid
  • HEPES HEPES aCSF filled with carbon dioxide at room temperature
  • HEPES which contains aCSF (mM): sodium chloride 92, potassium chloride 2.5, sodium dihydrogen phosphate 1.25, sodium bicarbonate 30, HEPES 20, Glucose 25, Thiourea 2, Sodium Ascorbate 5, Sodium Pyruvate 3, Calcium Chloride 2, Magnesium Sulfate 2].
  • aCSF (mM): sodium chloride 119, potassium chloride 2.5, sodium dihydrogen phosphate 1.25, sodium bicarbonate 24, glucose 12.5, chloride Calcium 2, magnesium sulfate 2].
  • the neuron-like mCherry positive cells were recorded under a microscope (Olympus BX51WI), and Clampex 10 was used to obtain the data.
  • mice were intraperitoneally injected with 0.5 mg/kg apomorphine (A4393, Sigma-Aldrich) 10 minutes before the test. After that, each of them was placed in an opaque cylinder (30 cm in diameter) and recorded on it by a camera for 20 minutes. Rotation is defined as a whole body turning with one hind paw as the center and no head orientation is switched. Calculate the number of rotations on the injection side and the contralateral side. The data was quantified as the number of contralateral reversals within 20 minutes.
  • apomorphine A4393, Sigma-Aldrich
  • Each mouse was gently put into a glass beaker (1000ml) and recorded with a camera for 10 minutes in front of it. Calculate the number of wall touches on the injection side and the contralateral paw respectively, and quantify the data as the ratio of the number of wall touches on the same side to the total number of wall touches.
  • mice All mice were trained for 2 days and tested on the third day. On the first day, the mice were trained 4 times on a rotating rod at a fixed speed of 4 laps/min, each for 300 seconds. On the 2nd and 3rd days, the mice were trained or tested 4 times at an acceleration of 4 to 40 laps/min. The time the mouse stayed on the rod before falling off was recorded as the stay period, and the average of the 3 longest stay periods was used for analysis.
  • N2a cells were seeded in 6-well plates. According to standard procedures, Lipofectamine 3000 (Thermo Fisher Scientific) was used, and cells were transfected with 7 ⁇ g gRNA-CasRx-GFP vector. The control plasmid does not express gRNA. Two days after transfection, about 50,000 GFP-positive cells were collected from each sample by fluorescence activated cell sorting (FACS), and lysed for qPCR analysis. At the same time, the retina was separated to determine the expression of AAV. The RNA was extracted using Trizol (Ambion) and converted into cDNA using a reverse transcription kit (HiScript QRT SuperMix for qPCR, Vazyme, Biotech). Use AceQ qPCR SYBR Green Master Mix (Vazyme, Biotech) to track the amplification process.
  • FACS fluorescence activated cell sorting
  • Ptbp1 qPCR primer is: upstream primer, 5'-AGAGGAGGCTGCCAACACTA-3' (SEQ ID NO: 7);
  • Downstream primer 5'-GTCCAGGGTCACTGGGTAGA-3' (SEQ ID NO: 8).
  • CasRx qPCR primer upstream primer, 5'-CCCTGGTGTCCGGCTCTAA-3' (SEQ ID NO: 9);
  • RNA-seq N2a cells were cultured in a 15-cm petri dish and transiently transfected with 70 ⁇ g plasmid. Collect ⁇ 500,000 GFP-positive (top 20% GFP) N2a cells by FACS, extract RNA, convert it into cDNA, and use it for full-transcriptome RNA-seq.
  • NMDA and AAV were introduced by intravitreal and subretinal injections, respectively.
  • high titer >1 ⁇ 10 13
  • AAV was injected into the eye using a Hamilton syringe (32G needle) under an Olympus microscope (Olympus, Tokyo, Japan).
  • GFAP-GFP-Cre 0.2 ⁇ l
  • GFAP-CasRx-Ptbp1 0.8 ⁇ l
  • dissolve NMDA to a concentration of 200 mM in PBS dissolve NMDA to a concentration of 200 mM in PBS, and then inject 1.5 ⁇ l of NMDA solution into 4-8 week old Ai9 mice or 5-6 week old C57BL/6 mice by intravitreal injection.
  • Mouse eyes (used for VEP and black and white scene preference testing).
  • GFAP-GFP-Cre and GFAP-CasRx-Ptbp1 or GFAP-CasRx were co-delivered to the retina by subretinal injection.
  • 5-6 weeks old mice were injected with NMDA to induce retinal damage, and GFAP-mCherry ( 0.2 ⁇ l) and GFAP-CasRx-Ptbp1 (0.8 ⁇ l) or GFAP-CasRx (0.8 ⁇ l) mixture.
  • the eyes, optic nerve and brain were taken, fixed with 4% paraformaldehyde (PFA) for 2 hours (eyes and optic nerve) or 24 hours (brain), and then stored in 30% sucrose solution 2 (eyes) And optic nerve) or 24 (brain) hours. After embedding and freezing, the eyes and brain were sliced at a thickness of 30 ⁇ m.
  • PFA paraformaldehyde
  • mice anti-Brn3a (1:100, MAB1585, Millipore), rabbit anti-RBPMS (1:500, 15187-1-AP, Proteintech), rabbit anti-Sox9 (1:500, AB5535, Millipore), rabbit anti-Pax6 (1:500, 901301, Biolegent), rabbit anti-Prox1 (1:500, AB5475, Millipore), and secondary antibody: Cy TM 5 AffiniPure Donkey mouse anti-IgG (H+L) (1: 500,715-175-150, Jackson ImmunoResearch), CyTM 5 AffiniPure Donkey rabbit anti-IgG (H+L) (1: 500, 711-175-152, Jackson ImmunoResearch). After applying the antibody, wash and mount the film. Use Olympus FV3000 microscope for imaging.
  • an oxygenated (95% O2 / 5% CO2) artificial cerebrospinal fluid (ACSF) containing 126mM NaCl, 2.5mM KCl, 1.25mM NaH2PO4, 2mM CaCl2, 2mM NaHCO3 and 10mM glucose. Place the RGC of the retina facing the cell recording groove on the table of the
  • ASCF and 0.25mM Alexa488 hydration were added to the pipette (4-7M ⁇ ) used for recording.
  • the signal is low-pass filtered at 1kHz and digitized at 10kHz.
  • the white LED light is used to deliver the full-field light stimulation. After recording, inject current pulses to fill the cells to visualize their morphology.
  • a mixture of fentanyl (0.05mg/kg), midazolam (5mg/kg) and medetomidine (0.5mg/kg) was injected intraperitoneally in mice.
  • the head of the mouse was fixed in a stereotaxic instrument, and the body temperature was maintained at 37°C through a heating blanket.
  • a craniotomy (approximately 1mm in diameter) was performed on both sides of the main visual cortex (V1) (AP-3.6 to -3.9mm, ML 2.2mm), and the dura mater was removed.
  • the visual stimulus is emitted by a 17-inch LCD display (Dell P170S, maximum brightness 69cd/m2), which is 8 cm away from the eyes at the recording end, and at the same time shields the side of the eye on the same side of the recording end from visual stimulation.
  • a 17-inch LCD display (Dell P170S, maximum brightness 69cd/m2)
  • Use a multi-point silicon probe (A1 ⁇ 16-5mm-50-177, NeuroNexus Technologies) to record at V1 (AP-3.6 to -3.9mm, ML 2.2mm), and the cortical depth reached by the electrode tip of each recording is about 900 ⁇ m.
  • Both the reference wire and the ground wire are placed in a small craniotomy at least 3 mm away from the recording point.
  • the Cerebus 32-channel system Blackrock microsystems
  • the Cerebus 32-channel system was used to amplify and filter neural responses.
  • Use broadband front-end filter (0.3 ⁇ 500Hz) to sample the local field potential (LFP) signal at 2kHz or 10kHz.
  • LFP response to stimulation is used to flash the full screen current source density (CSD) analysis to determine the location of the cortical layer 43.
  • CSD current source density
  • Layer 4 (granular layer) is defined as those recorded positions at the initial current receiver. We used the layer 4 channel showing the maximum average amplitude to analyze the visual evoked response of each mouse.
  • the equipment used for the light-dark box shuttle experiment includes a box with a door, which is divided into a small (one-third) dark box part and a large (two-thirds) lighting part (550 lumens).
  • the mouse can move freely between the two compartments for 10 minutes.
  • the time the mice spend in each compartment is recorded by the camera and then analyzed using Ethovision XT. After each test, clean the compartment with 70% ethanol to avoid olfactory cues.
  • AAV-GFAP-CasRx-Ptbp1 (gRNA 5+6) driven by the Mueller glial cell-specific promoter GFAP, hoping to specifically knock down Ptbp1 in Mueller glial cells and we also constructed The control virus AAV-GFAP-CasRx, which does not target Ptbp1 ( Figures 3A and 2D).
  • AAV-GFAP-CasRx-Ptbp1 and AAV-GFAP-Cre-GFP into the retina of Ai9 mice aged about 5 weeks. After 1 month, we found that many tdTomato-positive cells were specific to optic ganglion cells.
  • Example 4 Induced optic ganglion cells partially restore visual function
  • Example 5 The projection process of optic ganglion cells to the brain
  • mCherry+ cells also expressed two other midbrain dopamine neuron markers, namely dopa decarboxylase (DDC) and forkhead box protein A2 (FOXA2) (Figure 13J-13M), further verifying the transformed neurons Dopaminergic neurons.
  • DDC dopa decarboxylase
  • FOXA2 forkhead box protein A2
  • mice injected with AAV-GFAP-CasRx-Ptbp1 had significantly lower net spins (calculated as contralateral spins on the same side) and ipsilateral spin ratio (calculated on ipsilateral/total spins) ( Figures 14C, 14D and 13Q). These results indicate that neurons induced in the striatum can release enough dopamine to alleviate the motor dysfunction in PD model mice caused by the drug. In addition, we tested whether the two kinds of drug-free motor functions were improved, namely the asymmetry of forelimb use and motor coordination.
  • mice injected with AAV-GFAP-CasRx-Ptbp1 had a significant reduction in the cylinder ipsilateral touch percentage and a longer duration on the rotating tripod ( Figure 14E and 14F ).
  • Ptbp1 knockdown of astrocytes in the striatum mediated dopamine neuron transconversion reduces motor dysfunction in Parkinson's disease model mice.
  • glial cells can be effectively transformed into neurons through CasRx-mediated down-regulation of Ptbp1.
  • Ptbp1 knockdown can induce the conversion of MG in the injured retina to RGC, which partially restores the visual response and vision-dependent behavior.
  • a mouse model of Parkinson's disease induced by 6-OHDA it can induce astrocytes in the striatum into dopamine neurons and reduce motor dysfunction related to the loss of substantia nigra dopamine neurons.
  • RNA-binding protein Ptbp1 is sufficient to convert glial cells into specific types of neurons that are lost in different nervous systems, which provides a new method for future therapeutic applications.
  • Short hairpin RNA (shRNA) technology can cut or suppress the desired transcript, but it has a very serious off-target effect.
  • Cas13-mediated knockdown is not only more efficient than RNAi, but can also reduce off-target effects to a large extent, which has greater potential in therapeutic applications.
  • CasRx is the smallest one in the Cas13 protein family and can be packaged in an AAV with the CRISPR array (encoding multiple guide RNAs). In the field of gene editing, the RNA-targeted CRISPR system CasRx can avoid the risk of permanent DNA changes caused by the DNA-targeted CRISPR-Cas9 editing system, making it safer in clinical applications.

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Abstract

La présente invention concerne une application d'un inhibiteur de Ptbp1 dans la prévention et/ou le traitement d'une maladie du système nerveux liée à la mort neuronale fonctionnelle. Spécifiquement, la présente invention concerne l'utilisation d'un gène de Ptbp1 ou d'un ARN ou d'un inhibiteur de protéine codée associé dans la préparation d'une composition ou d'une préparation. La composition ou la préparation est utilisée pour prévenir et/ou traiter une maladie du système nerveux liée à la mort neuronale fonctionnelle. En inhibant l'expression ou l'activité du gène Ptbp1 ou de l'ARN ou d'une protéine codée associée dans les astrocytes dans le cerveau, la transdifférenciation des astrocytes en neurones dopaminergiques peut être efficacement induite. De plus, par inhibition de l'expression ou de l'activité du gène Ptbp1 ou de l'ARN ou d'une protéine codée associée dans des cellules gliales de Müller dans la rétine, la transdifférenciation des cellules gliales de Müller en cellules ganglionnaires de la rétine peut être efficacement induite, ce qui permet de prévenir et/ou de traiter une maladie du système nerveux liée à la mort neuronale fonctionnelle.
PCT/CN2020/105285 2019-08-16 2020-07-28 Application d'un inhibiteur de ptbp1 dans la prévention et/ou le traitement d'une maladie du système nerveux liée à la mort neuronale fonctionnelle WO2021031810A1 (fr)

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