Use of neurod1 in the preparation of a medicament for converting retinal mueller glial cells into neurons

Abstract

The present application provides use of NeuroD1 in preparation of a drug for converting retinal Muller glial cells into neurons. The present application also provides a method for converting mouse retinal Muller glial cells into neurons, which comprises overexpressing neurotranscription factor NeuroD1 in mouse retinal Muller glial cells. After overexpression of NeuroD1 in mouse retinas by using an adeno-associated virus vector, the present application induces conversion of retinal Muller glial cells into retinal neurons, which can be applied to repair retinas damaged or degenerated due to diseases, thereby restoring vision.

Description

Technical Field

[0001] This invention belongs to the field of neurobiology, specifically relating to the use of NeuroD1 in the preparation of drugs that convert retinal Müller glial cells into neurons. Background Technology

[0002] Retinal degenerative diseases are among the most common diseases worldwide, often associated with neuronal loss and frequently leading to blindness, yet no effective treatment has been found to date. Regeneration of retinal neurons may offer a new strategy for repairing degenerating retinas. How to regenerate new neurons to restore vision has become a crucial question.

[0003] Müller glial cells are the predominant type of glial cell in the retina and have been reported to regenerate neurons in the zebrafish retina (Powell et al., 2016). However, this regenerative capacity is rather limited in birds such as chicks and is completely lost in mammals like mice (Palazzo et al., 2020). Previous studies have employed various strategies to activate the regenerative capacity of mammalian Müller glial cells, including ectopic expression of Ascl1, Math5, and Brn3b, or knockout of Ptbp1 (Jorstad et al., 2017; Zhou et al., 2020; Xiao et al., 2021). However, most studies have not achieved high conversion efficiency and typically require more than one transcription factor or a combination of other factors to trigger transdifferentiation. It remains unclear whether a single transcription factor can efficiently reprogram Müller glial cells.

[0004] NeuroD1 (ND1) is a bHLH transcription factor that plays a crucial role in early brain development by inducing neuronal differentiation from neural stem cells (Goebbels et al., 2005; Pataskar et al., 2016). A series of studies have reported that NeuroD1 alone can reprogram brain astrocytes into neurons in various brain disease models, including Alzheimer's disease, Huntington's disease, stroke, and epilepsy. In the retina, NeuroD1 is involved in the development of photoreceptor cells (Ochocinska et al., 2012) and amacrine cells (Cho et al., 2007). Furthermore, these studies have shown that loss of NeuroD1 function leads to delayed amacrine cell development and loss of photoreceptor cells.

[0005] It remains unclear whether NeuroD1 can reprogram Müller glial cells into retinal neurons in adult mice. Summary of the Invention

[0006] The purpose of this invention is to address the above-mentioned technical problems by providing a method for converting retinal Müller glial cells into neurons to obtain regenerated new neurons.

[0007] To achieve the above-mentioned objectives, this invention provides the use of NeuroD1 in the preparation of a medicament for converting retinal Müller glial cells into neurons.

[0008] Preferably, the neuron is a retinal neuron. More preferably, the neuron is an inner retinal neuron. More preferably, the neuron is amacrine aneurysms and / or ganglion cells. Preferably, the neuron is an outer retinal neuron. More preferably, the neuron is a photoreceptor cell.

[0009] Preferably, NeuroD1 converts retinal Müller glial cells into neurons in a time-dependent manner.

[0010] Preferably, NeuroD1 converts retinal Müller glial cells into neurons in a dose-dependent manner.

[0011] This invention also provides a method for converting mouse retinal Müller glial cells into neurons, the method comprising: overexpressing the neural transcription factor NeuroD1 in mouse retinal Müller glial cells. This method can be used for non-disease treatment or non-diagnostic purposes. Delivery routes can include intraocular injection (such as subretinal injection, intravitreal injection) and blood injection.

[0012] Preferably, overexpression of the neural transcription factor NeuroD1 includes infecting mouse retinal Müller glial cells with a recombinant viral vector expressing NeuroD1.

[0013] According to aspects of the present invention, the expression vector can be a viral vector such as adenovirus, adeno-associated virus, retrovirus, or lentivirus, or a non-viral vector, such as liposome nanoparticles for RNA delivery. Preferably, the viral vector is an adeno-associated virus vector. More preferably, the adeno-associated virus vector is AAV7m8 or AAV9. Preferably, an enhancer is added to the adeno-associated virus vector to increase NeuroD1 expression.

[0014] This invention utilizes the adeno-associated virus vector AAV7m8 GFAP::GFP-ND1 to overexpress NeuroD1 in normal or NMDA-treated mice, inducing various types of retinal neurons, including amacrine cells and some ganglion cells. Furthermore, when an enhancer is added to increase NeuroD1 expression, the adeno-associated virus vector AAV9 GFAP104::ND1-GFP more efficiently converts Müller glial cells into outer retinal neurons (photoreceptor cells). Moreover, the NeuroD1-induced Müller glial cell conversion exhibits significant time- and dose-dependent characteristics. This invention can be applied to the repair of damaged or degenerative retinas, thereby restoring vision. Attached Figure Description

[0015] Figure 1 The diagram shows AAV7m8 GFAP::GFP-ND1 reprogramming a limited number of Müller glial cells into inner retinal neurons. (A) Schematic diagram of the recombinant viral vector structure; the top is the control AAV7m8 GFAP::GFP, and the bottom is the experimental group AAV7m8 GFAP::GFP-P2A-ND1. (B) Schematic diagram of intravitreal viral injection and description of the experimental protocol. (C) Images of retinal sections stained with GFP (green) and the Müller glial cell marker Sox9 (red) at 5 days and 28 days (dpi) after injection of the control or ND1 virus. DAPI (blue) staining of cell nuclei. (D) Images of retinal sections stained with GFP (green), the ganglion cell marker RBPMS (red, left experimental group), or the non-protruding cell marker Calretinin (red, right experimental group) at 28 dpi. The white square area in the ND1 group is magnified and shown on the right, showing individual color channels or combined into one. DAPI (blue) staining of cell nuclei. (E) Retinal tile images stained with GFP (green) and Calretinin (red, left column) in the nuclear layer (INL) or with RBPMS (red, right column) in the ganglion cell layer (GCL). GFP+ cells with oval cell bodies were observed in the ND1 group, while polygonal cell bodies and terminal feet were observed in the control group. (F) Percentage of Sox9+GFP+ cells out of all GFP+ cells at 5 and 28 dpi. Following ND1 infection, 14% of GFP+ cells lost expression of the Müller glial cell marker Sox9 at 28 dpi. (G) Percentage of GFP+ cells with either the RBPMS marker or the Calretinin marker out of all GFP+ cells at 28 dpi. ND1 infection converted 5.0% and 9.6% of GFP+ cells into ganglion cells and amacrine cells, respectively.

[0016] Figure 2The image shows ND1 in the retina damaged by NMDA, which transforms Müller glial cells into numerous amacrine cells. (A) Experimental protocol description. (B) Images of retinal sections stained with GFP (green) and the amacrine cell marker Calretinin or the ganglion cell marker Brn3a 8 weeks after viral injection. Yellow arrows point to GFP+ cells co-expressing neuronal markers. (C) Retinal tilings double-stained with GFP (green) and the amacrine cell marker Calretinin (red). Enlarged white square areas are shown on the right. Yellow arrows point to GFP+ cells co-expressing Calretinin, and white arrows point to GFP+ cells not expressing Calretinin. (D) Percentage of GFP+ cells with Calretinin markers among all GFP+ cells 8 weeks after viral injection.

[0017] Figure 3 The diagram shows that AAV9 GFAP104::ND1-GFP induced stronger ND1 expression than AAV7m8. (A) Schematic diagram of the AAV9 virus structure, with the upper image showing the control virus AAV9 GFAP104::GFP and the lower image showing the experimental group AAV9 GFAP104::ND1-GFP. (B) Illustration of subretinal injection and experimental protocol. (C) Retinal section images 5 dpi after intravitreal injection of AAV7m8 GFAP::ND1-GFP or control virus. Sections were stained with GFP (green), NeuroD1 antibody (red), and Müller glial cell marker GS (purple). The white square areas in each group are magnified and displayed on the right, with each color channel shown individually and then merged into one. In the ND1 group, GFP+ cells showed weaker ND1 and GS expression in their cell bodies. (D) Retinal section images 5 dpi after subretinal injection of AAV9 GFAP104::ND1-GFP or control virus. In the ND1 group, GFP+ cells showed strong ND1 and GS expression in their cell bodies. For the control virus, all GFP+ cells expressed GS, but not ND1.

[0018] Figure 4This shows the ability of ND1 to convert Müller glial cells into neurons in the outer retina as it increases over time. (A) Retinal sections stained with GFP (green) and the Müller glial cell marker Sox9 (red) at different time points after 28 dpi following control viral injection and after ND1 viral injection. The top row shows only the GFP channel, and the bottom row shows the merged channel. Cell nuclei are stained with DAPI (blue). A large number of GFP+ photoreceptor-like cells and some level-like cells were observed over time in the ND1 group. (B) Retinal sections stained with GFP (green), the photoreceptor marker Recoverin (red, left), or the level-like cell marker Calbindin (red, right) at 28 dpi. The white square area in the ND1 group is magnified and shown on the right, showing the individual color channels first and then a merged image. Cell nuclei are stained with DAPI (blue). (C) Retinal tile images stained with GFP (green) and Opsin (red, left) above the ONL layer, or Calbindin (red, middle) at the top of the INL, or RBPMS (red, right) at the GCL. In the ND1 group, clear extracellular structures of GFP+ photoreceptor cells were observed (arrows). All GFP+ cells in the INL were co-labeled with Calbindin (asterisk), but no GFP+ cells were observed in the GCL layer. In the control group, extracellular processes, intracellular processes, and terminal feet of Müller glial cells were observed in each layer. (D) Percentage of Sox9+GFP+ cells (red line) or GFP+ photoreceptor-like cells (green line) to all GFP+ cells at different time points. After ND1 infection, 23.3%, 54.7%, and 96.0% of GFP+ cells lost expression of Müller glial cell markers at days 7, 14, and 28, respectively. (E) 28 dpi, the percentage of GFP+ cells with either the Recoverin or Calbindin markers. ND1 infection converted 81.3% and 5.4% of GFP+ cells into photoreceptor cells and horizontal cells, respectively.

[0019] Figure 5 The images show how different titers of AAV9-ND1 virus convert Müller glial cells into neurons in the outer retinal layer. (A) Experimental protocol. (B) Retinal slice images after subretinal injection of AAV9GFAP104::ND1-GFP or control virus, titers from 10... 11 Increase to 10 13GC / ml. Sections were stained with GFP (green) and Sox9 (red). The number of GFP+ cells increased with increasing viral titer. In the control group, GFP+ cells remained Müller glial cells, while in the ND1 group, a large number of GFP+ cell bodies and outer segments were observed in the ONL containing photoreceptor cells. At high titers, some GFP+ cells with horizontal cell morphology were also observed. (C) The number of GFP+ cells in the ONL layer increased with increasing ND1 viral titer. In the control group, even at high titers, very few GFP+ cells were seen in the ONL layer. (D) Retinal sections stained with GFP (green) and the cone cell outer segment marker Opsin (red) at both titers showed that most GFP+ fibers did not co-label with red Opsin, indicating that the GFP+ structure was not a cone.

[0020] Figure 6 This study shows that viruses with both GFAP promoters do not infect retinal astrocytes. Images of retinal tiles stained with GFP (green) and GFAP (red) in the GCL layer containing astrocytes are shown. Images were collected 7 weeks after injection of AAV7m8GFAP-GFP or AAV9GFAP104-GFP. For both viruses, astrocytes without GFAP+ expressed GFP. Detailed Implementation

[0021] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0022] Unless otherwise specified, the materials, reagents, concentrations, experimental operations, and steps used in the embodiments of the present invention are all conventional materials, reagents, concentrations, experimental operations, and steps well known to those skilled in the art.

[0023] The term "NeuroD1 protein" refers to the pre-bHLH neural transcription factor involved in embryonic brain development and adult neurogenesis; see Cho, J He et al., Mol, Neurobiol., 30:35-47, 2004; Kuwabara, T. et al., Nature Neurosci., 12:1097-1105, 2009; and Gao, Z. et al., Nature Neurosci., 12:1090-1092, 2009. NeuroD1 is expressed late in development—primarily in the nervous system—and is involved in neuronal differentiation, maturation, and survival.

[0024] The term "NeuroD1 protein" includes the human NeuroD1 protein (amino acid sequence as shown in SEQ ID NO: 2) and the mouse NeuroD1 protein (amino acid sequence as shown in SEQ ID NO: 4). In addition to the NeuroD1 proteins of SEQ ID NO: 2 and SEQ ID NO: 4, the term "NeuroD1 protein" also includes variants of the NeuroD1 protein, such as those of SEQ ID NO: 2 and SEQ ID NO: 4, which may be included in the methods of the present invention. As used herein, the term "variant" refers to naturally occurring genetic variations and variations prepared by recombination methods, each variant containing one or more changes in its amino acid sequence compared to a reference NeuroD1 protein (such as SEQ ID NO: 2 or SEQ ID NO: 4). Such changes include those in which one or more amino acid residues have been modified by amino acid substitution, addition, or deletion. The term "variant" includes orthologs of human NeuroD1, including, for example, mammalian and avian NeuroD1, such as, but not limited to, NeuroD1 orthologs from non-human primates, cats, dogs, sheep, goats, horses, cattle, pigs, birds, poultry, and rodents (e.g., but not limited to mice and rats). In a non-limiting embodiment, mouse NeuroD1 (which is exemplified herein as having the amino acid sequence SEQ ID NO:4) is an ortholog of human NeuroD1.

[0025] Preferred variants have at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:2 or SEQ ID NO:4.

[0026] Mutations can be introduced using standard molecular biology techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Those skilled in the art will understand that one or more amino acid mutations can be introduced without altering the functional properties of the NeuroD1 protein. For example, one or more amino acid substitutions, additions, or deletions can be made without changing the functional properties of the NeuroD1 protein in SEQ ID NO:2 or SEQ ID NO:4.

[0027] Conserved amino acid substitutions can be performed on the NeuroD1 protein to generate NeuroD1 protein variants. A conserved amino acid substitution is a substitution in which an amino acid is replaced by another amino acid with similar characteristics—a substitution recognized in the art. For example, each amino acid can be described as having one or more of the following characteristics: positively charged, negatively charged, aliphatic, aromatic, polar, hydrophobic, and hydrophilic. A conserved substitution is the replacement of an amino acid with a specific structural or functional characteristic by another amino acid with the same characteristic. Acidic amino acids include aspartic acid and glutamic acid; basic amino acids include histidine, lysine, and arginine; aliphatic amino acids include isoleucine, leucine, and valine; aromatic amino acids include phenylalanine, glycine, tyrosine, and tryptophan; polar amino acids include aspartic acid, glutamic acid, histidine, lysine, asparagine, glutamine, arginine, serine, threonine, and tyrosine; hydrophobic amino acids include alanine, cysteine, phenylalanine, glycine, isoleucine, leucine, methionine, proline, valine, and tryptophan; conserved substitutions include substitutions between amino acids within each group. Amino acids can also be described according to their relative size; alanine, cysteine, aspartic acid, glycine, asparagine, proline, threonine, serine, and valine are generally considered small amino acids.

[0028] NeuroD1 variants may include synthetic amino acid analogs, amino acid derivatives, and / or non-standard amino acids, illustratively including but not limited to α-aminobutyric acid, citrulline, canavonine, cyanalanine, diaminobutyric acid, diaminopimelic acid, dihydroxyphenylalanine, quinolone, homoarginine, hydroxyproline, ortholeucine, orthovaline, 3-phosphoserine, homoserine, 5-hydroxytryptophan, 1-methylhistidine, 3-methylhistidine, and ornithine.

[0029] To determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., vacancies may be introduced in the first amino acid sequence or nucleic acid sequence to optimize alignment with the second amino acid sequence or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. The molecules are considered identical at that position when a position on the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence. The percentage of identity between the two sequences is a function of the number of shared positions (i.e., percentage of identity (%) = number of overlapping positions / total number of positions × 100%). In one embodiment, the two sequences are of the same length.

[0030] Determining the percentage of identity between two sequences can also be accomplished using mathematical algorithms. A preferred, non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm in Karlin and Altschul, 1990, PNAS 87:2264-2268, which was improved in Karlin and Altschul, 1993, PNAS.90:5873-5877. This algorithm is incorporated into the NBLAST and XBLAST procedures in Altschul et al., 1990, J.Mol.Biol.215:403. A BLAST nucleotide search is performed using the NBLAST nucleotide procedure parameter set (e.g., score = 100, word length = 12) to obtain nucleotide sequences homologous to the nucleic acid molecules of the present invention. A BLAST protein search is performed using the XBLAST procedure parameter set (e.g., score = 50, word length = 3) to obtain amino acid sequences homologous to the protein molecules of the present invention. To obtain vacancy alignments for comparative purposes, Gapped BLAST is used as described in Altschul et al., 1997, Nucleic Acids Res. 25:33893402. Alternatively, iterative searching is performed using PSI BLAST—which detects distant relationships between molecules (as above). When using BLAST, Gapped BLAST, and PSI BLAST programs, the default parameters of each program (e.g., XBLAST and NBLAST) are used (see, for example, the NCBI website). Another preferred, non-limiting example of a mathematical algorithm for sequence comparison is the algorithm in Myers and Miller, 1988, CABIOS 4:1117. This algorithm is incorporated into the ALIGN program (version 2.0)—part of the GCG sequence alignment software package. When using the ALIGN program to compare amino acid sequences, it uses a PAM120 weighted residue table, a vacancy length penalty of 12, and a vacancy penalty of 4.

[0031] Whether gaps are allowed or not, a technique similar to the one described above is used to determine the percentage of identity between two sequences. When calculating the percentage of identity, typically only exact matches are counted.

[0032] The term "NeuroD1 protein" includes fragments of the NeuroD1 protein that can be used in the methods and compositions of the present invention, such as fragments of SEQ ID NO:2 and SEQ ID NO:4 and their variants.

[0033] NeuroD1 protein and nucleic acid can be isolated from natural sources, such as cells in the brain or cell lines of organisms expressing NeuroD1. Alternatively, NeuroD1 protein or nucleic acid can be produced through recombinant methods, such as expression in vitro or in vivo using expression constructs. NeuroD1 protein and nucleic acid can also be synthesized using known methods.

[0034] Recombinant nucleic acid technology is preferably used to generate NeuroD1 included in the methods and compositions of the present invention. The production of recombinant NeuroD1 involves introducing a recombinant expression vector containing a DNA sequence encoding NeuroD1 into a host cell.

[0035] Preferably, the nucleic acid sequence encoding NeuroD1 introduced into the host cell to produce NeuroD1 encodes SEQ ID NO:2, SEQ ID NO:4, or a variant thereof.

[0036] According to an aspect of the invention, the nucleic acid sequence of SEQ ID NO:1 encodes SEQ ID NO:2, and is contained in an expression vector and expressed to generate NeuroD1. According to an aspect of the invention, the nucleic acid sequence of SEQ ID NO:3 encodes SEQ ID NO:4, and is contained in an expression vector and expressed to generate NeuroD1.

[0037] It should be understood that, due to the degeneracy of the genetic code, nucleic acid sequences substantially identical to SEQ ID NO:1 and SEQ ID NO:3 encode NeuroD1 and its variants, and such alternative nucleic acids can be included in expression vectors and expressed to produce NeuroD1 and its variants. Those skilled in the art will appreciate that nucleic acid fragments encoding the NeuroD1 protein can be used to produce fragments of the NeuroD1 protein.

[0038] The term "expression vector" refers to a recombinant vector used to introduce a nucleic acid encoding NeuroD1 into a host cell, either in vitro or in vivo (wherein the nucleic acid is expressed to produce NeuroD1). In a specific embodiment, an expression vector containing the nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:3 or substantially the same nucleic acid sequence is expressed in cells containing this expression vector to produce NeuroD1. The term "recombinant" is used to refer to a nucleic acid construct in which two or more nucleic acids are linked together and they are not linked together in nature. Expression vectors include, but are not limited to, plasmids, viruses, BACs, and YACs. Specific viral expression vectors exemplarily include those derived from adenoviruses, adeno-associated viruses, retroviruses, and lentiviruses, and can also be non-viral vectors, including those using liposome nanoparticles to deliver RNA.

[0039] Expression vectors contain nucleic acids containing fragments encoding a target polypeptide, which are operatively linked to one or more regulatory elements that provide for transcription of fragments encoding said target polypeptide. As used herein, the term "operatively linked" refers to a nucleic acid in a functional relationship with another nucleic acid. The term "operatively linked" includes the functional connection of two or more nucleic acid molecules (such as the nucleic acid to be transcribed and a regulatory element). As used herein, the term "regulatory element" refers to a nucleotide sequence that controls certain aspects of the expression of the operatively linked nucleic acid. Exemplary regulatory elements include enhancers, such as, but not limited to: marmot hepatitis virus posttranscriptional regulatory elements (WPREs), internal ribosome entry sites (IRES) or 2A domains, introns, origins of replication, polyadenylation signals (pA), promoters, transcription termination sequences, and upstream regulatory regions that facilitate the replication, transcription, and posttranscriptional processing of the operatively linked nucleic acid sequence. Those skilled in the art can select and use the above and other regulatory elements in expression vectors using only routine experiments.

[0040] As used herein, the term "promoter" refers to a DNA sequence that is operatively linked to a nucleic acid sequence to be transcribed (such as the nucleic acid sequence encoding NeuroD1). Promoters are typically located upstream of the nucleic acid sequence to be transcribed and provide sites for the specific binding of RNA polymerases and other transcription factors. In specific embodiments, promoters are typically located upstream of the nucleic acid sequence to be transcribed to generate the desired molecule and provide sites for the specific binding of RNA polymerases and other transcription factors.

[0041] Those skilled in the art understand that the 5' untranslated region of a gene can be isolated and used as a whole as a promoter to drive the expression of operably linked nucleic acids. Alternatively, a portion of the 5' untranslated region can be isolated or used to drive the expression of operably linked nucleic acids. Typically, about 500-6000 bp of the 5' untranslated region of a gene is used to drive the expression of operably linked nucleic acids. Optionally, a portion of the 5' untranslated region of a gene is used, containing the minimum amount of 5' untranslated region necessary to drive the expression of operably linked nucleic acids. Experiments used to determine the ability of a specific portion of the 5' untranslated region of a gene to drive the expression of operably linked nucleic acids are well known in the art.

[0042] Promoters can be entirely derived from a single gene or they can be chimeric, meaning they have parts derived from more than one gene.

[0043] According to aspects of the invention, delivery routes may include intraocular injection and blood injection.

[0044] I. Methods

[0045] 1. Animals

[0046] Wild-type (C57BL / 6J) mice were purchased from the Guangdong Laboratory Animal Center, China. These animals were housed under standard conditions with a 12 / 12-hour light / dark cycle and had free access to regular food and water.

[0047] The animal experimental procedures were conducted in accordance with ARVO’s Statement on the Use of Animals for Ophthalmological and Visual Research and were approved by the Ethics Committee of Jinan University (Approval No. #IACUC-20201118-03).

[0048] 2. Virus construction and injection

[0049] The virus was created by OBiO Technology (Shanghai).

[0050] The constructed virus includes AAV7m8 GFAP::GFP-P2A-ND1 (containing the mouse NeuroD1 gene sequence SEQ ID NO:3) and its control AAV7m8 GFAP::GFP ( Figure 1 A) AAV9 GFAP104::ND1-P2A-GFP (containing mouse NeuroD1 gene sequence SEQ ID NO:3) and its control AAV9 GFAP104::GFP ( Figure 3 A).

[0051] For adeno-associated virus AAV7m8, the GFAP promoter is a synthetic 681-bpgfaABC1D derived from the 2.2-kb gfa2 promoter (Lee Y, Messing A, Su M, Brenner M (2008) GFAP promoter elements required for region-specific and astrocyte-specific expression. Glia 56:481-493.).

[0052] For AAV9, the GFAP104 promoter consists of the ef1α enhancer and the subsequent gfaABC1D promoter (Perea G, Yang A, Boyden ES, Sur M (2014) Optogenetic astrocyte activation modulates response selectivity of visual cortex neurons in vivo. Nat Commun 5:3262.).

[0053] For the titer used in the experiment, except for the titer experiment, AAV7m8 is 3×10. 12 GC / mL, AAV9 is 1×1012 GC / mL. The AAV9 titer used in the titer experiment was 10. 11 10 12 and 10 13 GC / mL.

[0054] For viral injection, animals were anesthetized by intraperitoneal injection of 1.25% tribromoethanol (0.1 ml / 10 g body weight), and the pupils were dilated with 0.5% tropicamide solution (Santen Pharmaceutical Co., Ltd., Japan). A 3 mm diameter slide was placed on the mouse cornea to make the fundus clearly visible. After puncturing a hole in the limbus with a sharp 30-gauge needle, 0.8–1.2 μl of virus was injected into the vitreous body (1.2 μl) or subretinal space (0.8 μl) using a 34-gauge flat-tipped needle. The needle remained in the vitreous body for an additional 10 seconds before being slowly withdrawn, and a drop of antibiotic gel was instilled into the eye. The mice were heated on a safe heating pad until fully awake. Animals exhibiting cataracts or inflammation were excluded from future experiments. To minimize inter-animal variability, control virus and virus expressing the ND1 protein were injected into the right and left eyes of the same animal, respectively.

[0055] To induce NMDA (N-methyl-D-aspartic acid) damage in the mouse retinas, 1.2 μl of NMDA (N-methyl-D-aspartic acid) solution (20 mM) was injected intravitreally into the eyes of adult mice one day before viral injection.

[0056] 3. Organizational handling

[0057] To examine the effects of viral infection, animals were euthanized by overdose anesthesia at different time points after viral injection, their eyes were removed, and they were fixed in 4% paraformaldehyde (PFA) at room temperature for 30 minutes.

[0058] For retinal sections, the eyecups containing the lens were then washed with PBS and cryoprotected overnight at 4°C in 0.01M PBS containing 30% sucrose, and finally embedded in OCT embedding medium (Tissue Tek, Torrance, Canada). The retina was longitudinally cryosectioned through the optic disc (OD) at a thickness of 10 μm using a microtome (Leica Microsystems, Buenos Aires, Argentina). Subsequently, the retinal sections were mounted on glass slides for future processing. For retinal tilings, after PFA fixation, the retina was removed from the other layers, flattened on a glass slide, and sealed with Vectashield Antifade mounting medium, or stored directly in PBS at 4°C for future immunostaining.

[0059] 4. Immunofluorescence

[0060] To perform immunostaining of markers for retinal glial cells and neurons, retinal sections were blocked for 1 hour in 0.01M PBS containing 10% normal donkey serum, 3% bovine serum albumin, and 0.3% Triton X-100, and then incubated overnight at 4°C with primary antibody. After thorough washing, the retinal sections were incubated with secondary antibody at room temperature for 2 hours, followed by washing, mounting, and coverslipping.

[0061] For DAPI staining, sections were incubated with DAPI (1:1000, Electron Microcopy Sciences, Hatfield, PA) at room temperature for 5 minutes before embedding. For fully embedded retina, the retina was blocked for 1 hour with 0.01M PBS containing 10% normal donkey serum, 3% bovine serum albumin, and 3% Triton X-100, and incubated overnight at 4°C with primary antibody, then washed and incubated with secondary antibody at room temperature for 2 hours.

[0062] The primary antibodies used included: chicken anti-GFP (1:1000, Aveslabs, GFP-1020), mouse anti-glutamine synthase (GS) (1:1000, Millipore, MAB302), rabbit anti-Sox9 (1:1000, Millipore, AB5535), rabbit anti-Calretinin (1:1000, SWant, CR7697), rabbit anti-RPBMS (1:1000, Invitrogen, PA5-31231), rabbit anti-Recoverin (1:1000, Millipore, AB5585), rabbit anti-Opsin (1:1000, Millipore, AB5405), rabbit anti-Calbindin (1:1000, Abcam, ab49899), and rabbit anti-NeuroD1 (1:1000, Invitrogen, PA5-78075).

[0063] The secondary antibodies used were as follows: donkey anti-chicken 488 (1:1000, Jackson Immuno Research, 703-545-155), donkey anti-rabbit 647 (1:1000, Invitrogen, A-31573), and goat anti-mouse 594 (1:1000, Invitrogen, A-32744).

[0064] 5. Image acquisition, processing, and analysis

[0065] Immunofluorescence stained tissue was imaged using a confocal microscope (Carl Zeiss LSM700).

[0066] Since the virus did not infect the entire retina, only the regions with GFP signals were sampled.

[0067] For retinal slices, Z-stacks with a step size of 1 μm were collected to cover the GFP+ region.

[0068] For retinal tiling, Z-stacks with a step size of 1 μm are collected in the inner nuclear layer (INL) (the region containing the cell bodies of Müller glial cells and most amacrine cells), the ganglion cell layer (GCL) (the region containing ganglion cells and some amacrine cells), or the outer nuclear layer (ONL) (the region containing photoreceptor cells).

[0069] GFP+ cells and those double-stained with glial or neuronal markers were manually counted by independent observers using Zen software (NIH, Bethesda, MD). For each retina, a data point was obtained by averaging 3–6 images, and then the average of all data points across a set of retinas was taken.

[0070] To estimate the efficiency of local infection, the number of GFP+ cells was counted on each image. To analyze the occurrence of reprogramming, GFP+ cells lacking expression of the Müller glial cell marker Sox9 (Sox9-GFP+) were counted and divided by the total number of GFP+ cells. To estimate the reprogramming efficiency of a certain type of neuron, GFP+ cells co-expressing the marker for that type of neuron were counted and then divided by the total number of GFP+ cells.

[0071] II. Results

[0072] Previous studies have shown that overexpression of a single neural transcription factor ND1 in astrocytes can convert them into neurons in vitro and in vivo, using retroviruses that infect only dividing glial cells or AAV (Guo et al., 2014) (Wu et al., 2020) (Chen et al., 2020) (Ge et al., 2020) (Zheng Jiajun et al., Prog Neurobiol, 2021). Here, this invention further investigates whether ND1 can convert Müller glial cells in the retina into neurons to develop a neuroregenerative therapy for the treatment of retinal degenerative diseases.

[0073] 1. NeuroD1 reprograms Müller glial cells into internal retinal neurons.

[0074] To test whether NeuroD1 (ND1) can convert Müller glial cells in the retina into neurons, AAV7m8 GFAP::GFP-ND1 (ND1 group) or a control virus expressing GFP alone (AAV7m8GFAP::GFP) (control group) were first injected intravitreal. Figure 1 A).

[0075] The retina was examined 5 or 28 days after injection (dpi) using a series of immunostaining assays targeting glial cell and neuronal markers (experimental protocol as follows). Figure 1 (As shown in B).

[0076] At 5 dpi, for both the control and ND1 groups, virtually all GFP+ cells expressed the glial cell marker Sox9 in the Müller glial cell layer. Figure 1 (C, left column), confirming AAV's specific infection of Müller glial cells.

[0077] At 28 dpi, all GFP+ cells in the control group maintained typical Müller glial cell morphology, with cell processes spanning the entire retina, and cell bodies expressing Sox9 (…). Figure 1 (C, top right figure). In contrast, in the ND1 group at 28 dpi, although most GFP+ cells were still Sox9+ Müller glial cells, a small subset of GFP+ cells lost Sox9 signaling and were located off-center from the Müller glial cell layer. Figure 1 (C, bottom right corner, white arrow).

[0078] Further immunostaining with neuronal markers from the inner retinal layer was performed to observe whether GFP+ cells expressed neuronal markers. The results showed that 28 days after ND1 infection, some GFP+ cells expressing the ganglion cell marker RBPMS were identified in the ganglion cell layer (GCL). Figure 1 D; D1 to D4 show enlarged cells in the ND1 group). Furthermore, some GFP+ cells expressing the amacrine marker Calretinin in the nuclear layer (INL) were identified in the ND1 group at 28 dpi. Figure 1 (D; D'1 to D'4 show enlarged cells in the ND1 group). However, almost no GFP+ cells expressing neuronal markers were found in the control virus group.

[0079] Immunostaining of retinal tilings also revealed significant differences between the control group and the ND1 group. Figure 1 In the control group, cells infected with AAV7m8 GFAP::GFP showed a typical polygonal shape of Müller glial cell bodies in both the INL and GCL layers. Figure 1(E, top row), but in the ND1 group, cells infected with AAV7m8 GFAP::GFP-ND1 showed oval or round cell bodies in the INL or GCL layers ( Figure 1 Quantitative analysis revealed that in the control group, the percentage of Sox9+GFP+ double-positive cells in all GFP+ cells was close to 100% from 5 dpi to 28 dpi. Figure 1 (The blue bar in F). Conversely, in the ND1 group, the percentage of Sox9+GFP+ double-positive cells in all GFP+ cells decreased from nearly 100% at 5 dpi to 86% at 28 dpi. Figure 1 The green bars in F indicate that some GFP+ cells gradually lose the characteristics of Müller glial cells after ND1 infection. This percentage decrease in Müller glial cell markers in the ND1 group matches very well with the percentage increase in internal retinal markers (…). Figure 1 (G, 5% GFP+RBPMS+ double positive cells and GFP+Calretinin+ double positive cells). Similarly, in 28 dpi ND1 group retinal tilings, 11.6% of cells showed neuronal morphology in the INL layer and 6.8% of cells showed neuronal morphology in the GCL layer.

[0080] Therefore, intravitreal injection of AAV7m8 GFAP::GFP-ND1 can convert Müller glial cells into inner retinal neurons, but the conversion efficiency is limited.

[0081] 2. Mueller glial cell transformation in the NMDA injury model

[0082] Previous studies have shown that zebrafish Müller glial cells can regenerate ganglion cells in a ganglion cell injury model and photoreceptor cells in a photoreceptor cell injury model (Nagashima et al., 2013), indicating that injury itself is a factor stimulating regeneration. Therefore, this invention investigates whether Müller glial cells can also be transformed more effectively in injury models.

[0083] Cell death was induced by intravitreal injection of NMDA one day before AAV infection, and AAV7m8-infected cells were examined 8 weeks after viral injection (procedure as follows). Figure 2 (As shown in Figure A).

[0084] In the control group, most GFP+ cells remained Müller glial cells with typical Müller glial cell morphology, with only a very small number of cells at the base of the INL expressing the non-protruding cell marker Calretinin. Figure 2B, left control column, yellow arrow points to a co-labeled cell). However, in the ND1 group, most ND1-infected cells had transformed into neuronal morphology and expressed neuronal markers such as calretinin (…). Figure 2 B, middle column) or Brn3a ( Figure 2 (B, right column). The reprogramming effect is even more pronounced when examining the entire retina in areas of viral infection.

[0085] In the control group, the cell bodies of GFP+ cells were well embedded among the Calretinin+ cell bodies in the INL. Figure 2 (C, left image, white arrow in enlarged C1 inset). Conversely, in the ND1 group, many GFP+ cell bodies overlapped with Calretinin+ cell bodies, indicating successful ND1 reprogramming. Figure 2 (C, right figure, yellow arrow in enlarged C2 illustration).

[0086] Quantitative analysis showed that in the NMDA injury model, ND1 transformed Müller glial cells into Calretinin+ amacrine cells with a transformation efficiency of 46.6±7.1% (n=3 mice), while the control group (n=3 mice) had an efficiency of 5.8±1.5%. Figure 2 D).

[0087] Therefore, experimental data show that ND1 can reprogram Müller glial cells into inner retinal neurons in the retina of NMDA-damaged mice, which appears to be more effective than in undamaged retinas.

[0088] 3. Enhanced NeuroD1 expression converts Müller glial cells into outer retinal neurons in a time-dependent manner.

[0089] Because intravitreal injection of AAV7m8 GFP-ND1 resulted in low transformation efficiency of virus-infected Müller glial cells, we performed immunostaining to investigate ND1 expression levels. Surprisingly, although GFP signaling was clearly detectable, almost no significant ND1 signaling was detected in ND1-infected cells.

[0090] To this end, this invention designs a novel vector, AAV9 GFAP104::ND1-P2A-GFP, which is a different serotype of AAV9 with the addition of an EF1a enhancer to increase ND1 expression, and the sequence is converted to ND1-P2A-GFP. Figure 3 A). This invention also changes the injection site from the vitreous body to subretinal injection ( Figure 3 B).

[0091] Following these changes, subretinal injection of AAV9 ND1-GFP resulted in efficient expression of ND1 (red) in infected Müller glial cells (green), which was confirmed by immunostaining with the Müller glial cell marker glutamine synthase (GS, purple). Figure 3 In contrast, for intravitreal injection of AAV7m8GFAP::ND1-GFP, in the ND1 group, GFP+ cells showed weaker expression of ND1 and GS (D). Figure 3 C).

[0092] Therefore, this new AAV9 vector was used for further experimental analysis to investigate whether it could convert Müller glial cells into neurons.

[0093] To understand how ND1 causes changes in Müller glial cells, a series of different time points were examined at 3, 5, 7, 14, and 28 dpi.

[0094] In the control group, from 3 dpi to 28 dpi, GFP+ cells maintained the Müller glial cell morphology and expressed Sox9. Figure 4 (A, left column). In the ND1 group, cells infected with AAV9 ND1-GFP initially also exhibited typical Müller glial cell morphology, and from 3 dpi to 7 dpi, most GFP+ cells expressed Sox9 ( Figure 4 (A, ND1 group, 3-7 dpi). However, by 14 dpi, the Müller glial cell morphology disappeared in ND1-GFP infected cells; instead, most GFP+ cells appeared in the ONL (outer nuclear layer), where GFP+ photoreceptor cell bodies appeared ( Figure 4 (Group A, ND1, 14 dpi). Furthermore, numerous GFP+ fibers were observed protruding outside the ONL, resembling the segmental structure of photoreceptor cells. Figure 4 (A, ND1 group, 14 dpi). By 28 dpi, the ND1 group showed more GFP+ cells in the ONL and an increased number of ciliate-like processes outside the ONL. Figure 4 , A, ND1 group, 28dpi).

[0095] To verify the cellular characteristics of ND1-transformed cells, immunostaining was performed on the photoreceptor marker protein Recoverin. The results showed that most ND1-transformed cells were Recoverin+ photoreceptor cells. Figure 4 (B, left column). In addition to photoreceptor cell markers, a small amount of the ND1-transformed cell marker calcium-binding protein (Calbindin) was also found to be expressed at low levels. Figure 4 (B, right column).

[0096] ND1-induced cell transformation can also be clearly seen in retinal tile images. Figure 4 In the control group, GFP+ cells did not overlap with opsin, calbindin, or RBPMS. Figure 4 (C, top row); conversely, in the ND1 group, many GFP+ cilia and some GFP+ cell bodies colocalize with calcium-binding proteins, but rarely with RBPMS ( Figure 4 Quantitative analysis at different time points showed that in the control group, almost all virus-infected cells were Sox9+ Müller glial cells, which were rarely found in ONL (C, bottom row). Figure 4 (D, top figure). In the ND1 group, ND1-GFP-infected cells showed a gradual loss of the Müller glial cell marker Sox9, accompanied by a gradual increase in photoreceptor cells in the ONL ( Figure 4 (D, see diagram below).

[0097] Quantitative immunostaining results (28 dpi) also showed that approximately 80% of ND1-GFP-infected cells expressed recovery protein, and approximately 5% expressed calcium-binding protein (CBP). Figure 4 These results indicate that ND1-induced conversion of Müller glial cells in the retina of adult mice into photoreceptor cells is time-dependent, meaning the conversion increases over time.

[0098] 4. NeuroD1-induced Müller glial cell transformation is dose-dependent. Next, a series of different AAV doses were investigated to find the appropriate dose for Müller glial cell transformation.

[0099] With 3 different doses 10 11 10 12 and 10 13 Subretinal injections of GC / ml (of the same volume) were performed on control AAV9GFAP104::GFP and experimental AAV9GFAP104::ND1-P2A-GFP, and retinal samples were collected at 28 dpi for immunostaining. Figure 5 A).

[0100] In the control group ( Figure 5 In the top row (B), a small number of GFP-infected Müller glial cells were observed in 10 11 Sox9 is expressed at low doses of GC / ml, but at 10... 12 At a moderate dose of GC / ml, the number of GFP+ cells increased significantly, and at 10 13 The increase was greater at higher doses of GC / ml. At all three doses, virtually no significant GFP+ cells were found in the ONL of the control group. Figure 5 B, top row). In contrast, in ND1 group ( Figure 5 In B, bottom row), in 10 11 At low doses of GC / ml, GFP+ Müller glial cells were rarely detected, except for a few GFP+ cells in the ONL. At 10 12 At moderate doses of GC / ml, ONL contains more ND1-GFP-transformed cells, and there are many GFP+ cilia in the outer segment of ONL. Figure 5 (B, bottom row). In 10 13 At high doses of GC / ml, a greater number of ND1-GFP-transformed cells were detected in ONL. Figure 5 (C). Medium dose 10 12 GC / ml and high dose 10 13 Immunostaining with GC / ml of opsin showed that, with a few exceptions, most GFP+ cilia in the ND1 group did not express opsin ( Figure 5 (D) indicates that these cilia may have originated from transdifferentiated rod cells.

[0101] The above experimental results indicate that high expression of ND1 can convert Müller glial cells into photoreceptor cells in a dose-dependent manner.

[0102] also, Figure 6 This study demonstrated that viruses with the GFAP promoter do not infect retinal astrocytes. Retinal slices were collected 7 weeks after injection of AAV7m8 GFAP-GFP or AAV9 GFAP104-GFP, and the GCL layer containing astrocytes was double-stained with GFP (green) and GFAP (red). The results showed that no GFAP+ astrocytes expressed GFP for either virus.

[0103] This demonstrates that specific overexpression of NeuroD1 in Müller glial cells can convert them into neurons in a time- and dose-dependent manner.

[0104] This invention tested two different AAV serotypes (AAV7m8 and AAV9), with two different GFAP promoters (GFAP681 and GFAP104) and different sequences of NeuroD1 and GFP (GFP-NeuroD1 and NeuroD1-GFP).

[0105] AAV7m8 infected Müller glial cells well, but their NeuroD1 expression levels were relatively low. NeuroD1, through the weak expression of AAV7m8, caused Müller glial cells to transform into amacrine cells in the INL.

[0106] In contrast, NeuroD1, through high expression of AAV9, leads to the primary conversion of Müller glial cells into photoreceptor cells. Subretinal injection of AAV9 GFAP104::NeuroD1-P2A-GFP not only effectively infects Müller glial cells, but also exhibits higher ND1 expression levels compared to AAV7m8 GFAP681::GFP-P2A-ND1, and AAV9 GFAP104::NeuroD1-P2A-GFP converts Müller glial cells into photoreceptor cells in the ONL.

[0107] Related studies have shown that different transcription factors can reprogram Müller glial cells into different neuronal subtypes.

[0108] This invention can be applied to the in situ regeneration of new neurons from retinal Müller glial cells to restore vision in damaged or degenerative retinas. AAV9 GFAP104::NeuroD1-GFP can be used to reprogram Müller glial cells into photoreceptor cells after subretinal injection.

Claims

1. Use of NeuroD1 in the preparation of drugs that convert retinal Müller glial cells into neurons.

2. The use according to claim 1, characterized in that, The neurons in question are neurons in the inner retina.

3. The use according to claim 2, characterized in that, The neurons are amacrine cells and / or ganglion cells.

4. The use according to claim 1, characterized in that, The neurons in question are neurons in the outer layer of the retina.

5. The use according to claim 4, characterized in that, The neurons are photoreceptor cells.

6. The use according to claim 1, characterized in that, Overexpression of the neural transcription factor NeuroD1 in mouse retinal Müller glial cells.

7. The use according to claim 6, characterized in that, Overexpression of the neural transcription factor NeuroD1 includes: infection of mouse retinal Müller glial cells with a recombinant viral vector expressing NeuroD1.

8. The use according to claim 7, characterized in that, The viral vector is an adeno-associated virus vector.

9. The use according to claim 8, characterized in that, The adeno-associated virus vector is AAV7m8 or AAV9.

10. The use according to claim 8 or 9, characterized in that, An enhancer was added to the adeno-associated virus vector to increase NeuroD1 expression.

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