Retinal ganglion cell regeneration

Abstract

Provided herein are compositions and methods for regenerating retinal ganglion cells (RGCs) from retinal neuronal cells by activating one or more transcription factors, such as Atoh7, Brn3B, Sox4, Sox11, or Ils1. Retinal neuronal cells can be interneuronal cells, such as amacrine cells, horizontal cells, and bipolar cells. Regenerated RGCs can project axons to dispersed subcortical brain regions and establish retina-brain connections. They can respond to visual stimuli and transmit electrical signals to the brain. Thus, regenerated RGCs can replace damaged or degenerated RGCs, thereby treating visual dysfunction or blindness. This method is equally applicable to degenerated, damaged, or aged RGCs, rejuvenating them by stimulating them to regrow functional axons.

Description

[Technical Field]

[0001] This application claims priority to Application No. 202010047628.2, filed January 16, 2020, the contents of which are incorporated herein in their entirety.

[0002] Retinal ganglion cells (RGCs) are the final output neurons in the retina, processing visual information and transmitting it to the distributed visual cortex of the brain to form vision. Loss of RGCs is a major cause of blindness in a group of diseases broadly classified as optic neuropathies, including glaucoma, hereditary optic neuropathies, and disorders caused by toxins, malnutrition, and trauma. Because humans and all mammals lose the ability to generate RGCs at adulthood, vision loss in these patients is irreversible. There is considerable interest in developing regenerative therapies to restore lost vision in these patients. [Background technology]

[0003] One attractive approach to developing regenerative therapies for optic nerve disorders is to replace lost ganglion cells and reconnect the retina to the brain using endogenous cells. Significant efforts have been made to identify retinal stem / progenitor cells and understand how retinal neurons are generated in various model organisms. Previous studies have demonstrated that lower vertebrates, such as fish and amphibians, can functionally regenerate the retina after injury, with Müller glia serving as the cellular source of regenerating retinal neurons. In contrast, mammalian Müller glia lack this ability, and mammals, including humans, lack other retinal stem / progenitor cell reservoirs poised to regenerate retinal neurons in adulthood. The current consensus is that continuous neuronal addition to the mature mammalian retina is typically rare.

[0004] There is a great need to treat these diseases and conditions and restore vision to patients. Summary of the Invention [Problem to be solved by the invention]

[0005] The present disclosure relates to Atoh7, Brn3B, Sox4, Sox11, or Isl1 We report the discovery that retinal ganglion cells (RGCs) can be regenerated from retinal neurons by activating one or more of the transcription factors. Regenerated RGCs can project axons to dispersed subcortical brain regions and establish retina-brain connections. They can respond to visual stimuli and transmit electrical signals to the brain. Therefore, regenerated RGCs can treat visual dysfunction or blindness by replacing damaged or degenerated RGCs.

[0006] Another unexpected finding is that activating these transcription factors can reactivate degenerated, damaged, or senescent RGCs, resulting in functional axon regeneration. Therefore, administering therapeutic agents capable of activating these transcription factors to a subject can rejuvenate degenerated, damaged, or senescent RGCs while simultaneously reprogramming nearby interneuron cells into regenerative RGCs. This dual effect of the agent can more effectively achieve the desired therapeutic effect.

[0007] According to one embodiment of the present disclosure, there is provided a method for preparing mammalian cells that respond to visual signals, comprising: expressing POU class 4 homeobox 2 (Brn3B), SRY box transcription factor 4 (Sox4), atonal BHLH transcription factor 7 (Atoh7), SRY box transcription factor 11 (Sox11), and ISL LIM homeobox 1 (ISL LIM homeobox 1) in retinal neuron cells; Isl1 In one embodiment, methods are provided that include increasing the biological activity of one or more genes selected from the group consisting of:

[0008] In some embodiments, the one or more genes include Brn3B and Sox4. In some embodiments, the one or more genes further include Atoh7.

[0009] In some embodiments, the retinal neuron cells are interneuron cells, such as amacrine cells, horizontal cells, or bipolar cells. In some embodiments, the retinal neuron cells are degenerated, damaged, or senescent retinal ganglion cells (RGCs). In some embodiments, the retinal neuron cells are Lgr5 + In some embodiments, the retinal neuron cells are amacrine cells. + These are replacement amacrine cells.

[0010] In another embodiment, the present disclosure provides a method for improving the function of retinal ganglion cells (RGCs), which may be degenerated, damaged, aged, or normal / healthy retinal ganglion cells for which improved function is desired. In some embodiments, the method comprises increasing the expression of Atoh7, Brn3B, Sox4, Sox11, and Isl1 The method involves increasing the biological activity of one or more genes selected from the group consisting of:

[0011] In some embodiments, increasing the biological activity of one or more genes comprises introducing one or more polynucleotides encoding the genes, such as cDNAs, into the retinal neuron cells, which can be provided in a plasmid or viral vector, such as an adeno-associated viral (AAV) vector.

[0012] Also, Brn3B, Sox4, Atoh7, Sox11 and Isl1 A method of treating visual impairment or blindness in a subject in need thereof is provided, comprising administering to the retina of the subject an agent capable of increasing the biological activity of one or more genes selected from the group consisting of:

[0013] In some embodiments, the visual dysfunction or blindness is caused by degenerated retinal ganglion cells (RGCs). In some embodiments, the visual dysfunction or blindness is associated with a condition selected from the group consisting of optic neuropathies, including glaucoma, hereditary optic neuropathies, and disorders caused by toxins, malnutrition, and trauma.

[0014] Also provided in one embodiment is a nucleic acid construct comprising coding sequences encoding Brn3B and Sox4 proteins and a promoter associated with each coding sequence, wherein each promoter is active in retinal neuron cells.

[0015] Another embodiment provides a cell transfected with the nucleic acid construct. Yet another embodiment provides a cell responsive to a visual signal prepared by the methods disclosed herein.

[0016] These and other embodiments are further described in the following text. [Brief explanation of the drawings]

[0017] [Figure 1]This figure shows that Lgr5+ amacrine interneurons differentiate into other neuronal subtypes in adult mice. a) Retinal cross-section showing Lgr5+ amacrine interneurons in the inner nuclear layer. b, c) Retinal cross-sections from an Lgr5EGFP-IRES-CreERT2;Rosa26-tdTomato mouse. Arrows highlight the generation of bipolar (b) and horizontal (c) cells from Lgr5+ amacrine interneurons. d) Image of a flat-mounted retina sample focused on the retinal ganglion cell layer. Lgr5+ amacrine interneurons that migrated from the inner nuclear layer to the ganglion cell layer are labeled green. e) Representative membrane potential of an Lgr5+ amacrine cell in response to a full-field light flash. Inset: Fluorescence image of the recorded cell after dye filling. f) Representative excitatory postsynaptic currents (EPSCs, blue) and inhibitory postsynaptic currents (IPSCs, red) from an Lgr5+ amacrine cell in response to a full-field light flash. Overall, five of six recorded cells responded to full-field LED light stimulation. Arrows in panels e and f: stimulation artifact. ONL: external nuclear layer; OPL: outer plexiform layer; INL: inner nuclear layer; IPL: inner plexiform layer; GCL: ganglion cell layer. Scale bars = 30 μm in panels a, b, and c; = 200 μm in panel d; = 10 μm in panel e. [Figure 2]Figure 1. In vivo reprogramming of Lgr5+ amacrine interneurons into RGCs. a) In vivo neuronal reprogramming strategy. Lgr5EGFP-IRES-CreERT2;Rosa26-tdTomato mice were first given five doses of tamoxifen (TM) (from day -11 (D-11) to day -7 (D-7)) to label Lgr5+ amacrine interneurons with the Rosa26-tdTomato reporter and aid in tracking their identity. One week later, mice were intravitreally injected with AAV expressing Cre-dependent transcription factors on D1 and then given TM from D3 to D7 to specifically activate the AAV-delivered genes in Lgr5+ amacrine interneurons. Mice were sacrificed 6 weeks after viral injection for analysis. b) Diagram of an AAV expression vector using the Cre-dependent inverted open reading frame (DIO) system. c) Reprogramming efficiency of single and various combinations of transcription factors. Data shown are the number of tdTomato+ axons in the optic nerve (n = 8 from 4 mice per group). No tdTomato+ axons were detected in the optic nerves of mice injected with AAV-DIO-EGFP. (D-F) Images of flat-mounted retinal samples from experimental mice, focusing on the retinal ganglion cell layer. e, Higher magnification of the area in panel D; the arrow points to a tdTomato+ axon of a regenerating RGC. f, Highlighting a single regenerating RGC. gi, Immunohistochemical staining of regenerating RGCs using antibodies specific for RPBMS (g), Brn3A (h), and CART (i). **P < 0.001; NS, not significant. Abbreviations in panel c: B = Brn3B, S4 = Sox4, BS4 = Brn3B + Sox4, ABS4 = Atoh7 + Brn3B + Sox4, ABS4S11Is = Atoh7 + Brn3B + Sox4 + Sox11 + Isl1. Scale bars = 200 μm in panel d, = 150 μm in panel e, = 50 μm in panel f, and = 40 μm in panels g, h, and i. [Figure 3]This figure shows that regenerating RGCs project their axons into the brain. a) Confocal images of regenerating RGC axons within the optic nerve. b) and f) Regenerating RGC axon projections in the visual cortex. tdTomato+ axon terminals are shown in the dorsal and lateral ventral geniculate nuclei (dLGN and vLGN, panels b) and c), the pretectal olivary nucleus (OPN, panel d), and the superior colliculus (SC, panels e) and f). The arrow in panel f highlights bouton-like structures in regenerating RGC axon terminals in the SC region. g) PSD-95 staining of SC brain sections. tdTomato+ varicosities are juxtaposed near but not overlapping the PSD-95 staining. The staining patterns are identical in brain samples from three different animals. Scale bars: 200 μm in panels a-e, 40 μm in panel f, and 10 μm in panels g and h. [Figure 4] Reprogramming of Prokr2+ replacement amacrine interneurons into RGCs. a) Confocal image of a flat-mounted retinal sample from a Prokr2CreERT2;Rosa26-tdTomato mouse. tdTomato+ replacement amacrine cells are shown in red. Prokr2-tdTomato+ replacement amacrine cells lack axons in the flat-mounted retinal sample. b) Highlighted area in panel a. c) Confocal image of a flat-mounted retinal sample from a Prokr2CreERT2 mouse injected with an AAV co-expressing transcription factors and EGFP. Regenerated RGCs extend axons to the optic nerve head. d) Highlighted area in panel c. e) Regenerated RGC axons within the optic nerve. f-k) Regenerated RGC axons project to various cerebral retinal receptor regions, including the dLGN and vLGN (f), OPN (g), and the upper (panels h and i) and lower (panels j and k) SC regions. Images in panels i and k are shown at higher magnification. Scale bars: 1000 μm in panels a and c, 200 μm in panels e–g and j, 100 μm in panel h, and 40 μm in panels i and k. [Figure 5]Regenerating RGCs transmit visual information to the brain and establish functional connections with postsynaptic neurons. a) Calcium signal traces from three example axon terminals in response to drifting gratings of different orientations. The cyan patches indicate the duration of stimulus presentation, and the numbers at the bottom indicate the stimulus direction. Note that the three terminals shown in this panel have strong "on" responses. b) The same plot as panel A, but the terminal shown here has strong "off" responses. c, d) Calcium signal traces from three axon terminals with orientation (c) and direction (d) selectivity. e) Representative EPSCs of light-evoked postsynaptic AMPA receptor responses in an SC neuron. Arrows indicate postsynaptic responses to multiple presynaptic inputs. f) Representative EPSCs of light-evoked postsynaptic NMDA receptor responses in an SC neuron. g) An example of a light-evoked postsynaptic action potential in an SC neuron. h, i) Summary of EPSC amplitude (h) and peak number (i) of light-evoked AMPA receptor responses (n = 11 from 7 animals). j, Summary of EPSC amplitudes of light-evoked NMDA receptor responses (n = 3 from 3 animals). Data are mean ± sem. [Figure 6]Figure 1 shows the regeneration of functional RGCs in a mouse model of glaucoma. (a) Confocal images of retinal samples. (a) Retina from a normal Lgr5EGFP-IRES-CreERT2;Rosa26-tdTomato mouse. (b) Retina from a mouse damaged by intraocular pressure elevation (IPI) 7 days prior. (c) Retina from a mouse damaged by IPI 7 days prior but receiving daily ripasudil treatment. (d) Retina from a mouse damaged by IPI but receiving both ripasudil treatment and injection of an AAV expressing an RGC fate-specifying transcription factor (AAV-DIO-TF). The mouse was sacrificed 6 weeks after AAV injection. (e) and (f) Confocal images of the optic nerve from eyes treated with AAV-DIO-EGFP (e) and AAV-DIO-TF (f). gk, Confocal images of brain sections from Lgr5EGFP-IRES-CreERT2;Rosa26-tdTomato mice injected with AAV-DIO-EGFP in the left eye and AAV-DIO-TF in the right eye. The majority of regenerated RGC axons projected contralaterally (left), while a small proportion projected ipsilaterally (right). The visual areas shown are the optic tract immediately adjacent to the optic chiasm (g), the optic tract (h), projections to the dLGN, vLGN, and pretectum (i), the dLGN (j), and SC (k). ln, Light-evoked postsynaptic responses of SC neurons, including AMPA receptor-mediated EPSCs (l), NMDA receptor-mediated EPSCs (n), and action potentials (n). Scale bars: 100 μm in panels d, j, and k, 200 μm in panels e and f, 400 μm in panel g, and 600 μm in panels h and i. [Figure 7]Morphology of Lgr5+ amacrine interneurons and their migration into the ganglion cell layer. a) Confocal images of Lgr5+ amacrine interneurons sparsely labeled with the tdTomato reporter in Lgr5EGFP-IRES-CreERT2;Rosa26-tdTomato mice. Sparse labeling of Lgr5+ amacrine cells with the tdTomato reporter was achieved by administering a single dose of tamoxifen to the mice. Images were taken from flat-mounted retinal samples, focusing on the inner nuclear layer where Lgr5+ amacrine cells are located. d) Confocal image of a retinal cross-section from an Lgr5EGFP-IRES-CreERT2;Rosa26-tdTomato mouse. The arrow highlights an Lgr5+ amacrine cell labeled with the tdTomato reporter. Its dendrites reach the ganglion cell layer. e) Confocal image of a flat-mounted retinal sample from an Lgr5EGFP-IRES-CreERT2 mouse, focusing on the ganglion cell layer. The arrows highlight the presence of Lgr5+ amacrine cells in the ganglion cell layer. g) Groups of Lgr5+ amacrine cells in the ganglion cell layer of a 20-month-old mouse. h) Number of Lgr5+ amacrine cells in the ganglion cell layer per retina. An age-dependent increase in Lgr5+ amacrine cells in the ganglion cell layer was observed, suggesting that these cells may migrate from the internal nuclear layer to the ganglion cell layer. Scale bars: 20 μm in panels a-c, 30 μm in panel d, and 50 μm in panels e-f. [Figure 8]Reprogramming of neuronal identity in Lgr5EGFP-IRES-CreERT2;Rosa26-tdTomato mice. a, b, Representative images of flat-mounted retina samples (a) and optic nerves (b) from mice injected with AAV-DIO-EGFP. No tdTomato axons were detected in these mice. c, Representative images of the optic nerve from a mouse injected with a high dose of AAV-DIO-EGFP (7 x 10 pfu, 2 µL). Because the AAV-DIO plasmid self-recombines during DNA amplification and viral vector generation (flipped vector), injection of a large number of AAV particles may result in a small number (within one order of magnitude) of original RGCs and their axons being labeled by the injected AAV-DIO-EGFP via Cre-independent transgene expression. However, these EGFP axons do not express tdTomato, suggesting that they are not derived from regenerating RGCs. f-h: Highlighting of Lgr5-EGFP and tdTomato double-positive cells in the inner plexiform layer (IPL), suggesting that migration of Lgr5+ amacrine cells from the inner nuclear layer (INL) to the retinal ganglion layer (RGL) is triggered by programming. il: Confocal image of regenerating RGCs expressing the αRGC marker SMI-32. m-r: Confocal image of a retinal cross-section from an Lgr5EGFP-IRES-CreERT2 mouse intravitreally injected with AAV-DIO-tdTomato to examine the specificity and efficiency of AAV-delivered gene expression. After five doses of tamoxifen, the AAV-delivered tdTomato gene specifically labels Lgr5+ amacrine cells. pr: Higher magnification images taken from the same eye as panels m-o. s: Statistics on EGFP+ / tdTomato+ cells in panels m-r. With this expression system, approximately 21.3% of Lgr5+ amacrine cells were labeled by AAV-delivered tdTomato. t, Statistics on the expression levels of Brn3B and Sox4 in Lgr5EGFP-IRES-CreERT2;Rosa26-tdTomato mice intravitreally injected with AAV-DIO-Brn3B and AAV-DIO-Sox4.Brn3B and Sox4 expression was measured by quantitative PCR and normalized to that of control mice intravitreally injected with AAV-DIO-EGFP. Scale bars: 150 μm in panel a, 200 μm in panel b, 300 μm in panels c–e, 40 μm in panels f–l, 400 μm in panel o, and 50 μm in panel r. [Figure 9] This figure shows the time it takes for regenerating RGCs to grow axons into the dispersed visual cortex. Lgr5EGFP-IRES-CreERT2;Rosa26-tdTomato mice received intravitreal injections of AAV-DIO-Brn3B and AAV-DIO-Sox4 in one eye, followed by five doses of tamoxifen to activate gene expression. Mice were sacrificed at different time points, and brain sections were prepared and examined for the presence of tdTomato+ axons by confocal microscopy. a) Confocal image of a brain section from a mouse sacrificed 30 days after virus injection. tdTomato+ axons in the contralateral brain (left side of the photo) pass through the lateral geniculate nucleus. No tdTomato+ axons were detected in the ipsilateral brain section. b) Confocal image of the superior colliculus (SC) region of the same mouse as in panel a. tdTomato+ axons had not yet reached the SC at this time point. c) Confocal image of a brain slide from a mouse sacrificed 35 days after virus injection. Highlighted are the posterior region of the LGN (lateral geniculate nucleus) and the anterior region of the SC in the contralateral brain (left side of the photograph). tdTomato+ axons were also detected in the ipsilateral brain, but their numbers were dramatically lower. d) Time course of regenerated RGC axonal projections. The time it took for regenerated RGC axons to reach the OC (optic chiasm), LGN, and SC was determined by counting the time at which regenerated RGC axons were first observed in these locations after virus injection. Regenerated RGC axons reached the OC at approximately day 18, the LGN at day 28, and the SC at day 35 (n = 8 per group). [Figure 10]Construction of Prokr2 knock-in mouse lines and in vivo reprogramming of Prokr2+ replacement amacrine interneurons into RGCs. a) Schematic of the targeting strategy for generating the Prokr2CreERT2 mouse line. The CreERT2 coding region was knocked into the start codon of the Prokr2 locus. The positions of Southern blotting probes targeting the 5' arm and CreERT2 region are indicated. b) Images of Southern blotting membranes using the 5' arm probe (top) and CreERT2 probe (bottom). Founders 1, 2, 3, and 4 had correct genomic targeting and were used for further breeding. c) Immunohistochemical staining of retinal cross sections from Prokr2CreERT2;Rosa26-tdTomato mice using anti-RBPMS antibody. Prokr2-tdTomato+ cells do not express the RGC marker RBPMS. f-h: Confocal images of the coronal brain section (f), superior colliculus (g), and optic nerve (h) of a Prokr2CreERT2;Rosa26-tdTomato mouse. Prokr2-tdTomato+ cells are present in the brain and optic nerve. i, j: Images of a Prokr2CreERT2 mouse intravitreally injected with AAV-DIO-EGFP. Because the AAV-DIO plasmid self-recombines during DNA amplification and viral vector production, the inverted AAV-DIO-EGFP vector labels a small number of original retinal ganglion cells (i) and their axons (j). k: Diagram of the reprogramming strategy and AAV expression vectors in Prokr2CreERT2 mice. On day 1 (D1), mice were intravitreally injected with AAV and then given tamoxifen (TM) from D3 to D7 to activate the expression of the gene delivered by the Cre-dependent AAV-DIO system. Mice were sacrificed and analyzed after D42. l, Reprogramming efficiency of transcription factor combinations. In the control group, inverted AAV-DIO-EGFP labeled very few endogenous RGCs. Scale bars: panel d = 40 μm, panel f = 800 μm, panel g = 200 μm, panel h = 100 μm, panel i = 1000 μm, and panel j = 100 μm. [Figure 11]Figure 1 shows calcium imaging and optogenetic analysis of RGCs. Diagram of in vivo calcium imaging setup. b) Representative image of a regenerating RGC terminal in the SC region. c) Histogram distribution of the orientation selectivity index (OSI) of all responsive terminals recorded from three mice. d) Cumulative percentage plot of OSI for all terminal data shown in panel c. e) and f) Representative EPSCs of light-evoked postsynaptic AMPA receptor responses (e) and postsynaptic action potentials (f) in SC neurons from C57B6 / J mice with ChR2-labeled RGCs. Scale bar = 10 μm. [Figure 12]This figure shows in vivo reprogramming after injury to the original RGC. Because Pvalb is expressed in several subtypes of RGCs, we used PvalbCreERT2;Rosa26-tdTomato mice to establish the status of RGCs and their axons injured by intraocular pressure (IPI). a) Confocal image of the optic nerve of a PvalbCreERT2;Rosa26-tdTomato mouse. The axons of Pvalb-tdTomato+ RGCs are intact. b) Confocal image of the optic nerve of a PvalbCreERT2;Rosa26-tdTomato mouse 7 days after injury by intraocular pressure (IPI). All axons are damaged by IPI. c) Confocal image of a flat-mounted retinal sample from a PvalbCreERT2;Rosa26-tdTomato mouse. Pvalb-tdTomato labels RGCs and their axons in intact, uninjured mice (c). Seven days after injury due to elevated intraocular pressure (IPI), the mouse retina shows a dramatic loss of Pvalb-tdTomato+ RGCs (d). Ripasudil treatment slows the degeneration of Pvalb-tdTomato+ RGCs, but does not allow the remaining RGCs to retain intact axons (e). f. Schematic of in vivo neural reprogramming in Lgr5EGFP-IRES-CreERT2;Rosa26-tdTomato mice after injury of the original RGCs due to IPI. Lgr5EGFP-IRES-CreERT2;Rosa26-tdTomato mice were first treated with five doses of tamoxifen (TM) (from day -11 (D-11) to day -7 (D-7)), and Lgr5+ amacrine interneurons were labeled with the Rosa26-tdTomato reporter to aid in tracking their identity. One week later, at D1, mouse RGCs and their axons were injured by elevated intraocular pressure. Mice were intravitreally injected with an AAV expressing a Cre-dependent transcription factor on D7 and given tamoxifen (TM) from D9 to D13 to activate gene expression. Mice were sacrificed after D49 for analysis. To protect Lgr5+ amacrine interneurons and other retinal neurons, mice were treated with ripasudil eye drops daily from D1 to D49. Scale bar = 100 μm. [Figure 13]Confocal images of flat-mounted retinal samples and optic nerves. a) Pavalbumin (PV)-positive RGCs and their axons in flat-mounted retinas from PV-CreERT2;Rosa26-tdTomato mice. b) After elevated intraocular pressure, many RGCs die and their axons degenerate. c) When the remaining RGCs are overexpressed with Atoh7+Brn3B+Sox4, these cells regrow / regenerate axons. d) PV-positive RGC axons in the optic nerve of PV-CreERT2;Rosa26-tdTomato mice. e) After RGC damage due to elevated intraocular pressure, RGC axons degenerate within the optic nerve. F) Regenerated RGC axons in the optic nerve after overexpression of Atoh7+Brn3B+Sox4 in the remaining RGCs. [Figure 14] Projection of regenerated RGC axons to the visual cortex of the brain: a) Axons of regenerated RGCs in the dorsal and lateral ventral geniculate nuclei, b) Axons of regenerated RGCs in the superior colliculus. DETAILED DESCRIPTION OF THE INVENTION

[0018] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.As used herein, the following terms have the following meanings unless otherwise specified.Any methods, devices and materials similar or equivalent to those described herein can be used in the implementation of the compositions and methods described herein.The following definitions are provided to facilitate understanding of certain terms frequently used herein and are not intended to limit the scope of the present disclosure.All documents mentioned herein are incorporated by reference in their entirety.

[0019] As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements, but do not exclude others. When used to define compositions and methods, "consisting essentially of" is intended to mean excluding others that are essential to the combination. For example, a composition consisting essentially of the elements defined herein would not exclude other elements that do not materially affect the basic and novel characteristics of the claimed invention. "Consisting of" is intended to mean excluding more than trace amounts of other components and substantial method steps described. Embodiments defined by each of these transition terms are within the scope of the present invention.

[0020] The term "about" means within ±10%, ±5%, or ±1% of a given value or range. In one embodiment, about means ±10% of a given value or range. In another embodiment, about means ±5% of a given value or range. In another embodiment, about means ±1% of a given value or range.

[0021] An "expression control sequence" refers to a nucleic acid sequence that regulates the expression of a nucleotide sequence to which it is operably linked. An expression control sequence is "operably linked" to a nucleotide sequence if it controls and regulates the transcription and / or translation of the nucleotide sequence. Thus, expression control sequences can include promoters, enhancers, internal ribosome entry sites (IRES), transcription terminators, start codons in front of protein-coding genes, splicing signals for introns, and stop codons. The term "expression control sequence" is intended to include at least sequences designed whose presence affects expression and can also include advantageous components. For example, leader sequences and fusion partner sequences are expression control sequences. The term can also include designing a nucleic acid sequence to remove unwanted, in-frame, or out-of-frame potential start codons from the sequence. It can also include designing a nucleic acid sequence to remove unwanted, potential splice sites. The expression control sequence may also include a polyadenylation sequence (pA), which directs the addition of a polyA tail, a series of adenine residues at the 3' end of the mRNA, also referred to as a polyA sequence. It may also be designed to enhance mRNA stability. Expression control sequences that affect transcriptional and translational stability, such as promoters, and sequences that affect translation, such as Kozak sequences, suitable for use in insect cells are well known to those skilled in the art. The expression control sequence may be of a nature that regulates the nucleotide sequence to which it is operably linked, so as to achieve lower or higher expression levels.

[0022] As used herein, the term "promoter" or "transcriptional regulatory sequence" refers to a nucleic acid fragment that functions to control the transcription of one or more coding sequences, is located upstream of the transcription start site of the coding sequence in the direction of transcription, and is structurally defined by the presence of a DNA-dependent RNA polymerase binding site, a transcription start site, and any other DNA sequences, including transcription factor binding sites, repressor and activator protein binding sites, and any other nucleotide sequences known to those skilled in the art that act directly or indirectly to regulate the amount of transcription from the promoter, such as attenuators or enhancers, but including silencers. A "constitutive" promoter is a promoter that is active in most tissues and under most physiological and developmental conditions. An "inducible" promoter is a promoter that is physiologically or developmentally regulated, for example, by the application of a chemical inducer. A "tissue-specific" promoter is active only in specific types of tissues or cells.

[0023] A "vector" is a nucleic acid molecule (typically DNA or RNA) that serves to introduce passenger nucleic acid sequences (i.e., DNA or RNA) into a host cell. There are three common types of vectors: plasmids, phages, and viruses. Preferably, the vector is a virus. Vectors containing both a promoter and a cloning site to which a polynucleotide can be operably linked are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo and are commercially available from sources such as Stratagene (La Jolla, Calif.) and Promega Biotech (Madison, Wis.). To optimize expression and / or in vitro transcription, it may be useful to remove, add, or modify the 5' and / or 3' untranslated portions of the clone to eliminate extraneous, potentially inappropriate, alternative translation initiation codons or other sequences that could prevent or reduce expression at either the transcriptional or translational level. Alternatively, a consensus ribosome binding site can be inserted immediately 5' to the initiation codon to enhance expression.

[0024] A "viral vector" refers to a vector containing some or all of the viral genes encoding gene products, regulatory sequences, and viral packaging sequences. A "parvovirus vector" is defined as a recombinantly produced parvovirus or parvovirus particle containing a polynucleotide to be delivered to a host cell, whether in vivo, ex vivo, or in vitro. Examples of parvovirus vectors include adeno-associated virus vectors. Here, a parvovirus vector construct refers to a polynucleotide containing a viral genome or a portion thereof and a transgene.

[0025] The term "administration" refers to introducing an agent into a patient's body. An effective amount can be administered, which can be determined by a treating physician or the like. The related terms and phrases "administer" and "administration of," when used in reference to a compound or tablet (and grammatical equivalents), both refer to direct administration, which can be administration to a patient by a medical professional or self-administration by the patient.

[0026] A "therapeutically effective amount" or "effective amount" refers to the amount of a drug or agent that, when administered locally to a patient suffering from a condition via a pharmaceutical composition described herein, produces the intended therapeutic effect, e.g., relief, amelioration, alleviation, or elimination of one or more symptoms of the patient's condition. The full therapeutic effect does not necessarily occur immediately, but may occur only after a therapeutically effective amount has been continuously delivered over a period of time. In the case of sustained- or controlled-release formulations that are slowly released from a delivery vehicle to the disease site at a verifiable and controllable release rate that consistently provides an effective amount of drug at the disease site, a "therapeutically effective amount" or "effective amount" may refer to the total effective amount over a certain period of time. In some embodiments, a "therapeutically effective amount" or "effective amount" refers to the amount released to the disease site over a given period of time, e.g., per day.

[0027] The term "pharmaceutically acceptable" means generally safe and non-toxic for administration to humans.

[0028] "Treatment," "treating," and "treat" are defined as the application of an agent to a disease, disorder, or condition to reduce or ameliorate the harmful or other undesirable effects of the disease, disorder, or illness and / or its symptoms.

[0029] Regeneration of retinal ganglion cells (RGCs) from other retinal neuronal cells In glaucoma and various optic neuropathies, degeneration of retinal ganglion cells (RGCs) and their axons underlies vision loss. Currently, no treatments are available to restore lost vision in patients affected by these diseases. While regenerating RGCs and reconnecting the retina to the brain would be an ideal therapeutic strategy, mammals lack a reservoir of retinal stem / progenitor cells poised to generate new neurons in adulthood.

[0030] In the accompanying experimental example, we demonstrate that direct lineage reprogramming of retinal neurons results in the regeneration of RGCs. Amacrine and replacement amacrine interneurons were successfully converted into RGCs, which projected axons to cerebral retinal receptor areas. They were able to respond to visual stimuli, convey visual information to the brain, and transmit electrical signals to postsynaptic neurons in both normal animals and in an animal model of glaucoma in which the original RGCs were damaged by elevated intraocular pressure.

[0031] Therefore, according to one embodiment of the present disclosure, there is provided a method for reprogramming non-RGC neuronal cells to respond to visual signals. In one embodiment, the reprogramming involves activating (or increasing the biological activity of) one or more transcription factors in the non-RGC neuronal cells. In some embodiments, the transcription factor is a proneural transcription factor.

[0032] Examples of transcription factors include POU domain transcription factors such as Brn3B. Brn3B (POU class 4 homeobox 2, or POU4F2, BRN3.2, or Brn-3b) is a member of the POU domain transcription factor family and is involved in the maintenance of visual system neurons in the retina. The representative human Brn3B gene has the protein sequence NP_004566.2 and the mRNA sequence NM_004575.3. The representative mouse Brn3B gene has the protein sequence NP_620394.2 and the mRNA sequence NM_138944.3.

[0033] Another example of a transcription factor is a SOX (SRY-related HMG box) transcription factor, such as Sox4. Sox4 (SRY box transcription factor 4, or CSS10, or EVI16) is a member of the SOX (SRY-related HMG box) transcription factor family and is involved in regulating embryonic development and cell fate determination. A representative human Sox4 gene has the protein sequence NP_003098.1 and the mRNA sequence NM_003107.3. A representative mouse Sox4 gene has the protein sequence NP_033264.2 and the mRNA sequence NM_009238.3.

[0034] Another exemplary member of the SOX (SRY-related HMG box) transcription factor family is Sox11. Sox11 (SRY box transcription factor 11, or CSS9, or MRD27) is a member of the SOX (SRY-related HMG box) transcription factor family and is involved in regulating embryonic development and cell fate determination. The protein sequence of a representative human Sox11 gene is NP_003099.1, and the mRNA sequence is NM_003108.4. The protein sequence of a representative mouse Sox11 gene is NP_033260.4, and the mRNA sequence is NM_009234.6.

[0035] Another exemplary transcription factor is a basic helix-loop-helix transcription factor, such as Atoh7. Atoh7 (athonal bHLH transcription factor 7, also known as Math5, NCRNA, RNANC, PHPVAR, or bHLHa13) is a member of the basic helix-loop-helix family of transcription factors and regulates photoreceptor development. This gene plays a central role in the formation of retinal ganglion cells and the optic nerve. The representative human Atoh7 gene has the protein sequence NP_660161.1 and the mRNA sequence NM_145178.4. The representative mouse Atoh7 gene has the protein sequence NP_058560.1 or NP_001351577.1 and the mRNA sequence NM_016864.3 or NM_0013648.2.

[0036] Another example of a transcription factor is a LIM / homeodomain transcription factor, e.g., Isl1 is. Isl1 (ISL LIM homeobox 1, or Isl-1, or ISLET1) is a member of the LIM / homeodomain family of transcription factors, which binds to the enhancer region of the insulin gene, among other things, and may play an important role in regulating insulin gene expression. Isl1 is central to the development of pancreatic cell lineages and is required for motor neuron generation. Isl1 The gene has the protein sequence NP_002193.2 and the mRNA sequence NM_002202.3. Isl1 The gene has the protein sequence NP_067434.3 and the mRNA sequence NM_021459.4.

[0037] Exemplary protein and nucleic acid sequences for these exemplary transcription factors are shown in Table 1 below.

[0038] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0039] Methods for increasing the biological activity of a gene are known in the art. Increasing the biological activity can be increasing the expression of the protein, or increasing the function of the protein, or both.

[0040] In some embodiments, at least one transcription factor is activated in the cell. In one embodiment, the biological activity of Brn3B is increased. In one embodiment, the biological activity of Sox4 is increased. In one embodiment, the biological activity of Atoh7 is increased. In one embodiment, the biological activity of Sox11 is increased. In one embodiment, Isl1 The biological activity of

[0041] In some embodiments, the biological activity of at least two of the transcription factors is increased, the two being Brn3B and Sox4, Brn3B and Ato7, Brn3B and Sox11, or Brn3B and Isl1 , Sox4 and Ato7, Sox4 and Sox11, Sox4 and Isl1 , Ato7 and Sox11, Ato7 and Isl1 , or Sox11 Isl1 It could be.

[0042] In some embodiments, the biological activity of at least three of the transcription factors is increased, including, but not limited to, Brn3B, Sox4, and Atoh7; Brn3B, Sox4, and Sox11; or Brn3B, Sox4, and Isl1 In some embodiments, the biological activities of at least four of the transcription factors are increased. In some embodiments, the biological activities of all five of the transcription factors are increased.

[0043] Activation of endogenous transcription factors In one example, the expression of the corresponding endogenous gene is activated or enhanced. For example, the human cytomegalovirus (CMV) enhancer / promoter (CMV) is a naturally occurring mammalian promoter with high transcriptional activity. The CMV enhancer is a powerful enhancer in various mammalian cells and has been widely used to drive the ectopic expression of various genes in a wide range of mammalian cells and to drive the ectopic expression of foreign genes in a wide range of tissues in transgenic animals. In some examples, the transcriptional activity of the CMV enhancer can be further improved by changing the natural NF-κB binding site to an artificially selected NF-κB binding sequence with high binding affinity (Wang et al., Protein Expression and Purification 142:16-24, 2018). U.S. Patent No. 10,329,595 also reports the creation of two improved CMV promoters (SEQ ID NOs: 26 and 27). Other useful gene promoters and enhancers are also known in the art.

[0044] In some embodiments, the promoter or enhancer regulates the expression of a gene that is constitutively expressed in neurons. Examples of genes expressed in neurons, such as amacrine cells, include Pax6, Tcfap2B, Gad1, GlyT1, RBPMS, and Prox1. Another example gene is synapsin 1. Examples of promoters / enhancers are listed in Table 2.

[0045] [Table 2]

[0046] Gene expression promoters and enhancers can be introduced into target genes using traditional knock-in technology or CRISPR methods.

[0047] There are also a wealth of CRISPR-based gene activation technologies. In one example, an inactive Cas protein (e.g., Cas9) is fused to an appropriate transcription effector domain. Commonly used transcription activator domains include VP64, the p65 domain of NF-κB, the Epstein-Barr virus R transactivator (Rta), and the activator domain of heat shock factor 1 (HSF1). In the endogenous context, multiple transcription factors and cofactors work in concert to stimulate gene transcription. Indeed, CRISPR tools that recruit multiple unique transcription activators to a promoter are superior to tools with a single transcription activator domain or redundant copies of the same effector. Furthermore, targeting multiple sites on the same promoter increases CRISPR-mediated gene activation. One of the most effective CRISPR effectors is the CRISPR synergistic activation mediator (SAM) complex, which recruits three unique transcription activation domains to the target gene promoter. In this system, a single transcriptional activator, VP64 (a multimeric form of VP16), is fused directly to dCas9.

[0048] In another example, the dCas9-p300 CRISPR Gene Activator system (Signa Aldrich, Hilton, Isaac B., et al. Nature Biotechnology (2015)) is based on the fusion of dCas9 with the catalytic histone acetyltransferase (HAT) core domain of the human E1A-associated protein p300. This approach activates genes both proximal and distal to the transcription start site (TSS).

[0049] Introduction of exogenous transcription factors A more common method for increasing the biological activity (or expression) of a transcription factor is to introduce an exogenous sequence encoding the transcription factor, or the transcription factor protein. Proteins can be introduced into cells by encapsulating them in a vehicle such as a liposome. Examples of transcription factor protein sequences are provided in Table 1.

[0050] Alternatively, coding sequences such as cDNA or mRNA can be introduced into target cells. Examples of coding sequences for transcription factors are provided in Table 1. In some embodiments, nucleic acid constructs containing one or more coding sequences for these transcription factors are prepared. The coding sequence can be operably connected to an appropriate promoter or enhancer. In some embodiments, the promoter or enhancer is specific to target cells such as retinal interneurons. Examples of promoters are provided in Table 2.

[0051] The construct may be a plasmid or, preferably, is a viral vector. Suitable viral vectors include lentiviral vectors and AAV vectors.

[0052] As used herein, a "recombinant adeno-associated virus (AAV) vector" (or "rAAV vector") refers to a vector containing one or more polynucleotide sequences of interest, a gene product of interest, and a gene of interest, or "transgene," flanked by at least one parvovirus or AAV inverted terminal repeat (ITR). Such rAAV vectors can replicate and be packaged into infectious viral particles in insect host cells expressing the AAV rep and cap gene products (i.e., AAV Rep and Cap proteins). When an rAAV vector is integrated into a larger nucleic acid construct (e.g., in a chromosome or in another vector, such as a plasmid or baculovirus, used for cloning or gene transfer), the rAAV vector is typically referred to as a "provector" that can be "rescued" by replication and encapsidation in the presence of AAV packaging and necessary helper functions. Preferably, the gene product of interest is flanked on either side by AAV ITRs. AAV ITRs, including ITRs from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and / or AAV12, can be used in the constructs of the invention.

[0053] The AAV gene therapy vector used in this technology can be produced in mammalian cells or insect cells. Both methods have been described in the art. For example, Grimm et al. (2003 Molecular Therapy 7(6):839-850) disclose a strategy for producing AAV vectors in a helper virus-free and optically controllable manner based on the transfection of only two plasmids into 293T cells. They also disclose a method for producing a hybrid AAV vector containing AAV2 ITRs and AAV5 capsid proteins. Further information can be found in Blits et al. (2010) (Journal of Neuroscience Methods 185(2):257-263). The terms "hybrid" and "pseudotyped" are used interchangeably herein to refer to a vector in which the Rep protein, ITRs, and / or capsid protein are of different serotypes. For example, the ITRs and Rep protein are those of AAV2, and the capsid protein is that of AAV5. The term "chimera" is used herein to describe that a single gene, for example the capsid, is composed of at least two sequences derived from different serotypes.

[0054] AAV can be produced in mammalian cells according to the following method, but it is not limited to it: the vector genome contains a transgene expression cassette flanked by two inverted terminal repeats (ITRs) from AAV serotype 2. The total length of the viral vector genome can be kept below the wild-type genome size of 4.7 kB to maintain efficient packaging efficiency. One capsid consists of 60 viral proteins, either VP1 (62 kDa), VP2 (73 kDa), or VP3 (87 kDa), in a 1:1:10 ratio. The AAV vector production process is based on transfecting two plasmids into human embryonic kidney cells (HEK293) with Ca(PO4)2 in a roller bottle (surface area 850 cm2), followed by purification of the encapsidated vector genome by filtration and chromatography techniques. The first plasmid is the viral vector plasmid and contains an expression construct flanked by AAV2 ITRs. The second plasmid is a packaging plasmid, encoding the AAV rep type 2 and cap type 5 genes of the desired serotype and the adenovirus early helper genes E2A, VA, and E4 (pDP5). The genome of the resulting cell line contains adenovirus E1 to provide helper function. After cotransfection of the two plasmids in Iscove's modified Dulbecco's medium (IMDM) containing 10% fetal calf serum (FCS), the cells are incubated in serum-free Dulbecco's modified Eagle's medium (DMEM) for 3 days to allow vector production. Vector production in roller bottles averages 3 x 10 cells per cell. 3 vector genomes, or 4 x 10 per roller bottle 11 This yields vector genomes (quantified by qPCR). The cell culture is then lysed in a buffer containing Triton-X-100 and cell debris is removed by low-speed centrifugation. The clarified bulk is purified by AVB Sepharose affinity chromatography and formulated into PBS / 5% sucrose by concentration and diafiltration using a 400 kDa hollow fiber module (e.g., Spectrum Laboratories).

[0055] The AAV ITR and Rep sequences that can be used in the present invention for the construction of rAAV vectors in insect cells can be derived from the genome of any AAV serotype.Generally, AAV serotypes have genome sequences with significant homology at the amino acid level and nucleic acid level.This provides a set of identical genetic functions for producing essentially physically and functionally equivalent virions. For an overview of the genomic sequences and genomic similarities of various AAV serotypes, see, for example, GenBank Accession No. U89790; GenBank Accession No. J01901; GenBank Accession No. AF043303; GenBank Accession No. AF085716; Chiorini et al. (1997, J. Vir. 71: 6823-33); Srivastava et al. (1983, J. Vir. 45: 555-64); Chiorini et al. (1999, J. Vir. 73: 1309-1319); Rutledge et al. (1998, J. Vir. 72: 309-319); and Wu et al. (2000, J. Vir. 74: 8635-47). rAAV serotypes 1, 2, 3, 4, and 5 are preferred sources of AAV nucleic acid sequences for use in the context of the present invention. Preferably, the AAV ITR sequences for use in the context of the present invention are derived from AAV1, AAV2, and / or AAV5. More preferably, the ITR sequences for use in the present invention are AAV2 ITRs. Similarly, the Rep (Rep78 / 68 and Rep52 / 40) coding sequences are preferably derived from AAV1, AAV2, and / or AAV5, more preferably AAV2.

[0056] In particular, the sequences of AAV Rep and ITR are conserved among most serotypes. For example, the Rep78 proteins of various AAV serotypes are over 89% identical, and the overall nucleotide sequence identity at the genome level between AAV2, AAV3A, AAV3B, and AAV6 is approximately 82% (Bantel-Schaal et al., 1999, J. Virol., 73(2):939-947). Furthermore, the Rep sequences and ITRs of many AAV serotypes are known to efficiently cross-complement (i.e., functionally replace) corresponding sequences from other serotypes in the production of AAV particles in mammalian cells. Also, U.S. Patent Application Publication No. 2003148506 reports that AAV Rep and ITR sequences efficiently cross-complement with the Rep and ITR sequences of other AAVs in insect cells.

[0057] AAV VP proteins are known to determine the intracellular tropicity of AAV virions. The sequences encoding VP proteins are not as significantly conserved among different AAV serotypes as the Rep proteins and genes. The sequences encoding viral proteins (VP) VP1, VP2, and VP3 capsid proteins for use in the context of the present invention are derived from AAV5. Most preferably, VP1, VP2, and VP3 are VP1, VP2, and VP3 of AAV5. Alternatively, VP1, VP2, and VP3 are wild-type AAV5 sequences. The ability of Rep and ITR sequences to cross-complement corresponding sequences of other serotypes allows for the creation of pseudotyped rAAV particles containing capsid proteins of one serotype and ITR sequences of another AAV serotype. Such pseudotyped rAAV particles are part of the present invention.

[0058] Each AAV serotype may be more suitable for one or more specific tissues. For example, AAV2, AAV3, AAV4, AAV5, AAV7, and AAV8 may be suitable for the retina; AAV1, AAV2, AAV4, AAV5, AAV7, and AAV10 may be suitable for neurons; AAV2, AAV4, AAV8, and AAV9 may be suitable for the brain; AAV3, AAV5, AAV6, AAV9, and AAV10 may be suitable for the lung; AAV1, AAV6, AAV9, and AAV10 may be suitable for the heart; AAV2, AAV3, and AAV6-10 may be suitable for the liver; all serotypes except AAV5 may be suitable for muscle tissue; AAV2 and AAV10 may be suitable for the kidney; and AAV1, AAV7, and AAV9 may be suitable for the pancreas.

[0059] In one embodiment, the AAV is of serotype AAV2. In one embodiment, the AAV is of serotype AAV3. In one embodiment, the AAV is of serotype AAV4. In one embodiment, the AAV is of serotype AAV5. In one embodiment, the AAV is of serotype AAV7. In one embodiment, the AAV is of serotype AAV8.

[0060] In some embodiments, the AAV vector is an AAV2.7m8 vector, an engineered capsid with a 10 amino acid insertion in the adeno-associated virus (AAV) surface variable region VIII (VR-VIII), resulting in a modified antigenic region of AAV2 and the ability to efficiently transduce retinal cells after intravitreal administration (Bennett et al., J Struct Biol, 2020 Feb 1;209(2):107433. doi: 10.1016 / j.jsb.2019.107433. Epub 2019 Dec 16). In some embodiments, the AAV vector is an AAV-DJ (type 2 / type 8 / type 9 chimera) engineered by shuffling eight different wild-type native viruses (Katada Y, et al., 2019. PeerJ 7:e6317). In some embodiments, the AAV vector is an AAV7m8 vector (Ramachandran et al., Hum Gene Ther. 2017 Feb;28(2):154-167. doi: 10.1089 / hum.2016.111. Epub 2016 Oct 17).

[0061] target cell Reprogramming can be performed on non-RGC cells in the retina, such as any retinal neuron that is not an RGC. In some embodiments, such retinal neurons are interneuron cells. Examples of interneuron cells are amacrine cells, bipolar cells, and horizontal cells. In some embodiments, non-RGC cells are photoreceptors. In some embodiments, non-RGC cells can also be Müller cells.

[0062] In some embodiments, the amacrine cells are Lgr5 + In some embodiments, the amacrine cells are Prokr2 + In some embodiments, the amacrine cells are Lgr5 + Amacrine cells, where the biological activity (expression) of both Brn3B and Sox4 is limited to Lgr5 +In some embodiments, amacrine cells are increased in Prokr2 + In replacement amacrine cells, the biological activity (expression) of both Brn3B and Sox4 is suppressed by Prokr2 + In some embodiments, the amacrine cells are Prokr2 + In replacement amacrine cells, the biological activity (expression) of Brn3B, Sox4, and Ato7 is all suppressed by Prokr2. + Increased in replacement amacrine cells.

[0063] Target cells can be reprogrammed in vitro or in vivo. When reprogrammed in vitro, the cells are converted into regenerative RGCs and can be transplanted into a subject in need. When reprogrammed in vivo, the regenerative RGCs can replace damaged or degenerated RGCs, thereby treating visual dysfunction or blindness.

[0064] Retinal ganglion cell (RGC) rejuvenation In another surprising discovery, the present inventors demonstrated that activation of the transcription factor of the present disclosure was also effective in reactivating damaged RGCs (Example 2). The reactivated RGCs were able to regenerate functional axons, which projected to the optic nerve and connected to the brain.

[0065] Therefore, another embodiment of the present disclosure provides a method for improving the function of retinal ganglion cells (RGCs). The RGCs may be degenerated, damaged, aged, or normal / healthy RGCs, for which functional improvement is desired. In some embodiments, the method includes increasing the expression of Atoh7, Brn3B, Sox4, Sox11, and ATP in RGCs. Isl1 The method involves increasing the biological activity of one or more genes selected from the group consisting of:

[0066] Methods for increasing the biological activity of a gene are known in the art. Increasing the biological activity can be increasing the expression of the protein, increasing the function of the protein, or both.

[0067] In some embodiments, at least one of the transcription factors is activated in the cell. In one embodiment, the biological activity of Brn3B is increased. In one embodiment, the biological activity of Sox4 is increased. In one embodiment, the biological activity of Atoh7 is increased. In one embodiment, the biological activity of Sox11 is increased. In one embodiment, Isl1 The biological activity of

[0068] In some embodiments, the biological activity of at least two of the transcription factors is increased, the two being Brn3B and Sox4, Brn3B and Ato7, Brn3B and Sox11, or Brn3B and Isl1 , Sox4 and Ato7, Sox4 and Sox11, Sox4 and Isl1 , Ato7 and Sox11, Ato7 and Isl1 , or Sox11 Isl1 It could be.

[0069] In some embodiments, the biological activity of at least three of the transcription factors is increased, including but not limited to Brn3B, Sox4, and Atoh7, Brn3B, Sox4, and Sox11, or Brn3B, Sox4, and Isl1 In some embodiments, the biological activities of at least four of the transcription factors are increased. In some embodiments, the biological activities of all five of the transcription factors are increased.

[0070] Examples of methods for activating endogenous transcription factors and introducing exogenous transcription factors are described in more detail above. The methods may be in vitro or in vivo.

[0071] Composition and regenerative / rejuvenating cells Also provided are agents, reagents, and compositions that can facilitate the practice of the techniques disclosed herein. Also provided, in some embodiments, are RGC cells regenerated or rejuvenated by the techniques.

[0072] One embodiment of the present disclosure provides a nucleic acid construct that can be introduced into a target cell for the desired reprogramming of the cell. In some embodiments, the nucleic acid construct comprises a coding sequence encoding any one, two, three, four, or all of the transcription factors disclosed herein. In one embodiment, the nucleic acid construct comprises a coding sequence for Brn3B. In one embodiment, the nucleic acid construct comprises a coding sequence for Sox4. In one embodiment, the nucleic acid construct comprises a coding sequence for Atoh7. In one embodiment, the nucleic acid construct comprises a coding sequence for Sox11. In one embodiment, the nucleic acid construct comprises a coding sequence for Isl1 Examples of proteins and coding sequences for these transcription factors are provided in Table 1.

[0073] In some embodiments, the nucleic acid construct comprises Brn3B and Sox4, Brn3B and Ato7, Brn3B and Sox11, Brn3B and Isl1 , Sox4 and Ato7, Sox4 and Sox11, Sox4 and Isl1 , Ato7 and Sox11, Ato7 and Isl1 , or Sox11 Isl1 In some embodiments, the nucleic acid construct comprises coding sequences for at least two transcription factors, which may be, but are not limited to, Brn3B, Sox4, and Ato7, Brn3B, Sox4, and Sox11, or Brn3B, Sox4, and Ato7. Isl1 In some embodiments, the nucleic acid construct comprises coding sequences for at least four transcription factors. In some embodiments, the nucleic acid construct comprises coding sequences for all five transcription factors.

[0074] In some embodiments, the nucleic acid construct comprises a promoter or enhancer associated with each coding sequence. The promoter or enhancer is active in retinal interneuron cells. Non-limiting examples include the promoters of Pax6, Tcfap2B, Gad1, GlyT1, RBPMS, and Prox1, or the promoters provided in Table 2. In a specific example, the promoter is the synapsin 1 promoter.

[0075] In some examples, the nucleic acid construct comprises an expression vector, which may be a plasmid vector or a viral vector, such as an AAV vector. The AAV may be selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12.

[0076] In one embodiment, the AAV is of serotype AAV2. In one embodiment, the AAV is of serotype AAV3. In one embodiment, the AAV is of serotype AAV4. In one embodiment, the AAV is of serotype AAV5. In one embodiment, the AAV is of serotype AAV7. In one embodiment, the AAV is of serotype AAV8.

[0077] In some embodiments, the AAV vector is an AAV2.7m8 vector, an engineered capsid with a 10 amino acid insertion in the adeno-associated virus (AAV) surface variable region VIII (VR-VIII), resulting in a modified antigenic region of AAV2 and the ability to efficiently transduce retinal cells after intravitreal administration (Bennett et al., J Struct Biol, 2020 Feb 1;209(2):107433. doi: 10.1016 / j.jsb.2019.107433. Epub 2019 Dec 16). In some embodiments, the AAV vector is an AAV-DJ (type 2 / type 8 / type 9 chimera) engineered by shuffling eight different wild-type native viruses (Katada Y, et al., 2019. PeerJ 7:e6317). In some embodiments, the AAV vector is an AAV7m8 vector (Ramachandran et al., Hum Gene Ther. 2017 Feb;28(2):154-167. doi: 10.1089 / hum.2016.111. Epub 2016 Oct 17).

[0078] Also provided are cells transfected with vectors and cells reprogrammed or rejuvenated by the techniques of the present disclosure. In one embodiment, mammalian cells responsive to visual signals are provided. In one embodiment, the cells are prepared by increasing the biological activity of one or more genes disclosed herein in retinal cells, such as retinal interneuron cells or degenerated, damaged, or aged RGCs. In another embodiment, the retinal cells are Müller cells. In yet another embodiment, the retinal cells are photoreceptors. In some embodiments, the reprogrammed cells are regenerated retinal ganglion cells (RGCs). In some embodiments, the reprogrammed cells are rejuvenated retinal ganglion cells (RGCs).

[0079] In some embodiments, regenerated or rejuvenated RGCs can project axons to distributed subcortical brain regions.In some embodiments, regenerated or rejuvenated RGCs can establish retina-brain connection.In some embodiments, regenerated or rejuvenated RGCs can respond to visual stimuli and transmit electrical signals into the brain.

[0080] In some embodiments, the mammalian cell is an animal cell, hi some embodiments, the mammalian cell is a human cell.

[0081] Treatment and Use In diseases broadly classified as optic neuropathies, including glaucoma, hereditary optic neuropathies, and disorders caused by toxins, malnutrition, and trauma, loss of RGCs is a major cause of blindness. Therefore, this technology can be used to treat visual dysfunction or vision loss (blindness).

[0082] In some embodiments, the treatment or use comprises targeting Brn3B, Sox4, Atoh7, Sox11, and Isl1 In some embodiments, the biological activity of at least two of the transcription factors is increased, such as Brn3B and Sox4, Brn3B and Ato7, Brn3B and Sox11, or Brn3B and Sox12. Isl1 , Sox4 and Ato7, Sox4 and Sox11, Sox4 and Isl1 , Ato7 and Sox11, Ato7 and Isl1 , or Sox11 Isl1 In some embodiments, the biological activity of at least three of the transcription factors is increased, including, but not limited to, Brn3B, Sox4, and Atoh7; Brn3B, Sox4, and Sox11; or Brn3B, Sox4, and Isl1 In some embodiments, the biological activities of at least four of the transcription factors are increased. In some embodiments, the biological activities of all five of the transcription factors are increased.

[0083] Examples of agents such as nucleic acid constructs that introduce promoters and enhancers for one or more of the corresponding endogenous transcription factors (e.g., CRISPR systems), nucleic acid constructs that encode one or more of the transcription factors, and expressed proteins of the transcription factors have been described above.

[0084] Administration may be, but is not limited to, topical application, ophthalmic application, or intravitreal injection.

[0085] In some embodiments, the agent is an AAV vector or a pharmaceutical composition comprising an AAV vector. In some embodiments, the administered AAV vector or pharmaceutical composition is 1×10 6 ~1×10 20 genome copies (gc) / kg, or 1 × 10 7 ~1×10 20 , or 1 × 10 8 ~1×10 20 , or 1 × 10 8 ~1×10 19 , or 1 × 10 9 ~1×10 19 , or 1 × 10 9 ~1×10 18 , or 1 × 10 10 ~1×10 18 , or 1 × 10 11 ~1×10 17 , or 1 × 10 12 ~1×10 17 , or 1 × 10 13 ~1×10 16 , 2 × 10 13 ~2×10 15 , 8×10 13 ~6×10 14 The dosage may be gc / kg of subject body weight. Note that dosage values ​​may vary depending on the severity of the condition to be alleviated. For any particular subject, specific dosage regimen can be adjusted over time according to individual need and the professional judgment of the person who administers or supervises the administration of composition. The dosage ranges described herein are examples and do not limit the dosage range that can be selected by medical professionals.

[0086] In some embodiments, the treatment involves transplanting reprogrammed retinal cells (e.g., regenerated RPCs) disclosed herein into the patient's eye, where the retinal cells are reprogrammed in vitro. [Example]

[0087] Example 1. Reprogramming of retinal interneuron cells This example demonstrates that other retinal neurons can be used as an endogenous cell source for retinal ganglion cell regeneration. By ectopic expression of transcription factors important for RGC differentiation, amacrine and displaced amacrine interneurons can be reprogrammed into RGCs. The regenerated RGCs project axons to subcortical regions of the brain, where they can respond to visual stimuli and transmit electrical signals to the brain, both under normal conditions and in animal models of glaucoma in which the original RGCs are damaged by elevated intraocular pressure.

[0088] method Mice and husbandry. Lgr5 EGFP-IRES-CreERT2 Knock-in mouse line, Pvalb CreERT2 The knock-in mouse line, Rosa26-tdTomato reporter mouse line, was obtained from the Jackson laboratory. EGFP-IRES-CreERT2マウス and Pvalb CreERT2 The mice were crossed with Rosa26-tdTomato mice and Lgr 5EGFP-IRES-CreERT2 ;Rosa26-tdTomato mice and Pvalb CreERT2 ;Rosa26-tdTomato mice were generated.

[0089] Prokr2 CreERT2Mouse strains were generated by homologous recombination using CRISPR / Cas9 technology. Briefly, in vitro transcribed Cas9 mRNA, sgRNA, and a donor vector plasmid were mixed and injected into the pronuclei of fertilized C57BL / 6J mouse eggs. The donor vector plasmid was designed to insert the CreERT2 coding region followed by a PolyA sequence into the ATG start codon of the Prokr2 locus. Injected zygotes were cultured to the blastocyst stage by day 3.5 and then implanted into the uterus of pseudopregnant females. Correctly targeted F0 mice were crossed with C57BL / 6J mice to generate F1 Prokr2 mice. CreERT2 We generated Prokr2 mice. CreERT2 Mice were crossed with Rosa26-tdTomato mice to express Prokr2 CreERT2 The Rosa26-tdTomato mouse was generated. The DNA sequence surrounding the translation start site of Prokr2 is as follows: [ka] The translation start site is in bold, and the target sequence of the sgRNA used is underlined. The donor vector plasmid, which contains a 5' 4 kb-homology arm, a CreERT2-polyA cassette, and a 3' 4 kb-homology arm, was constructed using the In-Fusion cloning method.

[0090] All mice were housed in an animal facility with a 12-hour light / 12-hour dark cycle. Animal experiments were performed on both male and female mice aged 8–12 months. All animal experimental procedures were approved by the Animal Care and Use Committee of Shanghai University for Science and Technology.

[0091] Construction and production of AAV vectors. Mouse Atoh7, Brn3B, Sox4, Sox11, Isl1 The coding sequences for Arch and EGFP were subcloned into a CAG-driven, Cre-dependent expression vector (Addgene #22222) in place of the original Arch-GFP sequence. To coexpress transcription factors and EGFP from a single AAV vector, a P2A fragment was placed between the two coding sequences.

[0092] To produce AAV viral particles, HEK293T cells were transfected with the AAV transgene plasmid, pAAV7m8 serotype plasmid, and pHelper plasmid using PEI. Cells were harvested 48–72 hours later. Viral particles were purified by iodixanol density gradient centrifugation and titrated by qPCR.

[0093] Intravitreal injection of AAV. Mice were anesthetized by IP injection of a mixture of ketamine (80 mg / kg) and xylazine (8 mg / kg), and the pupils were dilated with topical administration of phenylephrine hydrochloride eye drops (2.5%). After brief local anesthesia with 0.5% proparacaine hydrochloride eye drops, the cornea was punctured to reduce intraocular pressure, and 1.5 μL of AAV viral particles were injected into the vitreous cavity using a 34-gauge needle. For injection of the AAV mixture, each AAV was first injected at 1 x 10 12 particles / mL and then mixed.

[0094] Glaucoma Model. Mouse RGCs were injured using an intraocular pressure elevation (IPI)-induced ischemia / reperfusion (I / R) model, which mimics clinical acute angle-closure glaucoma. A previously published protocol was slightly modified: the anterior chamber of the mouse eye was encircled with a needle, which was then connected via tubing to an elevated saline (0.1% heparin) reservoir. The reservoir was elevated to 150 cm above the eyeball, halting blood flow to the inner retina (ischemia). After 60 minutes, the needle was removed to restore circulation (reperfusion). This protocol resulted in the degeneration of all RGC axons and the death of other retinal neurons. To prevent apoptosis of other retinal neurons, a solution of the ROCK inhibitor ripasudil hydrochloride dihydrate (0.4% in PBS) was administered once daily to the ocular surface of the mouse.

[0095] Immunohistochemistry and imaging. After transcardial perfusion with saline (0.9% NaCl in ddH2O) followed by 4% PFA, mouse eyes, optic nerves, and brains were harvested and postfixed in 4% PFA for 24 hours. Eye and brain tissues were placed in 30% sucrose for cytoprotection and sectioned at 10 μm and 30 μm thicknesses, respectively, using a microtome cryostat. Immunohistochemical staining was performed according to standard protocols. The following antibodies were used: rabbit anti-RBPMS (Abcam, 1:400) to label RGCs, mouse anti-Brn3A (Santa Cruz Biotechnology, 1:200) to label RGCs, rabbit anti-SMI-32 (Abcam, 1:400) to label α-RGCs, rabbit anti-melanopsin (Abcam, 1:500) to target ipRGCs, rabbit anti-CART (cocaine- and amphetamine-regulated transcript) (Phoenix Peptide, 1:2500) to label on-off CART, and mouse anti-PSD95 (Abcam, 1:400) to label postsynaptic membranes. Secondary detection used Alexa Fluor 647 donkey anti-rabbit (IFKine™, 1:400), Alexa Fluor 647 donkey anti-mouse (IFKine™, 1:400), or Alexa Fluor 488 donkey anti-rabbit (Abcam, 1:400). Immunostained tissue sections were imaged with a Zeiss LSM880 confocal microscope, a Nikon spinning disk (CSU W1 Sora) confocal microscope, or a STED SP8 microscope.

[0096] In vivo calcium imaging. For surgery, mice were anesthetized with urethane (1.5 g / kg), and the eyes were covered with ophthalmic ointment and placed in a stereotaxic apparatus. A custom-made titanium head plate was glued to the skull parallel to the long axis of the mouse, approximately at the center of the occipital suture, using black dental cement (Fe3O4 added to block light). A 3 mm craniotomy was made above the posteromedial SC and inferior colliculus. A 3 mm diameter cover glass was gently pressed against the dura, and the craniotomy was sealed with black dental cement. A light-shielding cloth was attached to the head plate to avoid light contamination from visual stimuli during two-photon functional imaging.

[0097] Visual stimuli were generated using the Psychtoolbox function in Matlab (Mathworks) and displayed on a calibrated 17' LCD monitor (Dell, 1280 × 1024 pixels, 75 Hz refresh rate) 15 cm from the contralateral eye. The stimuli were full-screen sinusoidal drifting gratings (spatial frequency: 0.05 cycles / °, temporal frequency: 2 Hz) presented on a uniform gray background. The gratings were presented five times with a duration of 1 s and an inter-stimulus interval of 1–2 s. The stimuli drifted in eight directions orthogonal to the four azimuth directions at regular intervals of 45°.

[0098] Two-photon imaging of fluorescence from axon terminals was monitored using a custom-built LotosScan microscope (LotosScan, Suzhou Institute of Biomedical Engineering and Technology) coupled to a mode-locked Ti:Sa laser (Chameleon VISION-S, Coherent). The excitation wavelength was fixed at 920 nm. Imaging was performed using a 40X, 0.8NA objective (Nikon). The beam size was large enough to fill the rear aperture of the 40X objective. Images were acquired at a frame rate of 50 Hz (480 x 240 pixels, 0.225 μm / pixel).

[0099] Images were analyzed using Matlab (Mathworks) and ImageJ (National Institutes of Health). To correct for lateral motion in the image data, frame-by-frame alignment based on a shape-preserving transformation was applied using Turboreg software (an ImageJ plugin). Terminals were manually identified based on size, shape, and brightness. The time course of individual terminals was extracted by averaging pixel intensity values ​​within the terminal mask in each frame. If cerebral pulsation was evident during imaging, these data were not used. Neuropil signals were analyzed using a previously reported method. 40After this correction, the response to each stimulus presentation (Ft) was normalized to the response during the 0.2 seconds immediately preceding stimulus onset (F0). For each stimulus, the mean fluorescence change (ΔF / F) was calculated by averaging responses across all stimulus conditions and trials. Visually responsive cells were defined by ANOVA across the blank and stimulus presentation periods (P < 0.05).

[0100] Lgr5 + Whole-cell patch clamp recording of amacrine interneurons. Mice were dark-adapted for at least 2 hours and then euthanized. Retinal sections were then dissected under infrared light in artificial cerebrospinal fluid (ACSF) containing 126 mM NaCl, 1.25 mM NaH2PO4, 2.5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM glucose, and 26 mM NaHCO3. Retinal sections were manually cut with a razor blade, attached to a strip of paper, transferred to a recording chamber on the microscope stage, and perfused with oxygenated (95% O2 / 5% CO2) ACSF. Lgr5-tdTomato in the INL + Cells were identified using two-photon microscopy and targeted for whole-cell patch clamp recording under infrared light. Pipettes (4-7 MΩ) were filled with intracellular solution containing 120 mM Cs-methanesulfonate, 5 mM NaCl, 10 mM HEPES, 5 mM EGTA, 5 mM QX314, 0.5 mM CaCl2, 4 mM ATP, and 0.5 mM GTP for voltage-clamp recordings, or 123 mM K-gluconate, 10 mM KCl, 10 mM HEPES, 2 mM EGTA, 1 mM CaCl2, 1 mM MgCl2, 4 mM ATP, and 0.5 mM GTP for current-clamp recordings. All reagents used above were obtained from Sigma. Alexa488 hydrazide (0.2 mM, ThermoFisher) was added to the intracellular solution to visualize the morphology of recorded cells. Signals were acquired and processed using a Multiclamp700A amplifier and the pClamp10 software suite (Molecular Devices). Signals were filtered at 1 kHz and sampled at 10 kHz (Digidata 1440A, Molecular Devices). EPSCs were recorded using Cl -Recordings were made at a reversal potential of −67 mV, and IPSCs were recorded at 0 mV. Full-field light stimulation was delivered using a white LED light controlled by the recording computer.

[0101] In vitro whole-cell patch clamp recording of SC neurons. Deeply anesthetized mice were transcardially circulated with ice-cold oxygenated (95% O2, 5% CO2) cutting solution containing 92 mM choline chloride, 2.5 mM KCl, 1.2 mM NaH2PO4, 30 mM NaHCO3, 10 mM MgSO4, 0.5 mM CaCl2, 25 mM glucose, 5 mM Na-ascorbate, 3 mM sodium pyruvate, and 2 mM thiourea. The pH of the cutting solution was adjusted to 7.3-7.4 by adding concentrated HCl, and the osmolality was adjusted to 310-315 mOsm. After removal from the skull, brain tissue containing the SC region was cut into 300 μm coronal sections in the cutting solution using a vibrating blade microtome (VT1200 S, Leica Biosystems). The sections were then incubated in the same cutting solution for 15 min at 31–32°C before being transferred to a holding chamber containing oxygenated holding solution (92 mM NaCl, 30 mM NaHCO3, 1.25 mM NaH2PO4, 2.5 mM KCl, 2 mM MgSO4, 2 mM CaCl2, 25 mM glucose, 20 mM HEPES, 5 mM Na-ascorbate, 3 mM sodium pyruvate, and 2 mM thiourea, pH 7.3–7.4, osmolality 310–315 mOsm) at room temperature. After 1 h of storage, the sections were transferred to a recording chamber containing oxygenated recording solution (119 mM NaCl, 24 mM NaHCO3, 1.25 mM NaH2PO4, 2.5 mM KCl, 2 mM MgSO4, 2 mM CaCl2, and 12.5 mM glucose) at room temperature. Three to five slices encompassing the SC region were typically prepared from one animal, and recordings were taken from brain slices encompassing the central SC region.

[0102] Whole-cell patch-clamp recordings of synaptic responses were performed with 2-4 MΩ glass pipettes containing an internal solution of 125 mM K-gluconate, 20 mM KCl, 0.5 mM EGTA, 10 mM HEPES-NaOH, 10 mM P-creatine, 4 mM ATP-Mg, and 0.3 mM GTP (pH 7.3). Blue stimulus light was delivered by a 470 nm LED (Thorlabs, 35 mW / mm 2 ) and illuminated through a 40X objective (OLYMPUS). A 5-ms stimulus duration was found to saturate the recorded postsynaptic responses. The neuron's input resistance ranged from 1 to 5 GΩ, with series resistance below 20 MΩ. Recordings were performed using the following protocol: the membrane potential was first held at -70 mV to record light-evoked AMPA receptor-mediated synaptic currents (NMDA receptors were likely blocked by magnesium at this holding potential). The membrane holding potential was then switched to +55 mV to record a mixture of AMPA and NMDA receptor-mediated currents. Under these conditions, the AMPA receptor antagonist CNQX (10 mM) was then added to the recording solution to block AMPA receptor-mediated synaptic currents, allowing detection of NMDA receptor-mediated EPS. Recording was then switched to current-clamp mode to detect action potentials. The AMPA receptor antagonist CNQX (Tocris) and the NMDA receptor antagonist D-APV (Tocris) were applied by adding the respective drugs to the immersion recording solution. All recordings were made with an Axon 700B amplifier and digitized with a Digidata 1440 analog-to-digital board. Stimulation and data acquisition were performed with pClamp software and digitized at 50 kHz. All equipment and software was obtained from Axon Instruments / Molecular Devices (Molecular Devices, CA).

[0103] Statistics. Differences between two groups were compared using a two-tailed Student's t-test.

[0104] result Lgr5 +In vivo reprogramming of amacrine interneurons into RGCs. First, Lgr5 EGFP-IRES-CreERT2 We used the Rosa26-tdTomato mouse strain to test whether RGCs can be regenerated from amacrine interneurons. Lgr5 is expressed in a subset of retinal cells located on the vitreous side of the inner nuclear layer (Fig. 1a). + The cells not only exhibited the typical morphology of amacrine interneurons (Figures 1a-c and 7a-d), but also had active synaptic connections that responded to light stimulation (Figures 1e, f). They could receive and be depolarized or hyperpolarized by both excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs), as revealed by targeted patch-clamp recording (Figures 1e, f), suggesting that they were indeed mature amacrine interneurons. However, Lgr5 cells were not expressed in the Lgr5 subunits. + Amacrine interneurons were labeled with the tdTomato reporter and lineage-traced in adult mice. tdTomato was detected several months later. + Bipolar cells and horizontal cells (some Lgr5 + Amacrine cells (indicating that Lgr5 expression is lost and they transdifferentiate into other retinal lineages) were found in very low numbers (less than one cell per retina at any given time point) (Fig. 1b, c), indicating their limited regenerative potential. As mice aged, low numbers of Lgr5 cells were observed in the retinal ganglion cell layer. + Amacrine cells were detected, suggesting that they may have the ability to migrate from the inner nuclear layer to the retinal ganglion layer (Fig. 1d and Fig. 7eg). + The number of cells increases with age (Fig. 7h), and some of these cells stop expressing Lgr5 but do not become RGCs.

[0105] Lgr5 + To investigate whether amacrine interneurons can be reprogrammed into RGCs, we devised an in vivo lineage tracing and reprogramming strategy (Fig. 2a). +We labeled amacrine neurons with the Rosa26-tdTomato reporter and then ectopically expressed a gene essential for RGC fate determination specifically in these cells using a Cre-dependent, double-floxed, inverted open reading frame (DIO) expression system delivered via adenovirus-associated virus (AAV) (Figure 2b). Lgr5 + Regarding the generation of RGCs from amacrine cells, tdTomato shows the morphology of RGCs in flat-mounted retinal samples. + Presence of cells and tdTomato in the optic nerve at later time points + Analysis was performed by examining the presence of axons.

[0106] tdTomato in flat-mounted retinal samples + No RGC cells were observed, and tdTomato was not observed in the optic nerves of control mice injected intravitreally with AAV-DIO-EGFP. + However, in the retinal samples from mice injected with AAVs expressing genes important for RGC fate determination (Atoh7, Brn3B, Sox4, Sox11, and Isl1), tdTomato axons with RGC morphology were observed. + Six weeks after induction of gene expression, tdTomato cells with RGC morphology were detected in the retinal samples. + These cells extended axon-like processes toward the optic nerve head and into the optic nerve (Fig. 3a). Their cell bodies were located in the retinal ganglion cell layer and could be stained with RGC-specific markers RBPMS and Brn3A (Fig. 2g, h). Therefore, these cells may be considered newly generated RGCs.

[0107] On average, approximately 180 new RGCs were regenerated per retina 6 weeks after viral injection (Fig. 2c). This number was significantly higher than the Lgr5 RGCs present in the retinal ganglion layer at the time of initiating in vivo reprogramming. +The number of amacrine cells is much higher than that of the retinal ganglion cell layer of 2-3 month old mice (approximately 10-15 cells in the retinal ganglion cell layer) (Fig. 7h). + These results suggest that ectopic expression of RGC fate factors in amacrine cells may trigger migration of some of these cells from the inner nuclear layer to the ganglion cell layer. In support of this idea, tdTomato, which expresses lower levels of Lgr5-EGFP, + Cells were detected in the inner plexiform layer (Fig. 8 fh).

[0108] We investigated the reprogramming activity of single transcription factors and their combinations. We found that a single transcription factor (Brn3B or Sox4) could induce the reprogramming activity of Lgr5. + We found that reprogramming of amacrine interneurons into RGCs was possible, but the efficiency was very low (Fig. 2c). The combination of Brn3B and Sox4 dramatically synergized the reprogramming activity. Addition of Atoh7 to the Brn3B+Sox4 combination did not significantly improve the reprogramming efficiency (Fig. 2c). Therefore, unless otherwise noted, we used the Brn3B+Sox4 combination for the remaining experiments.

[0109] RGCs are a heterogeneous class of retinal neurons and can be classified into different subtypes. Immunohistological analysis using subtype-specific antibodies revealed that anti-CART (for on-off direction-selective ganglion cells) and anti-SMI-32 (for alpha ganglion cells) antibodies can identify regenerating RGCs (Figures 2i and 8il), but not melanopsin-expressing endogenous photosensitive ganglion cells. Collectively, these results suggest that ectopic expression of specific transcription factors, such as Lgr5, can alter the regeneration of RGCs. + These findings suggest that amacrine interneurons can be reprogrammed into RGCs and that the regenerated RGCs are subtype-specific.

[0110] Regenerated RGC Axon Projections to the Visual Nuclei of the Brain. To investigate whether regenerated RGCs can properly reconnect within the brain, we examined the axons of regenerated RGCs along the retinal pathway and their projections to the major retinal receptor areas of the brain. Six weeks after viral injection, many of the axons of regenerated RGCs traversed the entire optic nerve, passed through the optic chiasm, and entered the visual nuclei of the brain, including the dorsal and lateral ventral geniculate nuclei (dLGN and vLGN), pretectal area, and superior colliculus (SC) (Figure 3). No abnormal projections of regenerated RGC axons to brain regions unrelated to the visual pathway were observed. Within the retinal receptor areas, micron-sized nodular structures were observed along the axonal branches of regenerated RGCs. These nodular structures were closely associated with staining for the postsynaptic density protein PSD-95 (Figure 3g-i), suggesting that they represent virtual presynaptic boutons.

[0111] We investigated the time course of regenerating RGC axon projections to three key visual regions in the brain: the optic chiasm (OC), LGN, and SC, by analyzing the time when regenerating RGC axons were first detected in these regions in brain sections after viral injection. We found that it took approximately 18 days for RGC axons to reach the OC, 28 days for them to reach the LGN, and 35 days for them to reach the farthest visual target, the SC (Figure 9). Collectively, these data support the conclusion that Lgr5 + We demonstrate that amacrine interneuron-derived RGCs can project axons to appropriate brain regions and establish retina-brain connections.

[0112] Prokr2 + Reprogramming of replacement amacrine interneurons into RGCs. We wondered whether other retinal neurons could also be reprogrammed into RGCs. Because amacrine interneurons are located in the RGC layer, they may be a better cell source for RGC replacement. To test whether this neuronal subtype could be reprogrammed into RGCs, we used Prokr2. CreERT2 A knock-in mouse line was generated (Fig. 10a and b). CreERT2These mice express the tamoxifen-inducible CreERT2 recombinase under the endogenous transcriptional control of the Prokr2 gene, which is expressed in a subgroup of replacement amacrine interneurons. As expected, tamoxifen-treated adult Prokr2 CreERT2 ; In Rosa26-tdTomato mice, tdTomato + The cells are located in the retinal ganglion cell layer. They have no optic projections and do not express the RGC marker RBPMS (Fig. 4a, b and Fig. 10c, c).

[0113] In addition to being expressed in the retina, Prokr2 is also expressed in cells of the optic nerve and brain (Fig. 10f-h). This prevented us from tracing the axons of regenerating RGCs using the Rosa26-tdTomato reporter. To overcome this obstacle, we labeled regenerating RGCs by co-expressing EGFP with transcription factors during programming (Fig. 10k). We performed reprogramming using two combinations of transcription factors (Brn3B+Sox4 and Atoh7+Brn3B+Sox4), and found that Prokr2 expression was significantly reduced in both combinations. + We found that replacement amacrine interneurons could be efficiently reprogrammed into RGCs (Fig. 4c, d). However, Lgr5 + Unlike the case of amacrine interneurons, the inclusion of Atoh7 in the Brn3B+Sox4 combination dramatically improved reprogramming efficiency (Fig. 10l). + The RGCs derived from the replacement amacrine interneurons extended axons to the optic nerve and various visual targets in the brain (Figure 4E). Thus, these results demonstrate that RGCs can be regenerated by reprogramming multiple retinal neuron subtypes in vivo.

[0114] Transmission of visual information to the brain by regenerated RGCs. To examine whether regenerated RGCs can respond to visual stimuli and transmit visual information to downstream targets in the brain, we added AAV-DIO-GCamp6f to the reprogramming cocktail to transduce Lgr5 EGFP-IRES-CreERT2We labeled regenerating RGCs in mice with the calcium indicator GCamp6f. Next, 6 weeks after viral injection, we exposed the SCs of anesthetized mice and measured visually evoked calcium dynamics in the regenerating RGC axon terminals using in vivo functional calcium imaging (Figure 11a).

[0115] When mice were presented with drifting gratings, individual RGC boutons along the axonal branches of the SC exhibited stimulus-evoked calcium signals (Figure 11b), demonstrating that regenerated RGCs can respond to visual stimuli and transmit visual signals to the brain. Visually responsive boutons could be classified into different categories based on their response patterns. Boutons responded differently not only to on- and off-stimulus signals, but also to the orientation and direction of the drifting gratings (Figures 5a-d and 11c-d). Collectively, these data suggest that regenerated RGCs can transmit visual information to the brain and that in vivo reprogramming can generate functionally distinct RGC subtypes.

[0116] Establishment of functional synaptic connections with postsynaptic neurons by regenerated RGCs. To examine whether regenerated RGCs can transmit neural signals to postsynaptic neurons in the brain, we analyzed Lgr5 EGFP-IRES-CreERT2 We expressed channelrhodopsin-2 (ChR2) in regenerating RGCs from Rosa26-tdTomato mice and used whole-cell patch recording to detect light-evoked postsynaptic responses of SC neurons in brain slices 8–10 weeks after viral injection.

[0117] Light stimulation of the axon terminals of regenerated RGCs detected AMPA receptor-mediated excitatory postsynaptic currents (EPSCs) in SC neurons. A single light impulse evoked AMPA receptor-mediated EPSCs with multiple peaks (Figure 5e, h, and i), suggesting that the axons of regenerated RGCs formed multi-input synapses with SC neurons via activated AMPA glutamatergic receptors. NMDA receptor-mediated EPSCs and action potentials were also detected in postsynaptic SC neurons after light stimulation (Figure 5f, g, and j). Furthermore, the responses of SC neurons to regenerated ChR2-expressing RGCs were comparable to those of normal RGCs expressing ChR2 (Figure 11e, f). Collectively, these results suggest that in response to light stimulation, regenerated RGC axon terminals release glutamate as a neurotransmitter and establish functional synaptic connections with SC neurons.

[0118] Regeneration of functional RGCs in a mouse model of glaucoma. We next investigated whether regenerated RGCs could repair visual circuits under pathological conditions. In particular, we were interested in determining whether regenerated RGCs could send axons to the appropriate targets in the brain and reestablish retina-brain connections, even when the original RGCs and their axons had degenerated.

[0119] We optimized the conditions for damaging RGCs and their axons using an intraocular pressure-induced glaucoma model, which could induce degeneration of all RGC axons in the optic nerve and significant loss of RGC cell bodies in the retina (Figure 12a-e). However, under these damaging conditions, Lgr5 expression was not observed 7 days after intraocular pressure elevation. + Amacrine cells also disappeared dramatically (Fig. 6b). + When we searched for a reagent that could protect amacrine cells, we found that the Rock inhibitor ripasudil inhibited Lgr5 + We found that amacrine cells could be efficiently preserved (Fig. 6c).

[0120] We devised a protocol that combines neuroprotection and in vivo reprogramming to demonstrate that newly generated RGCs express Lgr5 EGFP-IRES-CreERT2We tested whether the damaged visual circuits of Rosa26-tdTomato mice could be repaired (Fig. 12f). EGFP-IRES-CreERT2 ;Rosa26-tdTomato mice were injured in both eyes and Lgr5 + Amacrine cells were labeled with the tdTomato reporter. After injury, both eyes were treated with Ripasudil once daily, and 7 days later, the left eye was injected with AAV-DIO-EGFP as a control, and the right eye was injected with a combination of AAVs expressing transcription factors including Brn3B, Sox4, and Ato7, and Lgr5. + We performed reprogramming of amacrine interneurons. Six weeks after viral injection, the mice were sacrificed for analysis. In the left eye injected with AAV-DIO-EGFP, tdTomato + RGCs did not regenerate, as no RGC axons were detected in the left optic nerve (Fig. 6e). On the other hand, overexpression of Brn3B, Sox4, and Atoh7 inhibited the regeneration of Lgr5. + Amacrine cells are reprogrammed into RGCs, and the regenerating RGCs are expressed as tdTomato cells. + Axons projected to the optic nerve and to various contralateral brain visual targets (Fig. 6 fk).

[0121] Regenerated RGCs established functional synaptic connections with postsynaptic brain neurons under pathological conditions. Light-evoked postsynaptic responses were detected in SC neurons on brain slices, where all RGC axon terminals were from RGCs that had regenerated after the original RGCs were damaged (Figure 6l-n). Collectively, these results demonstrate that regenerated RGCs can reconnect the retina and brain and transmit visual information to postsynaptic neurons under pathological conditions.

[0122] These results demonstrate that in vivo reprogramming of fully differentiated retinal interneurons can generate functional RGCs in adult mammals. Ectopic expression of essential transcription factors allows precise reprogramming of both amacrine and replacement amacrine interneurons into RGCs, and the newly generated RGCs can integrate into visual circuits and transmit visual information to the brain. While in vivo neuronal identity reprogramming has been achieved in other regions of the central nervous system (CNS), successful conversion between neuronal subtypes was limited to the first week after birth and, in adult mice, only in the presence of the chemical valproic acid. In contrast, in this study, we demonstrated that retinal neuron identity switching is possible in adults, even without chemical stimulation, and successful reprogramming even induces migration of amacrine interneurons from the inner nuclear layer to the RGC layer. These results suggest that neurons exhibit surprising and unexpected identity plasticity, which may be exploited for regenerative purposes.

[0123] The combination of Brn3B and Sox4 is Lgr5 + Amacrine interneurons and Prokr2 + We efficiently reprogrammed both replacement amacrine interneurons and Lgr5 into RGCs, demonstrating that these two transcription factors are sufficient for RGC fate determination. + Prokr2 does not improve amacrine cell reprogramming + The efficiency of regenerating RGCs from replacement amacrine cells was significantly improved, suggesting that direct lineage reprogramming is influenced by the intrinsic properties of the source cells.

[0124] Regenerating RGCs connect the retina and brain by projecting axons long distances to various visual areas, even in animals where the original RGCs and axons have been damaged. This finding reveals that the adult mammalian visual system maintains a remarkable ability to rewire neural circuits.

[0125] Example 2. Rejuvenation of degenerated RGCs This example shows that degenerated RGCs can be reactivated by transcription factors and once again grow functional axons.

[0126] In this study, we used elevated intraocular pressure to induce apoptosis of retinal ganglion cells (RGCs) and degenerate their axons in PV-CreERT2;Rosa26-tdTomato mice. In this animal model, expression of three transcription factors (Atoh7+Brn3B+Sox4) in the surviving RGCs stimulated axonal regrowth (regeneration) in these cells (Figure 13). Importantly, the axons of the regenerated RGCs projected into the optic nerve and reached the visual cortex in the brain (Figure 14).

[0127] These results demonstrate that a combination of transcription factors can not only reprogram interneuron cells into regenerative RGCs, but also rejuvenate degenerated, damaged, injured, or aged RGCs. Thus, when administered to subjects seeking vision repair, restoration, or improvement, these transcription factors can act cooperatively on both interneurons and RGCs to achieve the desired therapeutic effect.

[0128] While the present disclosure has been described with reference to disclosed embodiments, those skilled in the art will readily appreciate that the specific examples and studies detailed above are merely illustrative of the present disclosure. It is understood that various modifications can be made without departing from the spirit of the present disclosure. Accordingly, the present disclosure is limited only by the scope of the following claims.

Claims

1. A nucleic acid construct comprising coding sequences encoding Brn3B and Sox4 proteins and a promoter associated with each coding sequence, wherein each promoter is active in amacrine cells.

2. 2. The nucleic acid construct of claim 1, wherein at least one of the promoters is a promoter of a Pax6, Tcfap2b, Gad1, GlyT1, RBPMS, or Prox1 gene.

3. The nucleic acid construct of claim 1 , wherein at least one of the promoters is a synapsin 1 promoter.

4. The nucleic acid construct of any one of claims 1 to 3, further comprising an expression vector which is a plasmid vector or a viral vector.

5. The nucleic acid construct of claim 4 , wherein the expression vector is an AAV vector.

6. 6. The nucleic acid construct of claim 5, wherein the AAV is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12.

7. The nucleic acid construct of claim 5, wherein the AAV is AAV2.7m8 or AAV7m8.

8. An amacrine cell transfected with the nucleic acid construct according to any one of claims 1 to 7.

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