Regenerating functional neurons for treatment of neural injury caused by disruption of blood flow

Abstract

To provide methods and compositions of treating the effects of disruption of normal blood flow in the CNS in a subject.SOLUTION: Provided are methods including administering a therapeutically effective dose of exogenous NeuroD1 to an area where normal blood flow has been disrupted. Compositions are provided including 1) a recombinant adeno-associated adenovirus expression vector comprising a glial cell specific promoter operably linked to a nucleic acid encoding a site-specific recombinase and 2) a recombinant adeno-associated adenovirus expression vector comprising a ubiquitous promoter operably linked to a nucleic acid encoding NeuroD1, the nucleic acid encoding NeuroD1 being inverted, and flanked by two sets of site-specific recombinase recognition sites such that action of the recombinase irreversibly inverts the nucleic acid encoding NeuroD1 such that NeuroD1 is expressed in a mammalian cell.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 464,469, filed February 28, 2017; and U.S. Provisional Patent Application No. 62 / 518,914, filed June 13, 2017, the entire contents of each of which are incorporated herein by reference.

[0002] government support This invention was made with government support under Grant No. AG045656 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] FIELD OF THE INVENTION The present invention relates to compositions and methods for treating the effects of disruption of normal blood flow in the CNS of an individual subject. [Background technology]

[0004] The mammalian central nervous system (CNS) is largely unable to regenerate itself after injury. Neurons and other CNS cells often die or are damaged as a result of disease or injury that blocks blood flow to the area in which the cells are located. Furthermore, pathological changes near the blockage of blood flow are responsible for many deleterious effects, such as destruction or damage of neurons, destruction or damage of glial cells, destruction or damage of blood vessels, and destruction or damage of supporting cells in the CNS area affected by the disruption of normal blood flow. There remains a need for compositions and methods for treating the effects of disruption of normal blood flow in the CNS of an individual subject. Summary of the Invention

[0005] According to an embodiment of the present invention, there is provided a method for treating the effects of disruption of normal blood flow in the CNS of an individual subject in need thereof, comprising administering a therapeutically effective dose of exogenous NeuroD1 to an area where normal blood flow is disrupted. Optionally, administering exogenous NeuroD1 comprises delivering to the area an expression vector comprising a nucleic acid encoding NeuroD1. In a further option, administering exogenous NeuroD1 comprises delivering to the area a recombinant viral expression vector comprising a nucleic acid encoding NeuroD1. Optionally, NeuroD1 is the only exogenously expressed transcription factor delivered to the area.

[0006] According to an aspect of the present invention, there is provided a method of treating the effects of disruption of normal blood flow in the CNS of an individual subject in need thereof, comprising administering a therapeutically effective dose of exogenous NeuroD1 to an area where normal blood flow is disrupted, wherein expression of the exogenous NeuroD1 comprises delivering to the area a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding NeuroD1. Optionally, NeuroD1 is the only exogenously expressed transcription factor delivered to the area.

[0007] According to an embodiment of the present invention, the nucleic acid sequence encoding NeuroD1 is operably linked to a glial cell-specific promoter. According to an embodiment of the present invention, the glial cell-specific promoter is a GFAP promoter. According to an embodiment of the present invention, the GFAP promoter is a human GFAP promoter.

[0008] According to an aspect of the present invention, there is provided a method of treating the effects of disruption of normal blood flow in the CNS of an individual subject in need thereof, comprising administering a therapeutically effective dose of exogenous NeuroD1 to an area where normal blood flow is disrupted, wherein expression of the exogenous NeuroD1 comprises delivering to the area an effective "flip-excision" recombinant expression vector combination of 1) a recombinant adeno-associated viral expression vector comprising a nucleic acid encoding NeuroD1, and 2) a recombinant adeno-associated viral expression vector comprising a nucleic acid encoding a site-specific recombinase. Optionally, NeuroD1 is the only exogenously expressed transcription factor delivered to the area.

[0009] According to an embodiment of the present invention, there is provided a method of treating the effects of disruption of normal blood flow in the CNS of an individual subject in need thereof, comprising administering a therapeutically effective dose of exogenous NeuroD1 to the area where normal blood flow is disrupted, wherein expression of the exogenous NeuroD1 comprises delivering an effective "flip-excision" combination of recombinant expression vectors: 1) a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding NeuroD1, wherein the nucleic acid sequence encoding NeuroD1 is operably linked to a ubiquitous promoter; and 2) a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a site-specific recombinase, wherein the nucleic acid sequence encoding the site-specific recombinase is operably linked to a glial cell-specific promoter. According to an embodiment of the present invention, the glial cell-specific promoter is the GFAP promoter. According to an embodiment of the present invention, the GFAP promoter is the human GFAP promoter. Optionally, NeuroD1 is the only exogenously expressed transcription factor delivered to the area.

[0010] According to an embodiment of the present invention, there is provided a method of treating the effects of disruption of normal blood flow in the CNS of an individual subject in need thereof, comprising administering to the CNS in an area where normal blood flow has been disrupted adeno-associated viral particles comprising a nucleic acid encoding NeuroD1 in an amount of 10 10 ~10 14 The method includes administering to a subject 1 to 500 μl of a pharmaceutically acceptable carrier containing the adeno-associated virus particles at a concentration of 1 to 500 μl of the adeno-associated virus particles / 1 ml of carrier at a controlled flow rate of 0.1 to 5 μl / min.

[0011] According to an embodiment of the present invention, exogenous NeuroD1 is administered once reactive astrocytes are present, after normal blood flow in the CNS has been disrupted.

[0012] According to an embodiment of the present invention, exogenous NeuroD1 is administered once after normal blood flow in the CNS has been disrupted when glial scarring is present.

[0013] According to an embodiment of the present invention, the disruption of normal blood flow in the CNS is due to a disorder selected from the group consisting of ischemia, thrombosis, embolism, hemorrhage, concussion, brain penetration, blast, inflammation, infection, tumor, chronic disease-mediated restriction of blood vessels, and a combination of any two or more thereof.

[0014] According to aspects of the present invention, the disruption of normal blood flow in the CNS is due to a disorder selected from the group consisting of ischemic stroke; hemorrhagic stroke; cerebral aneurysm; traumatic brain injury; concussion; blast; brain invasion; inflammation; infection; tumor; traumatic spinal cord injury; ischemic or hemorrhagic myelopathy (spinal cord infarction); global cerebral ischemia caused by cardiac arrest or severe low blood pressure (shock); hypoxic-ischemic encephalopathy caused by hypoxia, hypoglycemia, or anemia; CNS embolism caused by infective endocarditis or atrial myxoma; fibrocartilaginous embolic myelopathy; CNS thrombosis caused by childhood leukemia; cerebral venous sinus thrombosis caused by nephrotic syndrome (kidney disease), chronic inflammatory disease, pregnancy, use of estrogen-based contraceptives, meningitis, dehydration; or a combination of any two or more thereof.

[0015] According to an aspect of the present invention, there is provided a method of treating the effects of disruption of normal blood flow in the CNS of an individual subject in need thereof, comprising administering a therapeutically effective dose of exogenous NeuroD1 to the area where normal blood flow is disrupted, wherein administering the therapeutically effective dose of NeuroD1 comprises administering a recombinant expression vector, wherein the expression vector comprises a nucleic acid sequence encoding a NeuroD1 protein, wherein the nucleic acid sequence encoding the NeuroD1 protein is selected from the group consisting of a nucleic acid sequence encoding SEQ ID NO:2 or a functional fragment thereof, a nucleic acid sequence encoding SEQ ID NO:4 or a functional fragment thereof, In some embodiments, the nucleic acid sequence comprises a nucleic acid sequence selected from the group consisting of a nucleic acid sequence encoding a protein having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a nucleic acid sequence encoding a protein having SEQ ID NO:1 or a functional fragment thereof, SEQ ID NO:3 or a functional fragment thereof, and SEQ ID NO:2 or SEQ ID NO:4 or a functional fragment thereof.

[0016] Optionally, administering the therapeutically effective dose of NeuroD1 comprises stereotactic injection within or near the glial scar.

[0017] Optionally, the method of treating the effects of disruption of normal blood flow in the CNS of an individual subject in need thereof according to the present invention further comprises evaluating the effectiveness of the treatment in the subject. Optionally, the method of treating the effects of disruption of normal blood flow in the CNS of an individual subject in need thereof according to the present invention further comprises evaluating the effectiveness of the treatment in the subject, wherein evaluating the effectiveness of the treatment comprises an assay selected from an electrophysiological assay, a blood flow assay, a tissue structure assay, a functional assay, and a combination of any two or more thereof.

[0018] Optionally, the method of treating the effects of disruption of normal blood flow in the CNS of an individual subject in need thereof according to the present invention further comprises assessing the effectiveness of the treatment in the subject, wherein assessing the effectiveness of the treatment comprises an electroencephalogram of the subject.

[0019] Optionally, the method of treating the effects of disruption of normal blood flow in the CNS of an individual subject in need thereof according to the present invention further comprises assessing the effectiveness of the treatment in the subject, wherein assessing the effectiveness of the treatment comprises an assay of blood flow selected from the group consisting of near-infrared spectroscopy and fMRI.

[0020] Optionally, the method of treating the effects of disruption of normal blood flow in the CNS of an individual subject in need thereof according to the present invention further comprises assessing the effectiveness of the treatment in the subject, wherein assessing the effectiveness of the treatment comprises a histological assay selected from the group consisting of MRI, CAT scan, PET scan and ultrasound.

[0021] Optionally, the method of treating the effects of disruption of normal blood flow in the CNS of an individual subject in need thereof according to the present invention further comprises assessing the effectiveness of the treatment in the subject, wherein assessing the effectiveness of the treatment comprises a behavioral assay.

[0022] Optionally, assessing the effectiveness of the subject's treatment includes an assay performed before administering a therapeutically effective dose of exogenous NeuroD1. Thus, for example, aspects of the present invention provide a method of treating the effects of disruption of normal blood flow in the CNS of an individual subject in need thereof, comprising: 1) performing on the subject a first assay selected from an electrophysiology assay, a blood flow assay, a tissue structure assay, a functional assay, or a combination of any two or more thereof, where the first assay is performed before administering a therapeutically effective dose of exogenous NeuroD1; 2) administering a therapeutically effective dose of exogenous NeuroD1 to an area where normal blood flow has been disrupted; and 3) performing on the subject a second assay selected from an electrophysiology assay, a blood flow assay, a tissue structure assay, a functional assay, or a combination of any two or more thereof, where the second assay is performed after administering the therapeutically effective dose of exogenous NeuroD1.

[0023] According to an aspect of the present invention, there is provided a composition comprising: 1) a recombinant adeno-associated adenovirus expression vector comprising a glial cell-specific promoter operably linked to a nucleic acid encoding a site-specific recombinase; and 2) a recombinant adeno-associated adenovirus expression vector comprising a ubiquitous promoter operably linked to a nucleic acid encoding NeuroD1, wherein the nucleic acid encoding NeuroD1 is inverted and flanked by two sets of site-specific recombinase recognition sites, such that action of the recombinase irreversibly inverts the nucleic acid encoding NeuroD1 such that NeuroD1 is expressed in mammalian cells.

[0024] The NeuroD1-mediated astrocyte-to-neuron conversion produced by the methods of the present invention results in 100-600 NeuN+ new neurons / mm in the stroke area. 2 This is 20 to 200 times more effective than the internal nerve regeneration capacity.

[0025] Along with neuronal regeneration, NeuroD1-treatment according to embodiments of the present invention also reduces reactive astrocytes, reduces neuroinflammation, enhances neuronal survival, repairs blood vessels, and restores the blood-brain barrier (BBB) ​​after stroke. Furthermore, NeuroD1-converted neurons according to embodiments of the present invention not only form local neural circuits but also integrate into global brain networks, achieving functional rescue of motor deficits induced by ischemic stroke. Thus, NeuroD1-mediated in vivo cell conversion according to the therapeutic methods of the present invention provides unprecedented neuronal regeneration efficiency for the treatment of neurological disorders.

[0026] Therapeutic methods according to embodiments of the present invention demonstrate that NeuroD1-mediated in vivo glial-to-neuronal conversion can functionally rescue motor deficits induced by ischemic stroke. In addition to neuronal regeneration following glial conversion, many damaged neurons are preserved following reduction in reactive astrocytes and decreased neuroinflammation. NeuroD1 treatment of the present invention also repairs damaged blood vessels, restores BBB integrity in the stroke area, and changes the characteristics of the environment from neuroinhibitory to neuropermissive. Therapeutic methods according to embodiments of the present invention regenerate and preserve over 50-80% of all neurons following injury caused by disruption of normal blood flow in the CNS.

[0027] Highly efficient nerve regeneration by in vivo cell conversion Brain function depends on a delicate balance between neurons and their surrounding glial cells. After severe ischemic injury, neurons die immediately or gradually due to secondary damage, while their neighboring glial cells become activated and may begin to proliferate, resulting in glial scar tissue that ultimately inhibits neuronal regeneration. The NeuroD1-mediated in vivo cell conversion technique described herein restores neuronal function in damaged areas resulting from the disruption of normal blood flow in the CNS by directly converting reactive glial cells into functional neurons. The in vivo glial-to-neuron conversion technique described herein has many advantages over the administration of "classical" exogenous stem cells for engraftment and treatment of stroke.

[0028] One advantage is that endogenous glial cells are used instead of foreign cells for neural regeneration, avoiding the immune rejection associated with cell transplantation. Using endogenous glial cells near lost neurons for regeneration is perhaps the most economical way to restore neuronal function in local circuits. Another advantage is that the conversion of dividing reactive glial cells into non-dividing neurons not only reduces the number of reactive glial cells, but also reduces the potential tumor risk associated with dividing glial cells. In contrast, "classical" stem cell therapy, which features the administration of exogenous stem cells, is inherently associated with tumor risk. Therefore, exogenous stem cells are not administered in the method of treating the effects of disruption of normal blood flow in the CNS of an individual subject in need thereof according to this embodiment of the present invention.

[0029] Furthermore, the NeuroD1-mediated glial-to-neuron conversion therapy described herein resulted in an average of 400 NeuN+ cells / mm 2 It can regenerate large numbers of new neurons in areas of the CNS characterized by disruption of normal blood flow, such as the stroke area, which is approximately 100 times the internal neuronal regeneration capacity in the adult mouse cortex or striatum after ischemic stroke.

[0030] Furthermore, this remarkable regenerative efficacy is incomparable to the typically low efficiency of regeneration after exogenous stem cell engraftment. Without being bound by theory, effective regeneration is important for functional restoration because the small number of new neurons may not survive in the injured environment or may be insufficient to reconstruct neural circuits.

[0031] In contrast to modulating endogenous neural stem cells, the in vivo cell conversion approach described herein utilizes reactive glial cells, which are closely associated with neural injury throughout the nervous system, for in situ regeneration and repair. Furthermore, the in vivo cell conversion approach described herein is distinctly different from many other approaches currently under development for the treatment of stroke, which primarily focus on short-term treatments immediately following ischemic injury, such as removing blood clots, promoting blood flow, anti-inflammation, antioxidants, or reducing excitotoxicity. While these approaches are necessary for short-term treatment, their long-term effectiveness is limited when large numbers of neurons are lost or functionally impaired after a stroke.

[0032] Complementing these short-term approaches, the in vivo cell conversion approach described herein offers a long-term solution by converting local glial cells and reconstructing local neural circuits disrupted by ischemic injury, thereby regenerating large numbers of functional neurons. In particular, the in vivo cell conversion approach described herein provides a broader time window for neural regeneration and tissue repair—days, weeks, or even months—after an ischemic stroke, rather than hours. Thus, in addition to administering a therapeutically effective dose of exogenous NeuroD1 to an area where normal blood flow has been disrupted according to embodiments of the present invention, short-term treatments, such as clot removal, blood flow promotion, anti-inflammation, anti-oxidation, or excitotoxicity reduction, are optionally administered immediately after ischemic injury.

[0033] The conversion of reactive glial cells into functional neurons by the therapeutic method of the present invention is different from the death of reactive glial cells. Reactive glial cells are a positive defensive response to neuronal injury, and the death of reactive glial cells may worsen the infarct area. However, it is well known that reactive glial cells release cytokines and inflammatory factors, including CSPGs, LCN2, TNFα, IL-1β, etc., which may inhibit axonal regeneration and nerve regrowth.

[0034] Instead of killing reactive glial cells, the NeuroD1-mediated in vivo cell conversion technique described herein converts reactive astrocytes into functional neurons, reducing the number of reactive astrocytes and the cytokines and inflammatory factors secreted by reactive glial cells. In particular, in the astrocyte-to-neuron conversion therapy described herein, astrocytes in the damaged area are replenished with newly generated astrocytes. While the death of reactive astrocytes after injury leads to adverse effects, converting reactive astrocytes into neurons by the therapeutic method of the present invention leads to neural repair. [Brief explanation of the drawings]

[0035] [Figure 1A] Figure 1 shows the establishment of a focal stroke model and NeuroD1-mediated glial-to-neuronal conversion. Injection of endothelin-1(1-31) into the mouse motor cortex caused progressive tissue loss starting one week after ischemic injury. PBS injection served as a sham control. The dashed line indicates the cortical area. Scale bar: (A) 3 mm. [Figure 1B] 1 shows the establishment of a focal stroke model and NeuroD1-mediated glial-to-neuronal conversion, and is a graph showing the results of quantitative analysis of cortical size from the midline to a 3 mm lateral region at 1, 4, and 10 weeks after stroke (n=3 animals for each time point). [Figure 1C]Figure 1C shows the establishment of a focal stroke model and NeuroD1-mediated glial-to-neuronal conversion. Immunostaining results for NeuN (neuronal marker) and GFAP (astrocyte marker) were obtained at 5 days post-stroke (dps) (Figure 1C) and 10 days post-stroke (Figure 1D), respectively. Note that NeuN signal was significantly impaired in the motor cortex, and GFAP signal was weak at 5 dps but significantly increased at 10 dps. The dashed line indicates the corpus callosum. Scale bar: 200 μm in the left panels of Figure 1C and Figure 1D; 40 μm in the right insets of Figure 1C and Figure 1D. [Figure 1D] Figure 1C shows the establishment of a focal stroke model and NeuroD1-mediated glial-to-neuronal conversion. Immunostaining results for NeuN (neuronal marker) and GFAP (astrocyte marker) were obtained at 5 days post-stroke (dps) (Figure 1C) and 10 days post-stroke (Figure 1D), respectively. Note that NeuN signal was significantly impaired in the motor cortex, and GFAP signal was weak at 5 dps but significantly increased at 10 dps. The dashed line indicates the corpus callosum. Scale bar: 200 μm in the left panels of Figure 1C and Figure 1D; 40 μm in the right insets of Figure 1C and Figure 1D. [Figure 1E] Figure 1 shows the establishment of a focal stroke model and NeuroD1-mediated glial-to-neuronal conversion. Figure 1 shows the results of injection of a retrovirus carrying CAG::GFP as a control or CAG::NeuroD1-IRES-GFP at 10 dps, followed by immunostaining at 17 days post-virus injection (dpi). Expression of GFP alone labels only glial cells, whereas NeuroD1-GFP-expressing cells showed immunopositive signals for NeuN. Scale bar: 20 μm. [Figure 1F] This figure shows the establishment of a focal stroke model and NeuroD1-mediated glial-to-neuronal conversion. It shows the results of injection of adeno-associated virus (AAV9) carrying hGFAP::NeuroD1-P2A-GFP or hGFAP::GFP as a control at 10 dps, and immunostaining results at 17 dpi. The GFP control group showed mainly glial cells without NeuN signal, while the NeuroD1 group showed numerous NeuN-immunopositive neurons. Scale bar: 40 μm. [Figure 1G] Figure 1 shows the establishment of a focal stroke model and NeuroD1-mediated glial-to-neuronal conversion. At 4 dpi, injection of AAV9 hGFAP::Cre and CAG::FLEX-NeuroD1-P2A-mCherry resulted in significant NeuroD1 expression in GFAP-labeled astrocytes (top row). Interestingly, some NeuroD1-mCherry-labeled cells showed both NeuN and GFAP signals (bottom row), suggesting a transitional stage from astrocytes to neurons. Scale bar: (G) 40 μm (inset 20 μm). [Figure 1H] Figure 1 shows the establishment of a focal stroke model and NeuroD1-mediated glial-to-neuronal conversion. This figure shows a schematic diagram of the experimental design for stroke induction, AAV injection, and immunostaining procedures. Viral injection in this scheme represents the Cre-FLEX system with NeuroD1 or GFP (mCherry) control in the remaining experiments in mice. [Figure 1I] Establishment of a focal stroke model and NeuroD1-mediated glial-to-neuronal conversion are shown. At 17 dpi, the GFP control group showed numerous GFAP+ reactive glial cells (top row, GFAP), whereas the majority of NeuroD1-GFP labeled cells became NeuN-positive neurons (bottom row). Scale bar: (I) 40 μm. [Figure 2A] Establishment of the focal stroke model and AAV Cre-FLEX system is shown. Representative images showing severe ischemic injury induced by ET-1(1-31) in FVB mice (left), as well as milder damage induced by ET-1(1-21) in FVB mice or ET-1(1-31) in B6 mice are also shown. NeuN immunostaining at 27 dps revealed that ET-1(1-31) induced greater NeuN loss and more severe cortical atrophy in FVB animals compared with the other two conditions. Scale bar: (A) 400 μm. [Figure 2B] FIG. 1 shows the establishment of a focal stroke model and an AAV Cre-FLEX system, and is a schematic diagram of the mechanism of action of the Cre-FLEX system according to an embodiment of the present invention. [Figure 3A] Figure 1 shows the identification of astrocytes by GFAP immunostaining at 4, 7, or 17 days post-virus injection (dpi) in both control and NeuroD1 groups, demonstrating that NeuroD1 efficiently converts reactive astrocytes into cortical neurons. Scale bar: (A) 40 μm. [Figure 3B] Figure 1 shows that NeuroD1 efficiently converts reactive astrocytes into cortical neurons. Identification of neurons by NeuN immunostaining at 4, 7, or 17 dpi. Arrows indicate some NeuroD1-converted neurons. Scale bar: (A) 40 μm. [Figure 3C] Figure 1 shows the quantification of GFAP+ cells in all virus-infected cells, demonstrating that NeuroD1 efficiently converts reactive astrocytes into cortical neurons. Note the significant reduction in astrocytes in the NeuroD1 group. **P<0.01, two-way ANOVA followed by Sidak's multiple comparison test. n=3 mice per group. Three randomly acquired images were taken in the virus-infected cortical region. Data are presented as mean ± sem. [Figure 3D] Figure 1 shows the quantification of NeuN+ cells in all virus-infected cells, demonstrating that NeuroD1 efficiently converts reactive astrocytes into cortical neurons. Note the significant increase in neurons in the NeuroD1 group. ****P<0.0001, two-way ANOVA followed by Sidak's multiple comparison test. n=3 mice per group. Three randomly acquired images were taken in the virus-infected cortical region. Data are presented as mean ± sem. [Figure 3E] Demonstrating that NeuroD1 efficiently converts reactive astrocytes into cortical neurons, representative images are shown showing NeuroD1-converted neurons (GFP+) expressing the cortical marker (Tbr1) at 60 dpi. Note that Tbr1+ cells include both converted and non-converted cells. Scale bar: (E) 40 µm. [Figure 3F]Figure 1 shows that NeuroD1 efficiently converts reactive astrocytes into cortical neurons and is a graph depicting the characterization of neuronal identity after NeuroD1-mediated in vivo cell conversion. Many NeuroD1-converted neurons were immunopositive for cortical neuron markers, but only 10% were GABAergic neurons. n = 3 mice per group. Data are presented as mean ± sem. [Figure 4A] This figure shows the Tri-AAV system for tracking NeuroD1-converted neurons and is a schematic diagram illustrating the mechanism of the Tri-AAV tracking system. When AAV-hGFAP::GFP alone is injected, only astrocytes are labeled green. When AAV-hGFAP::GFP is injected together with the Cre-FLEX-mCherry control system, astrocytes express both GFP and mCherry and are yellow. When AAV-hGFAP::GFP is injected together with the Cre-FLEX-NeuroD1-mCherry conversion system, astrocytes are converted into neurons, express both GFP and mCherry, and are yellow, but are NeuN+. [Figure 4B] Figure 1 shows the Tri-AAV system for tracking NeuroD1-converted neurons and shows quantification of the transduction efficiency using the Tri-AAV tracking system at both 17 and 60 dpi. For the hGFAP::GFP group, the percentage of GFP+ / NeuN+ cells among all GFP+ cells was quantified. For the hGFAP::GFP+Cre-FLEX-mCherry or Cre-FLEX-NeuroD1-mCherry groups, the percentage of GFP+ / mCherry+ / NeuN+ cells among all GFP+ / mCherry+ cells was quantified. (n = 2 per group, and >150 cells). [Figure 4C] Figure 1 shows the Tri-AAV system tracking NeuroD1-transduced neurons. Representative images are shown showing different levels of colocalization of GFP, mCherry, and NeuN signals in the three groups at 17 and 60 dpi. Scale bar: (C) 40 μm. [Figure 5A]Representative low-magnification images show GFAP-labeled reactive astrocytes in the stroke cortex of the control and NeuroD1 groups at 17 dpi, demonstrating that NeuroD1 treatment reduces neuroinflammation in the stroke area. The dashed lines indicate the cortical area. The core of the stroke injury is marked with an asterisk (*). Scale bar: (A) 400 μm. [Figure 5B] High-magnification images illustrating GFAP-labeled reactive astrocytes (red) in the peri-infarct region after stroke in the GFP control group (top row) and the NeuroD1 group (bottom row), demonstrating that NeuroD1 treatment reduces neuroinflammation in the stroke area. Note that astrocytes in the NeuroD1 group appear morphologically less reactive compared to the control group. The core of the injury is marked with an asterisk. Scale bar: (B) 40 μm. [Figure 5C] Images from transgenic GFAP-GFP mice demonstrate that NeuroD1 treatment reduces neuroinflammation in the stroke area. Significant numbers of astrocytes are maintained in the NeuroD1-converted area (bottom row), suggesting that astrocytes are not depleted after cell conversion. Scale bar: (C) 40 μm. [Figure 5D] Low-magnification images showing the microglial marker Iba1 in the control and NeuroD1 groups at 17 dpi, demonstrating that NeuroD1 treatment reduces neuroinflammation in the stroke area. Note the significant reduction in Iba1 signal in the NeuroD1 group. Scale bar: (D) 400 μm. [Figure 5E] Higher magnification images show that NeuroD1 treatment reduces neuroinflammation in the stroke area. The GFP control group shows reactive microglia (Iba1) with an amoeboid shape (top), while the NeuroD1 group shows a more ramified morphology (bottom). Scale bar: (E) 40 μm. [Figure 5F] Immunofluorescence images show that NeuroD1 treatment reduces neuroinflammation in the stroke area, demonstrating a significant reduction in CSPGs, important neuroinhibitory factors released by reactive glial cells, in the NeuroD1 group compared with the control group in the peri-infarct region at 17 dpi. Scale bar: (F) 40 μm. [Figure 5G] Immunofluorescence images showing a significant reduction in LCN2, an inflammatory factor secreted by reactive glial cells, in the NeuroD1 group compared to the control group at 17 dpi, demonstrating that NeuroD1 treatment reduces neuroinflammation in the stroke area. Scale bar: (G) 40 μm. [Figure 5H] Figure 1 shows that NeuroD1 treatment reduces neuroinflammation in the stroke area. Figure 2 shows the results of real-time quantitative PCR (RT-PCR) to validate NeuroD1 expression in NeuroD1-infected tissues, revealing a significant decrease in reactive astrocyte markers GFAP and LCN2 in the NeuroD1 group. *P<0.05, **P<0.01, ****P<0.0001, one-way ANOVA followed by Tukey's multiple comparison test. n=4 mice per group. Data are presented as mean ± sem. [Figure 5I] This graph shows that NeuroD1 treatment reduces neuroinflammation in the stroke area. RT-PCR revealed that the significant increase in post-stroke inflammatory factors IL-1β, IFNγ, IL-6, and TNFα was significantly reduced after NeuroD1 treatment. *P<0.05, **P<0.01, one-way ANOVA followed by Tukey's multiple comparison test. n=4 mice per group. Data are shown as mean ± sem. [Figure 6A] Representative images of astrocytes (GFAP) and microglia (Iba1) in the peri-infarct area at 7 dpi (Figure 6A) and 40 dpi (Figure 6B) demonstrate reduced reactive glial cells and inflammation after NeuroD1 treatment. Note the reduced number and morphologically less reactive astrocytes and microglia in the NeuroD1 group at 40 dpi compared to the control group. Scale bar: 40 μm. [Figure 6B]Representative images of astrocytes (GFAP) and microglia (Iba1) in the peri-infarct area at 7 dpi (Figure 6A) and 40 dpi (Figure 6B) demonstrate reduced reactive glial cells and inflammation after NeuroD1 treatment. Note the reduced number and morphologically less reactive astrocytes and microglia in the NeuroD1 group at 40 dpi compared to the control group. Scale bar: 40 μm. [Figure 6C] Representative images of GFP (NeuroD1-GFP) co-stained with GFAP and NeuN in the peri-infarct area at 40 dpi, showing reactive glial cells and reduced inflammation after NeuroD1 treatment. Note that in the NeuroD1-infected area, many astrocytes were detected and were less reactive, suggesting that they were not depleted after conversion. Scale bar: 40 μm. [Figure 6D] Graphs showing reduction of reactive glial cells and inflammation after NeuroD1 treatment show quantification of signal intensity (AU, artificial units) and area coverage (%) for the microglial marker Iba1 (FIG. 6D), the glial scar inhibitor CSPG (FIG. 6E), and the astrocytic inflammation marker LCN2 (FIG. 6F). *P<0.05, **P<0.01, ***P<0.001. One-way ANOVA followed by Tukey's multiple comparison test. n=3 mice per group. Data are presented as mean ± sem. [Figure 6E] Graphs showing reduction of reactive glial cells and inflammation after NeuroD1 treatment show quantification of signal intensity (AU, artificial units) and area coverage (%) for the microglial marker Iba1 (FIG. 6D), the glial scar inhibitor CSPG (FIG. 6E), and the astrocytic inflammation marker LCN2 (FIG. 6F). *P<0.05, **P<0.01, ***P<0.001. One-way ANOVA followed by Tukey's multiple comparison test. n=3 mice per group. Data are presented as mean ± sem. [Figure 6F]Graphs showing reduction of reactive glial cells and inflammation after NeuroD1 treatment show quantification of signal intensity (AU, artificial units) and area coverage (%) for the microglial marker Iba1 (FIG. 6D), the glial scar inhibitor CSPG (FIG. 6E), and the astrocytic inflammation marker LCN2 (FIG. 6F). *P<0.05, **P<0.01, ***P<0.001. One-way ANOVA followed by Tukey's multiple comparison test. n=3 mice per group. Data are presented as mean ± sem. [Figure 7A] This figure shows a comparison of the motor cortex (17 dpi) between the control group (top row) and the NeuroD1 group (bottom row), demonstrating that NeuroD1 regenerates new neurons and preserves damaged neurons in the stroke area. The low-magnification image on the left shows the overall cortical morphology after stroke, while the image on the right shows a magnified image of the peri-infarct area. Note that the NeuroD1 group exhibits not only converted neurons (GFP+) but also non-converted neurons (NeuN+ but GFP-, arrows). Scale bar: 500 µm for the low-magnification cortical image on the left, and 40 µm for the magnified inset. [Figure 7B] (B) Immunofluorescence staining of NeuroD1 and NeuN in the NeuroD1 group at 17 dpi illustrates the intermingling of both NeuroD1-converted and non-converted neurons, demonstrating that NeuroD1 regenerates new neurons and preserves damaged neurons in the stroke area. Arrows indicate non-converted neurons (NeuN+ but NeuroD1-). Scale bar: (A) 40 μm. [Figure 7C] Figure 1 shows that NeuroD1 regenerates new neurons and preserves damaged neurons in the stroke area. Figure 1 shows the quantification of NeuN+ cells in the peri-infarct area of ​​the control and NeuroD1 groups, the latter containing both NeuroD1+ and NeuroD1- neurons. Note that the number of non-converted neurons in the NeuroD1 group was more than twice that of the control group, suggesting the neuroprotective effect of NeuroD1 conversion. [Figure 7D]Immunofluorescence images of the neuronal dendritic markers SMI32 (Figure 7D) and MAP2 (Figure 7E) show that NeuroD1 regenerates new neurons and preserves damaged neurons in the stroke area. The neuronal morphology in the NeuroD1 group (bottom row) is significantly improved compared to the control group (top row). Scale bar: 40 μm. [Figure 7E] Immunofluorescence images of the neuronal dendritic markers SMI32 (Figure 7D) and MAP2 (Figure 7E) show that NeuroD1 regenerates new neurons and preserves damaged neurons in the stroke area. The neuronal morphology in the NeuroD1 group (bottom row) is significantly improved compared to the control group (top row). Scale bar: 40 μm. [Figure 7F] Figure 7 shows that NeuroD1 regenerates new neurons and preserves damaged neurons in the stroke area. Immunostaining for the axon marker SMI312 (Figure 7F), NF200 (Figure 7G), and the axon myelination marker MBP (Figure 7G) demonstrates increased axons and axon myelination in the NeuroD1 group (bottom row) compared to the control group (top row). Note the better colocalization of NF200 and MBP in the NeuroD1 group than in the control group. Scale bar: 20 μm. [Figure 7G] Figure 7 shows that NeuroD1 regenerates new neurons and preserves damaged neurons in the stroke area. Immunostaining for the axon marker SMI312 (Figure 7F), NF200 (Figure 7G), and the axon myelination marker MBP (Figure 7G) demonstrates increased axons and axon myelination in the NeuroD1 group (bottom row) compared to the control group (top row). Note the better colocalization of NF200 and MBP in the NeuroD1 group than in the control group. Scale bar: 20 μm. [Figure 7H]Figure 1 shows the results of RT-PCR, which demonstrated that NeuroD1 regenerates new neurons and preserves damaged neurons in the stroke area, and revealed that the expression levels of neural factors, including NeuN, Robo2, and Syn1, in the stroke area of ​​the NeuroD1 group were significantly increased compared with the control group. *P<0.05, one-way ANOVA followed by Tukey's multiple comparison test. n=4 mice per group. Data are presented as mean ± sem. [Figure 7I] Figure 1 shows a quantitative analysis of the total number of NeuN+ cells in the motor cortex region 500–2500 μm lateral to the midline, demonstrating that NeuroD1 regenerates new neurons and preserves damaged neurons in the stroke zone. Note the significant difference between the control and NeuroD1 groups up to 60 dpi. n = 3 mice per group. *P<0.05, **P<0.01, two-way ANOVA followed by Sidak's multiple comparison test. Data are shown as mean ± sem. [Figure 8A] Fig. 1 shows neuroregeneration and neuroprotection after NeuroD1 treatment. Quantification of NeuN-positive cells in the peri-infarct area of ​​the control and NeuroD1 groups. In the NeuroD1 group, NeuN-positive cells were further divided into NeuroD1-converted neurons (GFP+ / NeuN+ double positive) and non-converted neurons (GFP-negative). [Figure 8B] Representative images of the neuronal dendritic marker Map2 in the peri-infarct area at 40 dpi showing neuroregeneration and neuroprotection after NeuroD1 treatment. Scale bar: 40 μm. [Figure 8C] Graphs showing neuroregeneration and neuroprotection after NeuroD1 treatment show quantification of signal intensity (AU, artificial units) and area coverage (%) for the dendritic marker Map2 (FIG. 8C), the axonal marker SMI312 (FIG. 8D), and the myelination marker MBP (FIG. 8E). *P<0.05, **P<0.01. One-way ANOVA followed by Tukey's multiple comparison test, n=3 mice per group. Data are presented as mean ± sem. [Figure 8D]Graphs showing neuroregeneration and neuroprotection after NeuroD1 treatment show quantification of signal intensity (AU, artificial units) and area coverage (%) for the dendritic marker Map2 (FIG. 8C), the axonal marker SMI312 (FIG. 8D), and the myelination marker MBP (FIG. 8E). *P<0.05, **P<0.01. One-way ANOVA followed by Tukey's multiple comparison test, n=3 mice per group. Data are presented as mean ± sem. [Figure 8E] Graphs showing neuroregeneration and neuroprotection after NeuroD1 treatment show quantification of signal intensity (AU, artificial units) and area coverage (%) for the dendritic marker Map2 (FIG. 8C), the axonal marker SMI312 (FIG. 8D), and the myelination marker MBP (FIG. 8E). *P<0.05, **P<0.01. One-way ANOVA followed by Tukey's multiple comparison test, n=3 mice per group. Data are presented as mean ± sem. [Figure 9A] Low-magnification images show the reorganization of neural circuits through NeuroD1-mediated cellular transformation in the stroke area, illustrating cortical tissue loss in the GFP control group (top row) and rescue of tissue loss by NeuroD1 treatment (bottom row). GFP labels AAV-infected cells, and DAPI labels cell nuclei. The dashed line indicates the cortical area. Scale bar: 400 μm. [Figure 9B] Figure 1 shows the reorganization of neural circuits by NeuroD1-mediated cellular transformation in the stroke area. Graphs show the results of quantitative analysis of cortical area (up to 3 mm lateral from the midline) in the control group versus the NeuroD1 group at 7, 17, 40, and 60 dpi. Note that there is robust tissue loss in the control group, but clear cortical preservation in the NeuroD1 group. *P<0.01, **P<0.01, ***P<0.005, two-way ANOVA followed by Sidak's multiple comparison test. n=3 mice per group. Data are presented as mean ± sem. [Figure 9C]Representative images of consecutive sagittal sections from medial (bottom left) to lateral (top) locations showing reorganization of neural circuits by NeuroD1-mediated cellular transformation in the stroke area, illustrating axonal projections from NeuroD1-transformed neurons in the cortex (box 1) to the striatum (box 2), thalamus (box 3), and hypothalamus (box 4). Scale bar: 1000 μm for the sagittal image on the left, 40 μm for the inset image. [Figure 9D] Figure 1 shows the reorganization of neural circuits by NeuroD1-mediated cellular transformation in the stroke area. Fluorescent (GFP) and bright-field images show NeuroD1-infected neurons in a brain section (top). Representative traces (bottom) show repetitive action potentials evoked in NeuroD1-transduced neurons (60 dpi). [Figure 9E] Figure 1. Reorganization of neural circuits through NeuroD1-mediated cellular transformation in the stroke area. Images show biocytin injection of the recording pipette during whole-cell recording followed by immunostaining after fixation. Recorded neurons are both GFP+ and NeuroD1+ and have complex apical dendrites at 60 dpi. Scale bar: 200 μm for the upper low-magnification image and 5 μm for the lower inset image. [Figure 9F] Representative traces of excitatory (sEPSCs) and inhibitory synaptic events (sIPSCs) recorded in NeuroD1-GFP labeled neurons (60 dpi) show the reorganization of neural circuits through NeuroD1-mediated cellular transformation in the stroke area. [Figure 9G]Figure 1 shows the reorganization of neural circuits by NeuroD1-mediated cellular transformation in the stroke area. Quantification of both sEPSC and sIPSC frequencies in cortical slices without stroke (white bars) or with stroke (black bars, GFP control; striped bars, NeuroD1 group). Note that after stroke, the NeuroD1 group had significantly higher sEPSC and sIPSC frequencies than the control group (EPSC: control, 4.3 ± 0.6, n = 22; NeuroD1, 6.7 ± 0.8, n = 25; p = 0.023, Student's t-test) (IPSC: control, 8.6 ± 1.3, n = 22; NeuroD1, 14.0 ± 2.0, n = 25; p = 0.032, Student's t-test). [Figure 10A] Images show serial brain sections from anterior (A) to posterior (P) in the control versus NeuroD1 group, demonstrating brain tissue repair and long-range axonal projections after NeuroD1 treatment. Note the severe tissue damage in the control group. ctx, cortex; cc, corpus callosum. Scale bar: 400 μm. [Figure 10B] Images showing brain tissue repair and long-distance axonal projections after NeuroD1 treatment. H&E (hematoxylin and eosin) staining at 60 dpi also shows severe tissue damage in the control group. Note the enlarged lateral ventricles in the control group, which are typically associated with severe brain damage. Scale bar: 400 μm. [Figure 10C] Brain tissue repair and long-distance axonal projections after NeuroD1 treatment. Images show coronal sections of mouse cortex 9 months after NeuroD1 viral injection, demonstrating long-distance axonal projections through the corpus callosum to the contralateral side. The bottom row shows a magnified view of axonal projections within the corpus callosum (boxes 1 and 3 show the same side, box 4 shows the contralateral side) and subcortical projections through the striatum (box 2). Scale bar: top row: 1000 μm, bottom row: 40 μm. [Figure 11A]NeuroD1 treatment restores blood vessels and the blood-brain barrier (BBB) ​​following ischemic injury. The top panel shows astrocyte endfeet (water channel aquaporin 4, AQP4, shown in red) surrounding blood vessels (monocyte and endothelial cell marker Ly6C, shown in cyan) in a non-stroke brain, forming an intact BBB. Arrows indicate perivascular colocalization of AQP4 with GFAP. The bottom panel shows that 1 week after stroke (wps), AQP4 signal is separated from blood vessels, suggesting BBB disruption. Scale bar: 40 μm. [Figure 11B] This figure shows the restoration of blood vessels and the blood-brain barrier by NeuroD1 treatment. This figure compares the blood vessels and BBB between the control and NeuroD1 groups after stroke. The control group (top row) showed destroyed blood vessels and diffuse AQP4 signaling throughout the stroke area. The NeuroD1 group (bottom row) showed significant vascular improvement associated with AQP4 signaling, suggesting BBB restoration after cell conversion (17 dpi). Scale bar: 40 μm. [Figure 11C] NeuroD1 treatment restores blood vessels and the blood-brain barrier. Immunofluorescence images of another vascular marker, CD31, co-stained with AQP4 confirm the rescue of blood vessels and the BBB in the NeuroD1-treated group (17 dpi). Scale bar: 40 μm. [Figure 11D] NeuroD1 treatment restores blood vessels and the blood-brain barrier. Co-immunostaining of AQP4 and GFAP shows mislocalized AQP4 signal in reactive astrocytes in the control group compared with concentrated AQP4 signal in the endfeet of astrocytes in the NeuroD1 group (17 dpi). Scale bar: 40 μm. [Figure 11E] High-magnification images show the restoration of blood vessels and the blood-brain barrier by NeuroD1 treatment, illustrating the dissociation of astrocyte endfeet (GFAP) from blood vessels (CD31) in the control group (top row) compared to the successful reassociation of astrocyte endfeet with blood vessels in the NeuroD1 group (bottom row). Scale bar: 5 μm. [Figure 11F]11F) and coverage area (FIG. 11G) in non-stroke, stroke control, and stroke NeuroD1 groups (17 dpi), demonstrating vascular and blood-brain barrier restoration by NeuroD1 treatment. Graphs show quantification of AQP4 signal intensity (FIG. 11F) and coverage area (FIG. 11G) in non-stroke, stroke control, and stroke NeuroD1 groups (17 dpi). The NeuroD1 group is very close to the non-stroke group. **P<0.01. One-way ANOVA followed by Tukey's multiple comparison test, n=3 mice per group. Data are presented as mean±sem. [Figure 11G] 11F) and coverage area (FIG. 11G) in non-stroke, stroke control, and stroke NeuroD1 groups (17 dpi), demonstrating vascular and blood-brain barrier restoration by NeuroD1 treatment. Graphs show quantification of AQP4 signal intensity (FIG. 11F) and coverage area (FIG. 11G) in non-stroke, stroke control, and stroke NeuroD1 groups (17 dpi). The NeuroD1 group is very close to the non-stroke group. **P<0.01. One-way ANOVA followed by Tukey's multiple comparison test, n=3 mice per group. Data are presented as mean±sem. [Figure 11H] Figure 1 shows the restoration of blood vessels and the blood-brain barrier by NeuroD1 treatment. Graph showing quantification of blood vessels (Ly6C signal) in the peri-infarct area of ​​the control and NeuroD1 groups. The NeuroD1 group showed significantly more blood vessels than the control group. ****P<0.0001. One-way ANOVA followed by Tukey's multiple comparison test, n=3 mice per group. Data are presented as mean ± sem. [Figure 12A] Demonstrating the broad impact of NeuroD1-mediated cellular transformation on injured tissue, including synaptic restoration and rebalancing of excitation-inhibition, this image of biocytin injected during patch-clamp recording followed by post-fixation staining shows a high density of spines along dendrites in NeuroD1-infected neurons. Scale bar: 50 μm. [Figure 12B] Representative images of Golgi impregnation staining at 17 dpi show increased neuronal processes and dendritic spines in the peri-infarct region of the NeuroD1 group compared to controls, demonstrating the broad impact of NeuroD1-mediated cellular transformation on injured tissue, including synaptic restoration and rebalancing of excitation-inhibition. Scale bar: 20 μm. [Figure 12C]Representative images of immunofluorescent staining for VGluT1 at 17 dpi (Fig. 12C) and a graph showing quantification of signal intensity in control and NeuroD1 groups (Fig. 12D) demonstrate the broad impact of NeuroD1-mediated cellular transformation on injured tissue, including synaptic recovery and rebalancing of excitation-inhibition. **P<0.01, one-way ANOVA followed by Tukey's multiple comparison test, n=3 per group. Data are presented as mean ± sem. Scale bar: 40 μm. [Figure 12D] Representative images of immunofluorescent staining for VGluT1 at 17 dpi (Fig. 12C) and a graph showing quantification of signal intensity in control and NeuroD1 groups (Fig. 12D) demonstrate the broad impact of NeuroD1-mediated cellular transformation on injured tissue, including synaptic recovery and rebalancing of excitation-inhibition. **P<0.01, one-way ANOVA followed by Tukey's multiple comparison test, n=3 per group. Data are presented as mean ± sem. Scale bar: 40 μm. [Figure 12E] Representative images of PV-labeled interneurons in the NeuroD1-converted region (Figure 12E) and a graph showing quantification of PV-labeled interneurons in the NeuroD1-converted region (Figure 12F) demonstrate the broad impact of NeuroD1-mediated cell conversion on injured tissue, including synaptic restoration and rebalancing of excitation-inhibition. Note that PV neurons intermingle with NeuroD1-converted neurons (NeuroD1-GFP) at 60 dpi. More PV interneurons were preserved in the NeuroD1 group than in the control group. **P<0.01. One-way ANOVA followed by Tukey's multiple comparison test, n=3 per group. Data are presented as mean ± sem. Scale bar: 100 μm. [Figure 12F]Representative images of PV-labeled interneurons in the NeuroD1-converted region (Figure 12E) and a graph showing quantification of PV-labeled interneurons in the NeuroD1-converted region (Figure 12F) demonstrate the broad impact of NeuroD1-mediated cell conversion on injured tissue, including synaptic restoration and rebalancing of excitation-inhibition. Note that PV neurons intermingle with NeuroD1-converted neurons (NeuroD1-GFP) at 60 dpi. More PV interneurons were preserved in the NeuroD1 group than in the control group. **P<0.01. One-way ANOVA followed by Tukey's multiple comparison test, n=3 per group. Data are presented as mean ± sem. Scale bar: 100 μm. [Figure 12G] This image shows the broad impact of NeuroD1-mediated cellular transformation on injured tissue, including synaptic restoration and rebalancing of excitation-inhibition. In the NeuroD1 group, AQP4 signals showed clear association with blood vessels in areas of high density of NeuroD1-expressing cells, but were less abundant in adjacent areas of low density of NeuroD1-expressing cells (demarcated by dashed lines). Scale bar: 40 μm. [Figure 12H] Representative images of biotin perfusion analysis are shown, demonstrating the broad impact of NeuroD1-mediated cellular transformation on injured tissue, including synaptic restoration and rebalancing of excitation-inhibition. In the injured area of ​​the control group, biotin leaked outside the blood vessels (labeled by Ly6C, indicated by the dashed line), whereas in the injured area of ​​the NeuroD1 group, biotin remained largely within the blood vessels. Scale bar: 5 μm. [Figure 12I] Representative images of the astrocyte endfeet marker AQP4 in the peri-infarct region at 60 dpi demonstrate the broad impact of NeuroD1-mediated cellular transformation on injured tissue, including synaptic restoration and rebalancing of excitation-inhibition. Note the significant reassociation of AQP4 with blood vessels in the NeuroD1 group. Scale bar: 40 μm. [Figure 13A]Figure 1 shows a schematic diagram of the experimental design for forelimb motor function testing in mice, demonstrating behavioral improvement after NeuroD1 treatment. Behavioral testing was performed 9 days after stroke (dps) and 1 day before viral injection to assess motor function in mice injected with ET-1(1-31) or PBS as a sham control. AAV was injected 10 dps, and behavioral testing was performed at the indicated time points (20, 30, 50, and 70 dps) to assess functional recovery. [Figure 13B] This graph shows the results of the pellet retrieval test, demonstrating behavioral improvement after NeuroD1 treatment. The NeuroD1 group demonstrated accelerated recovery after stroke compared with the control group. Ischemic injury to the motor cortex significantly impaired pellet retrieval ability, reducing it from 5–6 pellets / 5 min before stroke to 1 pellet / 5 min at 9 dps. After NeuroD1 treatment, pellet retrieval ability showed stable recovery compared with the control group. Note that for the pellet retrieval test, only the motor cortex contralateral to the ipsilateral forelimb was tested. Two-way ANOVA followed by Tukey's multiple comparison test. n = 12 for the ET-1 + control AAV group, n = 11 for the NeuroD1 AAV group, n = 6 for the ET-1 + non-viral group, and n = 6 for the PBS group. **P < 0.01, ***P < 0.001. Data are shown as mean ± sem. [Figure 13C] This graph shows the results of the grid walking test, demonstrating behavioral improvement after NeuroD1 treatment. The NeuroD1 group exhibited a lower limb disability rate compared to the control group. Stroke injury significantly increased the limb disability rate. NeuroD1 treatment reduced the limb disability rate. Two-way ANOVA followed by Tukey's multiple comparison test. n = 9 for ET-1 + control AAV contralateral group, n = 11 for ET-1 + NeuroD1 AAV contralateral group, n = 5 for ET-1 + NeuroD1 ipsilateral group, n = 5 for ET-1 + control ipsilateral group, n = 6 for ET-1 + non-viral contralateral group, and n = 6 for PBS contralateral group. **P<0.01, ***P<0.001. Data are presented as mean ± sem. [Figure 13D]10A-10C are graphs showing the results of the cylinder test, demonstrating behavioral improvement after NeuroD1 treatment. NeuroD1-treated mice showed significant recovery in forelimb lateral wall elevation and touch function compared to control mice. Stroke injury impaired mouse forelimb function in terms of cylinder wall elevation and touch. Two-way ANOVA followed by Tukey's multiple comparison test. n = 9 for stroke + control AAV contralateral group, n = 11 for stroke + NeuroD1 AAV contralateral group, n = 9 for stroke + NeuroD1 ipsilateral group, n = 7 for stroke + control ipsilateral group, n = 6 for stroke + non-viral contralateral group, and n = 5 for PBS contralateral group. **P<0.01, ****P<0.0001. Data are presented as mean ± sem. [Figure 14A] This is a photograph of the modified pellet retrieval apparatus from the staircase, showing the assessment of tissue damage after behavioral testing. Mice in the resting chamber were trained to reach the sucrose pellets in the V-shaped recess after 18 hours of fasting. From this design, it is clear that mice had to exert a great effort in motor coordination and strength to successfully retrieve the food pellets. [Figure 14B] Representative photographs of mouse brains after the behavioral test (2 months after virus injection) show the assessment of tissue damage after behavioral testing. The control group showed severe cortical tissue loss on the stroke side after ET-1(1-31)-induced focal stroke, while NeuroD1 treatment significantly rescued tissue loss. [Figure 14C] Representative images of a mouse cortex in a coronal section illustrating severe tissue loss after ET-1(1-31)-induced ischemic stroke, showing the assessment of tissue damage after behavioral testing. Note that two ET-1(1-31) injections were performed in both the motor and sensory cortices on one side, inducing more severe damage for long-term behavioral testing. Therefore, tissue loss is also more severe than the single injection performed in most cell conversion studies. Nevertheless, the NeuroD1 repair effect remains effective. Scale bar: 1000 µm. [Figure 14D]Figure 1 shows the assessment of tissue damage after behavioral testing. Graph showing quantification of cortical tissue damage after behavioral testing. In the NeuroD1 group with two sites of ET-1(1-31) injection, the stroked side also showed tissue damage compared with the contralateral non-stroke side (P=0.012), but was significantly better preserved than the control group (****P<0.0001). Two-way ANOVA followed by Sidak's multiple comparison test, n=6 mice per group. Data are presented as mean ± sem. DETAILED DESCRIPTION OF THE INVENTION

[0036] Aspects of the present invention provide compositions and methods for treating the effects of disruption of normal blood flow in the CNS of an individual subject.

[0037] Embodiments of the present invention provide an effective method for reversing neuronal loss that results from disruption of normal blood flow in the CNS.

[0038] Unexpectedly, expression of the neural transcription factor NeuroD1 in reactive astrocytes treats the effects of disruption of normal blood flow in the CNS of an individual subject in need thereof. Thus, the present invention provides a method for treating the pathological effects of disruption of normal blood flow in the CNS of a subject, comprising administering a therapeutically effective amount of NeuroD1 to the subject.

[0039] Administration of a therapeutically effective amount of NeuroD1 to a subject affected by disruption of normal blood flow in the CNS mediates the following: generation of new glutamatergic neurons through conversion of reactive astrocytes into glutamatergic neurons, reduction in the number of reactive astrocytes, survival of damaged neurons including GABAergic and glutamatergic neurons, generation of new non-reactive astrocytes, reduction in the reactivity of non-converted reactive astrocytes, and reintegration of blood vessels into the damaged area.

[0040] Administration of a therapeutically effective amount of NeuroD1 to a subject affected by disruption of normal blood flow in the CNS mediates the following: reduction of inflammation at the injury site, reduction of neuronal inhibition at the injury site, re-establishment of normal microglial morphology at the injury site, re-establishment of neural circuitry at the injury site, increase in blood vessels at the injury site, re-establishment of the blood-brain barrier at the injury site, re-establishment of normal tissue architecture at the injury site, and improvement of motor deficits due to disruption of normal blood flow.

[0041] The subject in need of treatment may be a human or non-human mammal, but may also be a non-mammal.

[0042] Surprisingly, administration of a therapeutically effective amount of NeuroD1 that ameliorates the effects of disruption of normal blood flow in the CNS of an individual subject in need thereof, when administered to reactive astrocytes rather than quiescent astrocytes, typically has a greater beneficial effect 3 to 60 days, 5 to 45 days, or 8 to 30 days after onset of disruption of normal blood flow in the subject's CNS, although certain advantages may be achieved earlier or later, including 2 days to 1 year or longer after onset of the interruption of blood flow in the subject's CNS.

[0043] Administration of NeuroD1 to a subject can be used to treat damage resulting from disruption of normal blood flow to the CNS caused by ischemia, thrombosis, embolism, hemorrhage, concussion, tumor, infection, inflammation, traumatic brain injury and spinal cord injury, or by chronic disease-mediated restriction of blood vessels, which results in neuronal cell death and activation of reactive astrocytes. Administration of NeuroD1 to a subject can be used to treat damage resulting from disruption of normal blood flow to the CNS, including, but not limited to, ischemic or hemorrhagic stroke, cerebral aneurysm, tumor, infection, inflammation, concussion, traumatic brain injury, traumatic spinal cord injury, ischemic or hemorrhagic spinal cord injury (spinal cord infarction), global cerebral ischemia caused by cardiac arrest or severe low blood pressure (shock), hypoxic-ischemic encephalopathy caused by hypoxia, hypoglycemia, or anemia, CNS embolism caused by infective endocarditis or atrial myxoma, fibrocartilaginous embolic myelopathy, CNS thrombosis caused by childhood leukemia, nephrotic syndrome (kidney disease), chronic inflammatory disease, pregnancy, use of estrogen-based contraceptives, meningitis, and cerebral venous sinus thrombosis caused by dehydration.

[0044] A method of treating the pathological effects of disruption of normal blood flow in the CNS of a subject, according to an embodiment of the present invention, comprising administering to the subject a therapeutically effective amount of NeuroD1 to the area of ​​disruption of normal blood flow in the CNS.A method of treating the pathological effects of disruption of normal blood flow in a subject, according to an embodiment of the present invention, comprising administering to the subject a therapeutically effective amount of NeuroD1 to or near the site of disruption of normal blood flow in the CNS.A method of treating the pathological effects of disruption of normal blood flow in a subject, according to an embodiment of the present invention, comprising administering to the subject a therapeutically effective amount of NeuroD1 within or near a glial scar caused by disruption of normal blood flow in the CNS.

[0045] NeuroD1 treatment can be administered to the area of ​​injury diagnosed by MRI. Electrophysiology can assess functional changes in neural firing caused by neuronal cell death or injury. Non-invasive methods for assessing neuronal injury include EEG. Disruption of blood flow to the injury site can be non-invasively assessed via near-infrared spectroscopy and fMRI. Blood flow within the area can be increased, as seen in aneurysms, or decreased, as seen in ischemia. Damage to the CNS caused by disruption of blood flow also leads to short-term and long-term changes in tissue structure that can be used to diagnose the injury site. In the short term, injury causes localized swelling. In the long term, cell death causes tissue loss. Non-invasive methods for assessing structural changes caused by tissue death include MRI, PET scan, CAT scan, or ultrasound. These methods can be used alone or in any combination to pinpoint the primary site of injury.

[0046] In a specific embodiment of the present invention, NeuroD1 is administered to the injury site where glial scars will develop if the subject is untreated, or around the injury site where glial scars already exist.The location of glial scars can be determined by assaying tissue structure or function.As mentioned above, non-invasive methods for assaying the structural changes caused by tissue death include MRI, CAT scan, or ultrasound.Functional assays can include EEG recording.

[0047] In a particular embodiment according to the present invention, NeuroD1 is administered as an expression vector containing a DNA sequence encoding NeuroD1.

[0048] According to an embodiment of the present invention, a viral vector comprising a nucleic acid encoding NeuroD1 is delivered by injection into the central or peripheral nervous tissue of a subject, for example, by intracerebral injection, spinal injection, and / or injection into the cerebrospinal fluid or peripheral nerve ganglia. Alternative viral delivery methods include, but are not limited to, intravenous injection, intranasal injection, intramuscular injection, intrathecal injection, and intraperitoneal injection.

[0049] According to an embodiment of the present invention, a viral vector containing a nucleic acid encoding NeuroD1 is delivered by stereotactic injection into the brain of a subject.

[0050] The term "expression vector" refers to a recombinant vehicle by which a nucleic acid encoding NeuroD1 is introduced into a host cell in vitro or in vivo, where the nucleic acid is expressed to produce NeuroD1. In certain embodiments, an expression vector comprising SEQ ID NO:1 or SEQ ID NO:3, or a substantially identical nucleic acid sequence, is expressed to produce NeuroD1 in cells containing the expression vector. The term "recombinant" is used to refer to a nucleic acid construct in which two or more nucleic acids are linked together and are not found to be linked in nature. Expression vectors include, but are not limited to, plasmids, viruses, BACs, and YACs. Specific viral expression vectors illustratively include those derived from adenoviruses, adeno-associated viruses, retroviruses, and lentiviruses.

[0051] According to an embodiment of the present invention, there is provided a method for treating a neurological condition in a subject in need thereof, comprising providing a viral vector containing a nucleic acid encoding NeuroD1 and delivering the viral vector to the subject's central nervous system or peripheral nervous system, whereby the viral vector infects glial cells in the central nervous system or peripheral nervous system, respectively, to produce infected glial cells, and exogenous NeuroD1 is expressed in the infected glial cells at a therapeutically effective level, whereby expression of NeuroD1 in the infected cells results in more neurons in the subject compared to an untreated subject with the same neurological condition, thereby treating the neurological condition. In addition to the generation of new neurons, the number of reactive glial cells is also reduced, resulting in less neuroinhibitory factors released, less neuroinflammation, and more evenly distributed blood vessels, making the local environment more permissive for neuronal growth or axonal penetration, thus alleviating the neurological condition.

[0052] Adeno-associated vectors are particularly useful in methods according to embodiments of the present invention, infecting both dividing and non-dividing cells at the injection site. Adeno-associated viruses (AAVs) are ubiquitous, non-cytopathic, replication-incompetent members of the parvoviridae family of ssDNA animal viruses. According to embodiments of the present invention, any of a variety of recombinant adeno-associated viruses, such as serotypes 1-9, can be used.

[0053] A "FLEX" switch approach is used to express NeuroD1 in infected cells according to an embodiment of the present invention. The terms "FLEX" and "flip-excision" are used interchangeably to refer to a method in which two pairs of heterotypic antiparallel loxP recombination sites are placed on either side of an inverted NeuroD1 coding sequence; the coding sequence is first inverted, and then the two sites are excised, reversing one of the orthologous recombination sites and disabling further recombination, achieving a stable inversion. See, e.g., Schnutgen et al., Nature Biotechnology 21:562-565, 2003, and Atasoy et al., J. Neurosci. 28:7025-7030, 2008. Because site-specific recombinase under the control of a glial cell-specific promoter is strongly expressed in glial cells, including reactive astrocytes, NeuroD1 is also expressed in glial cells, including reactive astrocytes. Then, when the stop codon before NeuroD1 is removed by recombination, a constitutive or neuron-specific promoter drives high expression of NeuroD1, converting reactive astrocytes into functional neurons.

[0054] According to certain embodiments, NeuroD1 is administered to a subject in need thereof by administering 1) an adeno-associated virus expression vector comprising a DNA sequence encoding a site-specific recombinase under the transcriptional control of an astrocyte-specific promoter, such as GFAP or S100b or Aldh1L1, and 2) an adeno-associated virus expression vector comprising a DNA sequence encoding NeuroD1 under the transcriptional control of a ubiquitous (constitutive) promoter or a neuron-specific promoter, such that the DNA sequence encoding NeuroD1 is inverted and in an alternate orientation for expression of NeuroD1 until the site-specific recombinase is able to invert the inverted DNA sequence encoding NeuroD1, thereby expressing NeuroD1.

[0055] Site-specific recombinases and their recognition sites include, for example, Cre recombinase with recognition sites loxP and lox2272 sites, or FLP-FRT recombinase, or a combination thereof.

[0056] To achieve optimal infection, 10 10 ~10 14 A concentration of adeno-associated virus particles / ml, a volume of 1-500 μl, and a controlled flow rate of 0.1-5 μl / min should be injected.

[0057] According to an aspect of the invention, an adeno-associated viral vector comprising a nucleic acid encoding NeuroD1 under the transcriptional control of a ubiquitous (constitutive) promoter or a neuron-specific promoter, wherein the DNA sequence encoding NeuroD1 is inverted and in an alternate orientation for expression of NeuroD1 until a site-specific recombinase can invert the inverted DNA sequence encoding NeuroD1, thereby allowing expression of NeuroD1, and further comprising sites for recombinase activity by the site-specific recombinase, is delivered by stereotactic injection into the brain of a subject along with an adeno-associated virus encoding the site-specific recombinase.

[0058] According to an embodiment of the present invention, an adeno-associated viral vector comprising a nucleic acid encoding NeuroD1 under the transcriptional control of a ubiquitous (constitutive) promoter or a neuron-specific promoter, wherein the DNA sequence encoding NeuroD1 is inverted and in an alternative orientation for expression of NeuroD1 until a site-specific recombinase can invert the inverted DNA sequence encoding NeuroD1, thereby enabling expression of NeuroD1, and further comprising a site for recombinase activity by the site-specific recombinase, is delivered by stereotactic injection into the brain of a subject together with an adeno-associated virus encoding the site-specific recombinase at or near the area of ​​disruption of normal blood flow in the CNS. Optionally, the site of stereotactic injection is within or near the glial scar caused by disruption of normal blood flow in the CNS.

[0059] According to an embodiment of the present invention, the site-specific recombinase is Cre recombinase and the sites of recombinase activity are the recognition sites loxP and lox2272 sites.

[0060] According to embodiments of the present invention, a subject's NeuroD1 treatment is monitored during or after treatment to monitor the progress and / or final outcome of the treatment. Post-treatment assessments of successful restoration of neuronal cell integration and tissue microenvironment are diagnosed by restoration or near-restoration of normal electrophysiology, blood flow, tissue structure, and function. Non-invasive methods for assessing neural function include EEG. Blood flow can be non-invasively assessed by near-infrared spectroscopy and fMRI. Non-invasive methods for assessing tissue structure include MRI, CAT scan, PET scan, or ultrasound. Behavioral assays can be used to non-invasively assess the restoration of CNS function. The behavioral assay should be consistent with the loss of function caused by the original CNS injury. For example, if the injury causes paralysis, the patient's mobility and limb dexterity should be tested. If the injury causes speech loss or slowing, the patient's ability to communicate through spoken language should be assessed. Restoration of normal behavior after NeuroD1 treatment indicates successful generation and integration of effective neural circuits. These methods can be used alone or in any combination to assess neural function and tissue integrity. Assays to assess treatment can be performed at any time after NeuroD1 treatment, such as 1 day, 2 days, 3 days, 1 week, 2 weeks, 3 weeks, 1 month, or later. Such assays can be performed before NeuroD1 treatment, if desired, to establish a baseline comparison.

[0061] Scientific and technical terms used herein shall have the meanings commonly understood by those skilled in the art.Such terms are illustratively used in J. Sambrook and D.W. Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 3rd ed., 2001; F.M. Asubel (ed.), Short Protocols in Molecular Biology, Current Protocols, 5th ed., 2002; B. Alberts et al., Molecular Biology of the Cell, 4th ed., Garland, 2002; D.L. Nelson and M.M. Cox, Lehninger Principles of Biochemistry, 4th ed., W.H. Freeman & Company, 2004; Engelke, D.R., RNA Interference (RNAi): Nuts and Bolts of RNAi Technology, DNA Press LLC, Eagleville, PA, 2003; Herdewijn, P. (ed.), Oligonucleotide Synthesis: Methods and Applications, Methods in Molecular Biology, Humana Press, 2004; A. Nagy, M. Gertsenstein, K. Wintersten, R. It is confirmed as defined and used in the context of various standard references, including Behringer, Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 3rd Edition, December 15, 2002, ISBN-10:0879695919; Kursad Turksen (ed.), Embryonic Stem Cells: Methods and Protocols in Methods in Molecular Biology, 2002; 185, Human Press: Current Protocols in Stem Cell Biology, ISBN:9780470151808.

[0062] The singular terms "a," "an," and "the" are not intended to be limiting and include plural referents unless specifically stated otherwise or the context clearly indicates otherwise.

[0063] The term "NeuroD1 protein" refers to a bHLH proneural transcription factor involved in fetal brain development and adult neurogenesis; see Cho, JH et al., Mol. Neurobiol., 30:35-47, 2004; Kuwabara, T. et al., Nature Neurosci., 12:1097-1105, 2009; and Gao, Z. et al., Nature Neurosci., 12:1090-1092, 2009. NeuroD1 is expressed primarily in the nervous system late in development and is involved in neuronal differentiation, maturation, and survival.

[0064] The term "NeuroD1 protein" encompasses the human NeuroD1 protein, identified herein as SEQ ID NO:2, and the mouse NeuroD1 protein, identified herein as SEQ ID NO:4. In addition to the NeuroD1 proteins of SEQ ID NO:2 and SEQ ID NO:4, the term "NeuroD1 protein" encompasses variants of NeuroD1 proteins, such as variants of SEQ ID NO:2 and SEQ ID NO:4, which may be included in the methods of the present invention. As used herein, the term "variant" refers to naturally occurring genetic and recombinantly prepared variants that contain one or more changes in their amino acid sequence compared to a reference NeuroD1 protein, such as SEQ ID NO:2 or SEQ ID NO:4, respectively. Such changes include those in which one or more amino acid residues have been modified by amino acid substitution, addition, or deletion. The term "variant" encompasses orthologs of human NeuroD1, including, for example, mammalian and avian NeuroD1, including, but not limited to, NeuroD1 orthologs from non-human primates, cats, dogs, sheep, goats, horses, cows, pigs, birds, poultry, and rodents, including, but not limited to, mice and rats. In a non-limiting example, mouse NeuroD1, exemplified herein by the amino acid sequence of SEQ ID NO: 4, is an ortholog of human NeuroD1.

[0065] Preferred variants have at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:2 or SEQ ID NO:4.

[0066] Mutations can be introduced using standard molecular biology techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Those skilled in the art will recognize that one or more amino acid mutations can be introduced without altering the functional properties of the NeuroD1 protein. For example, one or more amino acid substitutions, additions, or deletions can be made without altering the functional properties of the NeuroD1 protein of SEQ ID NO:2 or SEQ ID NO:4.

[0067] Conservative amino acid substitutions can be made in the NeuroD1 protein to produce NeuroD1 protein variants. A conservative amino acid substitution is a technically recognized substitution of one amino acid for another amino acid with similar characteristics. For example, each amino acid can be described as having one or more of the following characteristics: electropositive, electronegative, aliphatic, aromatic, polar, hydrophobic, and hydrophilic. A conservative substitution is the replacement of one amino acid with a particular structural or functional characteristic with another amino acid with the same characteristic. Acidic amino acids include aspartic acid and glutamic acid; basic amino acids include histidine, lysine, and arginine; aliphatic amino acids include isoleucine, leucine, and valine; aromatic amino acids include phenylalanine, glycine, tyrosine, and tryptophan; polar amino acids include aspartic acid, glutamic acid, histidine, lysine, asparagine, glutamine, arginine, serine, threonine, and tyrosine; hydrophobic amino acids include alanine, cysteine, phenylalanine, glycine, isoleucine, leucine, methionine, proline, valine, and tryptophan; and conservative substitutions include substitutions between amino acids within each group. Amino acids can also be described in terms of relative size, with alanine, cysteine, aspartic acid, glycine, asparagine, proline, threonine, serine, and valine all typically considered small.

[0068] NeuroD1 variants may include synthetic amino acid analogs, amino acid derivatives and / or non-standard amino acids, including, by way of example only, alpha-aminobutyric acid, citrulline, canavanine, cyanoalanine, diaminobutyric acid, diaminopimelic acid, dihydroxyphenylalanine, djenkolic acid, homoarginine, hydroxyproline, norleucine, norvaline, 3-phosphoserine, homoserine, 5-hydroxytryptophan, 1-methylhistidine, 3-methylhistidine, and ornithine.

[0069] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced into the first amino acid sequence or nucleic acid sequence for optimal alignment with the second amino acid sequence or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical overlapping positions / total number of positions × 100%). In one embodiment, the two sequences are the same length.

[0070] The determination of percent identity between two sequences can also be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for comparing two sequences is the algorithm of Karlin and Altschul, 1990, PNAS 87:2264-2268; Karlin and Altschul (ed.), 1993, PNAS. 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403. BLAST nucleotide searches are performed using the NBLAST nucleotide program parameters set, for example, to score=100 and word length=12, to obtain nucleotide sequences homologous to the nucleic acid molecules of the present invention.

[0071] BLAST protein searches are performed with the XBLAST program parameters set, e.g., to score 50, word length=3, to obtain amino acid sequences homologous to the protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST, as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389 3402, is utilized. Alternatively, PSI BLAST is used to perform an iterated search that detects distant relationships between molecules. When utilizing BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the respective programs (e.g., for XBLAST and NBLAST) are used (see, e.g., the NCBI website).

[0072] Another preferred, non-limiting example of a mathematical algorithm utilized for sequence comparison is the algorithm of Myers and Miller, 1988, CABIOS 4:11 17. Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 are used.

[0073] The percent identity between two sequences is determined using techniques similar to those described above, with or without gaps. In calculating percent identity, typically only exact matches are counted.

[0074] The term "NeuroD1 protein" encompasses fragments of NeuroD1 protein, such as fragments of SEQ ID NO: 2 and SEQ ID NO: 4, and variants thereof, that are operable in the methods and compositions of the present invention.

[0075] NeuroD1 proteins and nucleic acids can be isolated from natural sources, such as the brain of an organism or cells of a cell line that expresses NeuroD1. Alternatively, NeuroD1 proteins or nucleic acids can be produced recombinantly, for example, by expression using an expression construct in vitro or in vivo. NeuroD1 proteins and nucleic acids can also be synthesized by well-known methods.

[0076] The NeuroD1 included in the methods and compositions of the present invention is preferably produced using recombinant nucleic acid technology. Recombinant NeuroD1 production involves introducing into a host cell a recombinant expression vector comprising a DNA sequence encoding NeuroD1.

[0077] According to an embodiment of the present invention, the nucleic acid sequence encoding NeuroD1 that is introduced into a host cell to produce NeuroD1 encodes SEQ ID NO: 2, SEQ ID NO: 4, or a variant thereof.

[0078] According to an embodiment of the invention, the nucleic acid sequence identified herein as SEQ ID NO: 1 encodes SEQ ID NO: 2 and is included in an expression vector and expressed to produce NeuroD1. According to an embodiment of the invention, the nucleic acid sequence identified herein as SEQ ID NO: 3 encodes SEQ ID NO: 4 and is included in an expression vector and expressed to produce NeuroD1.

[0079] It will be understood that due to the degenerate nature of the genetic code, nucleic acid sequences substantially identical to SEQ ID NOs: 1 and 3 encode NeuroD1 and variants of NeuroD1, and that such alternative nucleic acids can be included in expression vectors and expressed to produce NeuroD1 and variants of NeuroD1. It will be understood by those of skill in the art that fragments of nucleic acids encoding NeuroD1 protein can be used to produce fragments of NeuroD1 protein.

[0080] An expression vector contains a nucleic acid comprising a segment encoding a polypeptide of interest operably linked to one or more regulatory elements that provide for transcription of the segment encoding the polypeptide of interest. As used herein, the term "operably linked" refers to a nucleic acid in a functional relationship with a second nucleic acid. The term "operably linked" encompasses the functional connection of two or more nucleic acid molecules, such as a nucleic acid to be transcribed and a regulatory element. As used herein, the term "regulatory element" refers to a nucleotide sequence that regulates some aspect of the expression of an operably linked nucleic acid. Exemplary regulatory elements include enhancers, such as, but not limited to, the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), internal ribosome entry sites (IRES) or 2A domains, introns, origins of replication, polyadenylation signals (pA), promoters, transcription termination sequences, and upstream regulatory domains that contribute to the replication, transcription, and post-transcriptional processing of an operably linked nucleic acid sequence. One of ordinary skill in the art can select and use these and other regulatory elements in expression vectors without more than routine experimentation.

[0081] The term "promoter," as used herein, refers to a DNA sequence operably linked to a nucleic acid sequence to be transcribed, such as a nucleic acid sequence encoding NeuroD1. A promoter is generally placed upstream of the nucleic acid sequence to be transcribed and provides a site for specific binding by RNA polymerase and other transcription factors. In certain embodiments, a promoter is generally placed upstream of the nucleic acid sequence to be transcribed to produce a desired molecule and provides a site for specific binding by RNA polymerase and other transcription factors.

[0082] As will be appreciated by those skilled in the art, the 5' non-coding region of a gene can be isolated and used in its entirety as a promoter to drive expression of an operably linked nucleic acid. Alternatively, a portion of the 5' non-coding region can be isolated and used to drive expression of an operably linked nucleic acid. Generally, approximately 500-6000 bp of the 5' non-coding region of a gene is used to drive expression of an operably linked nucleic acid. Optionally, a portion of the 5' non-coding region of a gene containing the minimum amount of 5' non-coding region required to drive expression of an operably linked nucleic acid is used. Assays to determine the ability of a specified portion of a gene's 5' non-coding region to drive expression of an operably linked nucleic acid are well known in the art.

[0083] The particular promoter used to drive expression of NeuroD1 by the methods described herein is a "ubiquitous" or "constitutive" promoter, which drives expression in many, most, or all cell types of the organism into which the expression vector is introduced. Non-limiting examples of ubiquitous promoters that can be used to express NeuroD1 include the cytomegalovirus promoter, the simian virus 40 (SV40) early promoter, the rous sarcoma virus promoter, the adenovirus major late promoter, the beta-actin promoter, the glyceraldehyde 3-phosphate dehydrogenase promoter, the glucose-regulated protein 78 promoter, the glucose-regulated protein 94 promoter, the heat shock protein 70 promoter, the beta-kinesin promoter, the ROSA promoter, the ubiquitin B promoter, the eukaryotic initiation factor 4A1 promoter, and the elongation factor I promoter, all of which are well known in the art and can be isolated from primary sources or obtained from commercially available sources using routine methods. Promoters can be derived entirely from a single gene or can be chimeric, containing portions derived from multiple genes.

[0084] A combination of regulatory sequences can be included in an expression vector and used to drive expression of NeuroD1. A non-limiting example of an expression vector that can drive expression of NeuroD1 is the CAG promoter in combination with the cytomegalovirus CMV early enhancer element and the chicken beta actin promoter.

[0085] Particular promoters used to drive expression of NeuroD1 according to the methods described herein are those that drive expression preferentially in glial cells, particularly astrocytes and / or NG2 cells. Such promoters are referred to as "astrocyte-specific" and / or "NG2 cell-specific" promoters.

[0086] Non-limiting examples of astrocyte-specific promoters are the glial fibrillary acidic protein (GFAP) promoter and the aldehyde dehydrogenase 1 family, member L1 (Aldh1L1) promoter. The human GFAP promoter is shown herein as SEQ ID NO: 6. The mouse Aldh1L1 promoter is shown herein as SEQ ID NO: 7.

[0087] A non-limiting example of an NG2 cell-specific promoter is the promoter of the chondroitin sulfate proteoglycan 4 gene, also known as neuron-glial antigen 2 (NG2). The human NG2 promoter is set forth herein as SEQ ID NO:8.

[0088] The particular promoters used to drive expression of NeuroD1 according to the methods described herein are those that drive expression preferentially in reactive glial cells, particularly reactive astrocytes and / or reactive NG2 cells. Such promoters are referred to as "reactive astrocyte-specific" and / or "reactive NG2 cell-specific" promoters.

[0089] A non-limiting example of a "reactive astrocyte-specific" promoter is the promoter of the lipocalin 2 (lcn2) gene. The mouse lcn2 promoter is set forth herein as SEQ ID NO:5.

[0090] Homologs and variants of ubiquitous and cell type-specific promoters can be used for expression of NeuroD1.

[0091] Promoter homologs and promoter variants can be included in expression vectors expressing NeuroD1 according to the present invention. The terms "promoter homolog" and "promoter variant" refer to promoters that have substantially similar functional characteristics to those described herein to confer the desired type of expression, e.g., cell-type-specific expression of NeuroD1 or ubiquitous expression of NeuroD1, on an operably linked nucleic acid encoding NeuroD1. For example, promoter homologs or variants have substantially similar functional characteristics to GFAP, S100b, Aldh1L1, NG2, lcn2, and CAG promoters, conferring cell-type-specific expression on an operably linked nucleic acid encoding NeuroD1.

[0092] Those skilled in the art will recognize that one or more nucleic acid mutations can be introduced without changing the functional properties of a given promoter. Mutations can be introduced using standard molecular biology techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis, to create promoter variants. As used herein, the term "promoter variant" refers to either isolated naturally occurring mutations or recombinantly prepared mutations of reference promoters, such as, but not limited to, GFAP, S100b, AldhlL1, NG2, lcn2, and pCAG promoters.

[0093] It is known in the art that promoters from other species are functional, for example, the mouse Aldh1L1 promoter is functional in human cells. Homologs and homologous promoters from other species can be identified using bioinformatics tools known in the art, see, e.g., Xuan et al., 2005, Genome Biol 6:R72; Zhao et al., 2005, Nucl Acid Res 33:D103-107; and Halees et al., 2003, Nucl. Acids. Res. 2003 31:3554-3559.

[0094] Structurally, homologs and variants of the cell type-specific and / or ubiquitous promoters of NeuroD1 have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more nucleic acid sequence identity to a reference developmentally-regulated and / or ubiquitous promoter and contain a binding site for RNA polymerase and, optionally, one or more binding sites for transcription factors.

[0095] A nucleic acid sequence that is substantially identical to SEQ ID NO:1 or SEQ ID NO:3 is characterized by having a complementary nucleic acid sequence that is capable of hybridizing to SEQ ID NO:1 or SEQ ID NO:3 under high stringency hybridization conditions.

[0096] In addition to one or more nucleic acids encoding NeuroD1, an expression vector can include one or more nucleic acid sequences encoding additional proteins, such as non-NeuroD1 proteins, such as reporters, including, but not limited to, beta-galactosidase, green fluorescent protein, and antibiotic resistance reporters.

[0097] In certain embodiments, the recombinant expression vector encodes at least NeuroD1 of SEQ ID NO:2, a protein having at least 95% identity to SEQ ID NO:2, or a protein encoded by a nucleic acid sequence substantially identical to SEQ ID NO:1.

[0098] In certain embodiments, the recombinant expression vector encodes at least NeuroD1 of SEQ ID NO:4, a protein having at least 95% identity to SEQ ID NO:4, or a protein encoded by a nucleic acid sequence substantially identical to SEQ ID NO:2.

[0099] SEQ ID NO: 9 is an example of a nucleic acid comprising a CAG promoter operably linked to a nucleic acid encoding NeuroD1, and further comprising a nucleic acid sequence encoding EGFP and an enhancer, WPRE. An IRES separates the nucleic acid encoding NeuroD1 from the nucleic acid encoding EGFP. SEQ ID NO: 9 is inserted into an expression vector to express NeuroD1 and the reporter gene EGFP. Optionally, the IRES and nucleic acid encoding EGFP are removed, and the remaining CAG promoter and operably linked nucleic acid encoding NeuroD1 are inserted into an expression vector for expression of NeuroD1. A WPRE or another enhancer is optionally included.

[0100] Optionally, a reporter gene is included in the recombinant expression vector encoding NeuroD1. The inclusion of a reporter gene allows for the production of a peptide or protein that serves as a surrogate marker for the expression of NeuroD1 from the recombinant expression vector. As used herein, the term "reporter gene" refers to a gene that is easily detectable when expressed, for example, by chemiluminescence, fluorescence, colorimetric reaction, antibody binding, inducible marker, and / or ligand binding assays. Exemplary reporter genes include, but are not limited to, green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (eYFP), cyan fluorescent protein (CFP), enhanced cyan fluorescent protein (eCFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (eBFP), MmGFP (Zernicka-Goetz et al., Development, 124:1133-1137, 1997), dsRed, luciferase, and beta-galactosidase (lacZ).

[0101] For example, the method of introducing genetic material into a recipient host cell for transient or stable expression of a desired protein encoded by the genetic material in the host cell is called "transfection." Transfection techniques are well known in the art and include, but are not limited to, electroporation, particle-accelerated transformation, also known as "gene gun" technology, liposome-mediated transfection, calcium phosphate or calcium chloride co-precipitation-mediated transfection, DEAE-dextran-mediated transfection, microinjection, polyethylene glycol-mediated transfection, heat shock-mediated transfection, and virus-mediated transfection. As described herein, virus-mediated transfection can be achieved using viral vectors, such as those derived from adenoviruses, adeno-associated viruses, and lentiviruses.

[0102] Optionally, the host cells are transfected ex vivo and then reintroduced into the host organism. For example, cells or tissues can be removed from a subject, transfected with an expression vector encoding NeuroD1, and then returned to the subject.

[0103] To express exogenous NeuroD1 in host glial cells to convert the glial cells into neurons, a recombinant expression vector containing a nucleic acid encoding NeuroD1 or a functional fragment thereof is introduced into host glial cells in vitro or in vivo by any of a variety of transfection methodologies.

[0104] Expression of exogenous NeuroD1 in host glial cells to convert the glial cells into neurons is optionally achieved by introducing mRNA encoding NeuroD1 or a functional fragment thereof into the host glial cells in vitro or in vivo.

[0105] Expression of exogenous NeuroD1 in host glial cells to convert them into neurons is optionally achieved by introducing NeuroD1 protein into the host glial cells in vitro or in vivo. Details of these and other techniques are known in the art, e.g., J. Sambrook and DW Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 3rd ed., 2001; F. M. Ausubel (ed.), Short Protocols in Molecular Biology, Current Protocols, 5th ed., 2002; and Engelke, D. R., RNA Interference (RNAi): Nuts and Bolts of RNAi Technology, DNA Press LLC, Eagleville, PA, 2003.

[0106] The expression vector containing the nucleic acid encoding NeuroD1 or a functional fragment thereof, the mRNA encoding NeuroD1 or a functional fragment thereof, and / or the NeuroD1 protein, full-length or a functional fragment thereof, is optionally associated with a carrier for introduction into a host cell in vitro or in vivo.

[0107] In certain embodiments, the carrier is a particulate carrier, e.g., lipid particles, including liposomes, micelles, unilamellar or multilamellar vesicles; polymeric particles, e.g., hydrogel particles, polyglycolic acid particles, or polylactic acid particles; inorganic particles, e.g., calcium phosphate particles, such as those described in U.S. Pat. No. 5,648,097; and inorganic / organic particulate carriers, such as those described in U.S. Pat. No. 6,630,486.

[0108] Particulate carriers can be selected from lipid particles, polymeric particles, inorganic particles, and inorganic / organic particles. Mixtures of particle types can also be included as particulate pharmaceutically acceptable carriers.

[0109] Particulate carriers are typically formulated so that the particles have an average particle size in the range of about 1 nm to 10 microns. In certain embodiments, particulate carriers are formulated so that the particles have an average particle size in the range of about 1 nm to 100 nm.

[0110] Further description of liposomes and methods for their preparation and use can be found in Liposomes: A Practical Approach (The Practical Approach Series, 264), VP Torchilin and V. Weissig (eds.), Oxford University Press, 2nd edition, 2003. Further aspects of nanoparticles are described in SM Moghimi et al., FASEB J. 2005, 19, 311-30.

[0111] Expression of NeuroD1 using a recombinant expression vector is achieved by introducing the expression vector into a eukaryotic or prokaryotic host cell expression system, such as insect cells, mammalian cells, yeast cells, bacterial cells, or any other unicellular or multicellular organism recognized in the art. Host cells are optionally primary cells or immortalized cells. Immortalized cells are cells that can be maintained in vitro for at least five replicative passages.

[0112] Host cells containing the recombinant expression vector are maintained under conditions in which NeuroD1 is produced. Host cells can be cultured and maintained using known cell culture techniques, such as those described in Celis, Julio (ed.), 1994, Cell Biology Laboratory Handbook, Academic Press, NY. Various culture conditions for these cells, including media formulations with respect to specific nutrients, oxygen, tension, carbon dioxide, and reduced serum levels, can be selected and optimized by those skilled in the art.

[0113] According to an embodiment of the present invention, a recombinant expression vector containing a nucleic acid encoding NeuroD1 is introduced into glial cells of a subject. Expression of exogenous NeuroD1 in the glial cells "converts" the glial cells into neurons.

[0114] According to an embodiment of the present invention, a recombinant expression vector containing a nucleic acid encoding NeuroD1 or a functional fragment thereof is introduced into astrocytes of a subject. Expression of exogenous NeuroD1 in glial cells "converts" the astrocytes into neurons.

[0115] According to an embodiment of the present invention, a recombinant expression vector containing a nucleic acid encoding NeuroD1 or a functional fragment thereof is introduced into reactive astrocytes of a subject. Expression of exogenous NeuroD1 or a functional fragment thereof in reactive astrocytes "converts" the reactive astrocytes into neurons.

[0116] According to an embodiment of the present invention, a recombinant expression vector containing a nucleic acid encoding NeuroD1 or a functional fragment thereof is introduced into NG2 cells of a subject. Expression of exogenous NeuroD1 or a functional fragment thereof in NG2 cells "converts" the NG2 cells into neurons.

[0117] Detection of expression of exogenous NeuroD1 following introduction of a recombinant expression vector containing a nucleic acid encoding exogenous NeuroD1 or a functional fragment thereof is accomplished using any of a variety of standard methodologies, including, but not limited to, immunoassays to detect NeuroD1, nucleic acid assays to detect NeuroD1 nucleic acids, and detection of reporter genes co-expressed with exogenous NeuroD1.

[0118] The terms "convert" and "converted" are used herein to describe the effect of expression of NeuroD1 or a functional fragment thereof, which results in a change of glial, astrocyte, or reactive astrocyte phenotype to a neuronal phenotype. Similarly, the phrases "NeuroD1-converted neuron" and "converted neuron" are used herein to designate a cell containing exogenous NeuroD1 protein or a functional fragment thereof, which has a resulting neuronal phenotype.

[0119] The term "phenotype" refers to a known detectable characteristic of a cell as referred to herein. A neuronal phenotype can be one or more of, but is not limited to, neuronal morphology, expression of one or more neuronal markers, electrophysiological characteristics of a neuron, synapse formation, and neurotransmitter release. For example, a neuronal phenotype includes, but is not limited to, characteristic morphological aspects of a neuron, such as the presence of dendrites, axons, and dendritic spines; the expression and distribution of characteristic neuronal proteins, such as the presence of synaptic proteins in synaptic puncta and the presence of MAP2 in dendrites; and characteristic electrophysiological signatures, such as spontaneous and evoked synaptic events.

[0120] In further examples, glial phenotypes, e.g., astrocytic phenotypes and reactive astrocyte phenotypes, include, but are not limited to, characteristic morphological aspects of astrocytes and reactive astrocytes, such as a morphology commonly referred to as "star-shaped"; and the presence of characteristic astrocytic and reactive astrocyte protein expression, e.g., glial fibrillary acidic protein (GFAP).

[0121] The term "nucleic acid" refers to an RNA or DNA molecule having a plurality of nucleotides in any form, including single-stranded, double-stranded, oligonucleotide, or polynucleotide. The term "nucleotide sequence" refers to the order of nucleotides in a single-stranded form of a nucleic acid, an oligonucleotide, or a polynucleotide.

[0122] The term "NeuroD1 nucleic acid" refers to an isolated NeuroD1 nucleic acid molecule, and includes an isolated NeuroD1 nucleic acid or fragment thereof having a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the DNA sequence set forth in SEQ ID NO:1 or SEQ ID NO:3, or its complement, or an isolated DNA molecule or fragment thereof having a sequence that hybridizes under high stringency hybridization conditions to the nucleic acid set forth as SEQ ID NO:1 or SEQ ID NO:3, or its complement.

[0123] The nucleic acid of SEQ ID NO: 3 is an example of an isolated DNA molecule having a sequence that hybridizes under high stringency hybridization conditions to the nucleic acid set forth in SEQ ID NO: 1. A fragment of a NeuroD1 nucleic acid is any fragment of a NeuroD1 nucleic acid that is operable in embodiments of the invention that involve a NeuroD1 nucleic acid.

[0124] Nucleic acid probes or primers capable of hybridizing to target NeuroD1 mRNA or cDNA can be used to detect and / or quantify mRNA or cDNA encoding NeuroD1 protein. Nucleic acid probes can be at least 10, 15, 30, 50, or 100 nucleotides in length and can be oligonucleotides sufficient to specifically hybridize to NeuroD1 mRNA or cDNA or its complementary sequence under stringent conditions. Nucleic acid primers can be at least 10, 15, or 20 nucleotides in length and can be oligonucleotides sufficient to specifically hybridize to mRNA or cDNA or its complementary sequence under stringent conditions.

[0125] The terms "complement" and "complementary" refer to Watson-Crick base pairing between nucleotides, particularly nucleotides that are hydrogen-bonded to each other with a thymine or uracil residue linked to an adenine residue by two hydrogen bonds and a cytosine and guanine residue linked by three hydrogen bonds. Nucleic acids generally contain nucleotide sequences that are described as having a certain "percent complementarity" to a specific second nucleotide sequence. For example, a nucleotide sequence may have 80%, 90%, or 100% complementarity to a specific second nucleotide sequence, indicating that 8 out of 10 nucleotides, 9 out of 10 nucleotides, or 10 out of 10 nucleotides of the sequence are complementary to the specific second nucleotide sequence. For example, the nucleotide sequence 3'-TCGA-5' is 100% complementary to the nucleotide sequence 5'-AGCT-3'. Furthermore, the nucleotide sequence 3'-TCGA- is 100% complementary to a region of the nucleotide sequence 5'-TTAGCTGG-3'.

[0126] The terms "hybridization" and "hybridize" refer to the pairing and binding of complementary nucleic acids. Hybridization occurs to varying degrees between two nucleic acids, as is well known in the art, depending on factors such as the degree of complementarity of the nucleic acids, the melting temperature (Tm) of the nucleic acids, and the stringency of the hybridization conditions. The term "stringency of hybridization conditions" refers to conditions of temperature, ionic strength, and the composition of the hybridization medium with respect to certain common additives, such as formamide and Denhardt's solution.

[0127] Determining specific hybridization conditions associated with a particular nucleic acid is routine and well known in the art, as described, for example, in J. Sambrook and DW Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 3rd ed., 2001; and F. M. Ausubel, ed., Short Protocols in Molecular Biology, Current Protocols, 5th ed., 2002. High stringency hybridization conditions are conditions that allow only substantially complementary nucleic acids to hybridize. Typically, nucleic acids having about 85-100% complementarity are considered highly complementary and will hybridize under high stringency conditions. Intermediate stringency conditions are exemplified by conditions under which nucleic acids having intermediate complementarity, about 50-84% complementarity, as well as nucleic acids with a high degree of complementarity, will hybridize. Conversely, low stringency hybridization conditions are conditions under which nucleic acids with a low degree of complementarity will hybridize.

[0128] The terms "specific hybridization" and "specifically hybridize" refer to hybridization of a particular nucleic acid to a target nucleic acid without substantial hybridization to nucleic acids other than the target nucleic acid in a sample.

[0129] As is well known to those skilled in the art, hybridization stringency and washing conditions depend on several factors, including the Tm of the probe and target and the ionic strength of the hybridization and washing conditions. Hybridization protocols and conditions that achieve the desired hybridization stringency are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2001; and Ausubel, F. et al. (eds.), Short Protocols in Molecular Biology, Wiley, 2002.

[0130] An example of high stringency hybridization conditions is overnight hybridization of nucleic acids greater than about 100 nucleotides in length in a solution containing 6xSSC, 5xDenhardt's solution, 30% formamide, and 100 micrograms / ml denatured salmon sperm at 37°C, followed by a 15-minute wash at 60°C in a solution of 0.1xSSC and 0.1% SDS. SSC is 0.15M NaCl / 0.015M Na citrate. Denhardt's solution is 0.02% bovine serum albumin / 0.02% FICOLL / 0.02% polyvinylpyrrolidone. Under high stringency conditions, SEQ ID NO: 1 and SEQ ID NO: 3 hybridize to the complement of substantially identical targets and do not hybridize to unrelated sequences.

[0131] According to an aspect of the present invention, there is provided a method of treating a neurological condition in a subject in need thereof, comprising delivering a therapeutically effective amount of NeuroD1 to glial cells of the subject's central or peripheral nervous system, wherein the therapeutically effective amount of NeuroD1 in the glial cells results in more neurons in the subject compared to an untreated subject with the same neurological condition, thereby treating the neurological condition.

[0132] Additionally, the conversion of reactive glial cells into neurons reduces neuroinflammatory and neuroinhibitory factors associated with reactive glial cells, thereby allowing glial scar tissue to grow more neurons, resulting in alleviation of the neurological condition.

[0133] The term "neurological condition" as used herein refers to any condition of a subject's central and / or peripheral nervous system that is alleviated, ameliorated, or prevented by additional neurons. Injuries or diseases that result in loss or inhibition of neurons and / or loss or inhibition of neuronal function are neurological conditions for treatment by methods according to aspects of the present invention.

[0134] Injuries or diseases that result in loss or inhibition of glutamatergic neurons and / or loss or inhibition of glutamatergic neuron function are neurological conditions for treatment by methods according to embodiments of the invention. Loss or inhibition of other types of neurons, such as GABAergic, cholinergic, dopaminergic, norepinephrine, or serotonergic neurons, can also be treated in similar ways.

[0135] Thus, for example, injuries or diseases that result in loss or inhibition of neurons and / or loss or inhibition of neuronal function, including, but not limited to, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), stroke, epilepsy, physical injury, such as brain injury or spinal cord injury, and tumors, are neurological conditions for treatment by methods according to aspects of the present invention.

[0136] The term "therapeutically effective amount" as used herein is intended to mean an amount of a composition of the present invention effective to alleviate, ameliorate, or prevent the symptoms or signs of a neurological condition being treated. In certain embodiments, a therapeutically effective amount is an amount that has a beneficial effect in a subject having signs and / or symptoms of a neurological condition.

[0137] As used herein, the terms "treat," "treatment," "treating," and "NeuroD1 treatment," or grammatical equivalents, mean alleviating, inhibiting, or ameliorating a neurological condition, a symptom or sign of a neurological condition, and preventing a symptom or sign of a neurological condition, and includes, but is not limited to, therapeutic and / or prophylactic treatment.

[0138] The signs and symptoms of neurological conditions are well known in the art, along with methods for detecting and assessing such signs and symptoms.

[0139] A combination of therapies for a subject's neurological condition may be administered in accordance with aspects of the present invention.

[0140] According to certain embodiments, additional pharmaceutical agents or therapeutic treatments administered to a subject to treat the effects of disruption of normal blood flow in the CNS of an individual subject in need thereof include, for example, but are not limited to, treatments such as removal of blood clots, promotion of blood flow, administration of one or more anti-inflammatory agents, administration of one or more antioxidants, and administration of one or more agents effective in reducing excitotoxicity.

[0141] The term "subject" refers to humans and non-human mammals, including, but not limited to, non-human primates, cats, dogs, sheep, goats, horses, cows, pigs, and rodents, including, but not limited to, mice and rats, as well as non-mammals, including, but not limited to, birds, poultry, reptiles, and amphibians.

[0142] Embodiments of the compositions and methods of the present invention are described in the following examples, which are provided for illustrative purposes and are not to be construed as limitations on the scope of the compositions and methods of the present invention.

[0143] [Example] Materials and Methods Mouse model of stroke and viral injection Wild-type (WT) FVB / NJ and GFAP-GFP transgenic mice were used for most of the experiments described in this study. GFAP-GFP transgenic mice were purchased from Jackson Laboratory [FVB / N-Tg(GFAPGFP)14Mes / J] (Zhuo, L. et al., 1997, Dev Biol 187, 36-42) and crossed with FVB / NJ mice (Jackson Laboratory). Endothelin-1(1-31) was injected into the motor cortex of adult WT FVB / NJ or GFAPA-GFP transgenic mice (28-40 g, 5-10 months old) to produce focal ischemic injury as described in Horie, N. et al., 2008, J. Neurosci. Methods 173, pp. 286-290; and Roome, R.B. et al., 2014, J. Neurosci. Methods 233, pp. 34-44.

[0144] Under anesthesia with an intraperitoneal injection of ketamine / xylazine (100 mg / kg ketamine, 12 mg / kg xylazine), the mouse was placed in a stereotaxic apparatus, and a midline incision was made to expose the skull and bregma. A small hole approximately 1 mm in diameter was drilled in the skull at the coordinates of the forelimb motor cortex (relative to the bregma): +0.2 mm anterior-posterior (AP), +1.5 mm medial-lateral (ML). ET-1(1-31) (Peptide International, Inc., PED-4360-s) was dissolved at 2 μg / μl in phosphate-buffered saline (PBS; OSM approximately 320, pH approximately 7.3).

[0145] A total volume of 0.5 μl (1 μg) was injected into each site at a dorsoventral velocity (DV) of -1.6 mm. Injections were performed by an infusion pump over a 10-minute period, with the needle slowly withdrawn at 0.1 mm / min. After injection, the needle was maintained at a DV of 1.1 mm for an additional 3 minutes before being fully withdrawn.

[0146] Viral injections followed a similar procedure, except that the viral injections were typically performed approximately 10 days after the ET-1 injection through the same holes drilled for the ET-1 injections.

[0147] Mice aged 5 to 10 months were used. They were housed under a 12-hour light-dark cycle and provided with adequate food and water. Both male and female mice were used, except for the behavioral experiments, in which only male mice were used.

[0148] AAV vector construction The hGFAP promoter was obtained from the pDRIVE-hGFAP plasmid (InvivoGen, Inc.) and inserted between the MluI and SacII sites of pAAV-MCS (Cell Biolab) to replace the CMV promoter. The Cre gene was obtained by PCR from hGFAP-Cre (Addgene plasmid #40591) and inserted between the EcoRI and Sal1 sites of pAAV MCS to generate the pAAV-hGFAP::Cre vector.

[0149] To construct the pAAV-FLEX-mCherry-P2A-mCherry (or pAAV-FLEX-GFP-P2A-GFP) and pAAV-FLEX-NeuroD1-P2A-mCherry (or pAAV-FLEX-NeuroD1-P2A-GFP) vectors, cDNA encoding NeuroD1, mCherry, or GFP was obtained by PCR using the retroviral constructs described in Guo, Z. et al., 2014, Cell Stem Cell 14, pp. 188-202.

[0150] The NeuroD1 gene was fused to P2A-mCherry or P2A-GFP and subcloned into the pAAV-FLEX-GFP vector (Addgene plasmid #28304) between the Kpn1 and Xho1 sites. Plasmid constructs were sequenced for verification.

[0151] AAV virus production Recombinant AAV9 was produced in 293AAV cells (Cell Biolabs). Briefly, polyethyleneimine (PEI, linear, 25,000 molecular weight) was used for transfection of a triple plasmid: pAAV expression vector, pAAV9-RC (Cell Biolabs), and pHelper (Cell Biolabs).

[0152] Seventy-two hours after transfection, cells in culture were scraped from the plate, centrifuged, and freeze-thawed four times by alternating between dry ice / ethanol and a 37°C water bath. AAV crude lysates were purified by centrifugation in a discontinuous iodixanol gradient at 54,000 rpm in a Beckman SW55Ti rotor for 1 hour.

[0153] The virus-containing layer was extracted, and the virus was concentrated using a Millipore Amicon Ultra Centrifugal Filter. Viral titers were determined using a QuickTiter™ AAV Quantitation Kit (Cell Biolabs) and were 1.2 × 10 for hGFAP::Cre, hGFAP::NeuroD1-GFP, and hGFAP::GFP. 12 GC / ml, and 1.4 × 10 for CAG::FLEX-NeuroD1-P2A-GFP and CAG::FLEX-NeuroD1-P2A-mCherry. 12 GC / ml, and 1.6 × 10 for CAG::FLEX-mCherry-P2A-mCherry and CAG::FLEX-GFP-P2A-GFP. 12 It was GC / ml.

[0154] Retrovirus production pCAG-NeuroD1-IRES-GFP and pCAG-GFP are described in Guo, Z. et al., 2014, Cell Stem Cell 14, pp. 188-202. To package retroviral particles, gpg helper-free human embryonic kidney (HEK) cells were transfected with the target plasmid together with a vesicular stomatitis virus glycoprotein (VSV-G) vector to produce retroviruses expressing NeuroD1 or GFP. The titer of retroviral particles was determined after transduction of HEK cells and was approximately 10 7 particles / ml.

[0155] immunohistochemistry Immunohistochemistry of free-floating frozen sections of mouse brain was performed as described in Guo, Z. et al., 2014, Cell Stem Cell 14, pp. 188–202. Animals were anesthetized with 2.5% avertin and transcardially perfused with artificial cerebrospinal fluid (ACSF) to flush out blood from the brain tissue. Brains were then dissected, trimmed, and placed in 4% paraformaldehyde (PFA) overnight at 4°C for postfixation. After fixation, brain tissue was cut into 40 μm sections using a vibratome (Leica).

[0156] Brain sections were permeabilized with 2% Triton X-100 in PBS for 1 hour, followed by incubation in blocking buffer (2.5% normal goat serum, 2.5% normal donkey serum, and 0.1% Triton X-100 in PBS) for 1 hour. Primary antibodies were added to the brain sections in blocking buffer and incubated overnight at 4°C.

[0157] After rinsing the primary antibodies in PBS, the brain sections were incubated with secondary antibodies conjugated with different fluorophores (1:800, Jackson ImmunoResearch) for 1 h at room temperature, washed in Triton-PBS, and then mounted on glass slides using antifade mounting solution containing DAPI (Invitrogen). Images were obtained using a confocal microscope (Olympus FV1000 or Zeiss LSM800) and a Keyence microscope.

[0158] To test antibody specificity, the primary antibody was omitted and only the secondary antibody was used for immunostaining as a side-by-side control for all primary antibodies. No specific signal was detected in these controls.

[0159] BBB permeability test Deeply anesthetized mice were perfused with artificial cerebrospinal fluid as described above, followed by 15 ml of 0.5 mg / ml sulfo-NHS-LC-biotin in PBS. For immunohistochemistry, brain sections were incubated with Texas Red streptavidin (Vector SA-5006) diluted 1:800 in PBS + 0.3% Triton + 2.5% normal goat or donkey serum for 1 hour at room temperature, followed by a standard mounting procedure.

[0160] electrophysiology Brain slice recordings were performed similarly to the method described in Guo, Z. et al., 2014, Cell Stem Cell 14, pp. 188–202; and Wu, Z. et al., 2014, Nat. Commun. 5, pp. 4159. Two to three months after AAV injection, mice were anesthetized with 2.5% avertin and then perfused with an NMDG-based cutting solution (in mM): 93 NMDG, 93 HCl, 2.5 KCl, 1.25 NaH2PO4, 30 NaHCO3, 20 HEPES, 15 glucose, 12 N-acetyl-L-cysteine, 5 sodium ascorbate, 2 thiourea, 3 sodium pyruvate, 7 MgSO4, and 0.5 CaCl2, pH 7.3–7.4, 300 mOsm, 95% O2 / 5% CO2.

[0161] Coronal sections, 300 μm thick, were cut around the AAV-injected cortical area using a vibratome (VT1200S, Leica, Germany) at room temperature. Sections were collected and incubated for 10–15 min at 33.0 ± 1.0°C in oxygenated NMDG cutting solution. Sections were then transferred to a holding solution (in mM): 92 NaCl, 2.5 KCl, 1.25 NaH2PO4, 30 NaHCO3, 20 HEPES, 15 glucose, 12 N-acetyl-L-cysteine, 5 sodium ascorbate, 2 thiourea, 3 sodium pyruvate, 2 MgSO4, and 2 CaCl2, continuously aerated with 95% O2 / 5% CO2.

[0162] After recovery in the holding solution at room temperature for at least 0.5 h, single slices were transferred to a recording chamber continuously perfused with standard aCSF (artificial cerebrospinal fluid) saturated with 95% O2 / 5% CO2 at 33.0 ± 1.0 °C. Standard aCSF contained the following (in mM): 124 NaCl, 2.5 KCl, 1.25 NaH2PO4, 26 NaHCO3, 10 glucose, 1.3 MgSO4, 2.5 CaCl2.

[0163] To detect action potential firing in NeuroD1-GFP-infected neurons, whole-cell recordings were performed with a pipette solution containing the following (in mM): 135 K-gluconate, 10 KCl, 5 Na phosphocreatine, 10 HEPES, 2 EGTA, 4 MgATP, and 0.3 Na2GTP, pH 7.3 adjusted with KOH, 280–290 mOsm. Depolarizing currents were injected to evoke action potentials under the current-clamp model.

[0164] For recording spontaneous excitatory postsynaptic currents (sEPSCs) and spontaneous inhibitory postsynaptic currents (sIPSCs), the pipette solution contained the following (in mM): 120 Cs-methanesulfonate, 10 KCl, 10 Na-phosphocreatine, 10 HEPES, 5 QX-314, 1 EGTA, 4 MgATP, and 0.3 Na2GTP, pH 7.3 adjusted with KOH, 280–290 mOsm. For labeling of recorded neurons, 0.5% biocytin was added to the pipette solution.

[0165] The cell membrane potential was held at -70 mV (the reversal potential of GABAA receptors) for sEPSC recordings and 0 mV (the reversal potential of ionotropic glutamate receptors) for sIPSC recordings. Data were collected using a MultiClamp 700A amplifier and analyzed with pCLAMPIO software (Molecular Devices).

[0166] Mouse behavioral tests Food pellet recovery test For all behavioral experiments in mice, ET-1(1-31) was injected into two locations in the forelimb motor cortex to induce severe motor deficits: (i) +0.2 mm AP, + / - 1.35 mm ML, and (ii) +0.38 mm AP, + / - 2.45 mm ML (+ML or -ML was based on the location contralateral to the dominant forelimb during pre-stroke pellet retrieval training).

[0167] The food pellet retrieval chamber was modified from a mouse staircase apparatus as described in Baird, AL et al., 2001, Brain Res. Bull. 54, pp. 243–250; and Roome, RB et al., 2014, J. Neurosci. Methods 233, pp. 34–44. Briefly, the staircase was modified with a single V-shaped well and a small baffle at the end to prevent food pellets from falling out of the well during testing. As shown in Figure 14A, the well was 33 mm long, and its lowest point was 16 mm from the bottom of the tabletop.

[0168] Before the pellet retrieval test, mice were fasted for 18 hours to increase their motivation. During the first food deprivation training session, mice were given 30 sucrose pellets (14 mg, TestDiet, Inc.) in their home cages for habituation. During the first part of the training, the top of the central platform was removed to reduce the difficulty and allow the animals to more easily reach the food. Five pellets were placed on either the right or left side of the depression to determine the dominant forelimb. Mice were given 5 min to try, with each side tested twice. The dominant side was determined at this stage by counting the number of pellets retrieved. Most animals were able to retrieve approximately five pellets on one side, but occasionally both sides. If animals failed to retrieve any pellets, they were excluded from subsequent testing.

[0169] During the second part of training and all subsequent trials, the top plate was replaced on the central platform and eight pellets were placed in the well on the dominant side. Animals were tested three times on consecutive days, with 5 minutes allowed for pellet retrieval each time. If the average retrieval was greater than four pellets on three consecutive training days, the animal was used for further surgery and testing.

[0170] After training, an ischemic stroke was induced in the contralateral motor cortex controlling the dominant forelimb. A pellet retrieval test was performed on the 9th day after stroke and 1 day before virus injection to determine their functional level. If the number of retrieved pellets was less than half of the pre-stroke level, these animals were used for virus injection the following day (10 days after stroke).

[0171] After virus injection, pellet retrieval tests were performed at 20, 30, 50, and 70 dps to assess functional recovery. At each time point, the pellet retrieval test was repeated on two consecutive days, with training on day 1 and actual testing on day 2. Animals were coded, and a second experimenter blinded to the animals' identities performed the tests and pellet counts.

[0172] Grid walking test The grid walking test was similar to that described in Baskin, YK et al., 2003, J. Neurosci. Methods 129, 87-93; and Clarkson, AN et al., 2010, Nature 468, 305-309.

[0173] Height 24cm, length 24cm, width 20cm 1cm 2 A grid wire mesh was used. A video camera was placed under the grid, pointing upward at a 45° angle. Slow-motion footage was recorded and the animals' limb impairments were assessed. Individual mice were placed on the grid floor and allowed to walk freely for 5 minutes. Animals were coded, and video footage was analyzed offline by a second researcher blinded to the animals' identities.

[0174] The total number of limb defects and the total number of limb-free steps in the dominant limb were counted. The percentage of limb defect was calculated as follows: number of limb defects / total number of steps × 100.

[0175] Cylinder Test The cylinder test was based on the method described by Roome, RB et al., 2014, J. Neurosci. Methods, 233, pp. 34-44. This test involved video recording of a mouse rearing and touching the sidewall of a transparent cylinder (10 cm diameter, 15 cm high) with its forelimbs. Each animal was placed in the cylinder on a transparent board, and video recording was performed from below using a camera. The experiment was stopped after the mouse attempted to rear and touch the sidewall more than 30 times, which usually lasted approximately 3-4 minutes.

[0176] Videos were analyzed offline by an experimenter blinded to the animal's identity. A total of 30 trials of rearing and wall touching were analyzed. Normal limb touching was defined as the animal placing both limbs on the wall while rearing and using both limbs to push against the wall while descending. Abnormal touching included using only one limb to touch the wall or dragging one limb along the wall after touching.

[0177] Dragging behavior was defined as one limb, typically the injured limb, sliding along the side wall without maintaining stability against the wall. No-touch behavior was defined as the mouse not using the injured limb and only using the uninjured limb from the time of standing up to the time of standing down. No-touch is considered a more severe injury phenotype, as the mouse completely abandons the injured limb. Normal standing and touch behavior was quantified as follows: total normal limb touches / trial × 100.

[0178] Quantitative real-time PCR Cortical tissue surrounding the injury core (approximately 2 mm x 2 mm square) was obtained after perfusion and flash-frozen in liquid nitrogen. RNA extraction was performed using the Macherey-Nagel NucleoSpin RNA kit, and RNA concentration was measured by NanoDrop. cDNA was synthesized using Quanta Biosciences qScript cDNA supermix.

[0179] The reaction mixture was incubated at 25°C for 5 min, 42°C for 30 min, and 85°C for 5 min, and held at 4°C. The mixture was then diluted 5-fold with RNase / DNase-free water, and 2.5 μl was taken for qRT-PCR. Primers for qRT-PCR were designed using Applied Biosystems Primer Express software.

[0180] qRT-PCR was performed using Quanta Biosciences PerfeCTa SYBR Green Supermix, ROX. GAPDH was used as an internal standard, and non-stroke cortical tissue from healthy mice was used as a control. The comparative Ct method was used to calculate fold changes, and Prism 6 was used for statistical analysis and bar graphs.

[0181] Golgi impregnation staining A section Golgi kit (Bieoenno Tech, LLC) was used for Golgi staining. Briefly, mice were perfused with aCSF followed by the kit's fixative. Brains were removed, impregnated for 5 days, sectioned with a 200 μm vibratome, stained, mounted according to the kit's protocol, and covered with DPX mounting medium. 4x and 100x brightfield images were obtained using a Keyence BZ-9000 fluorescence microscope.

[0182] Quantification and statistical analysis Cortical area size analysis Cortical area was quantified using images acquired with a 4x lens on a Keyence BZ-9000 fluorescence microscope. Three sections around the injection point (+0.2 AP) with the most obvious stroke damage were selected for image acquisition. DAPI and NeuN signals were used to identify the superior and inferior cortical boundaries along with the cingulate cortex. Cortical area was measured 3 mm lateral from the midline using ImageJ.

[0183] Ratio of GFAP+ to NeuN+ for conversion efficiency analysis 40x confocal images of mCherry / GFP co-stained with GFAP or NeuN were used for quantification at 4, 7, and 17 dpi. For each animal, four images were acquired per animal in the virus-infected area near the cerebral infarct. For the GFAP ratio, cells that were double-positive for mCherry / GFP and GFAP were counted using the Zeiss confocal software Zen. The percentage of double-positive cells among all mCherry- or GFP-positive cells (all virus-infected cells) was calculated. A similar method was applied to the NeuN ratio.

[0184] Cortical neuron markers for analysis of transformed cell identity 40x confocal images of GFP (NeuroD1-GFP) co-stained with cortical neuron markers (Tbr1, Emx1, Satb2, PV, GABA) were used for quantification at 60 dpi. For each animal, three images were obtained in the virus-infected area showing GFP signal. The percentages of GFP and double-positive cells for each marker among all GFP-positive cells (NeuroD1-GFP-infected cells) were calculated.

[0185] Glial and neuronal marker intensity and coverage analysis Single-layer confocal images of glial markers (LCN2, CSPG, AQP4, and Iba1) and neuronal process markers (Map2, SMI312, VGluT1, and MBP) were used for quantification at 17 dpi. A 40x lens was used for most markers, except for SMI312, which was imaged using a 63x lens. Three images were obtained in the virally infected area proximal to the stroke infarct (within 500 µm of the stroke core border) and midway through the cortical thickness. One image was obtained in a relatively healthy area for normalization. After setting a threshold based on the background of the healthy area, ImageJ was used to quantify intensity and coverage across the entire image area. The results from the three images were used to obtain the average data for each animal.

[0186] Analysis of overall NeuN and PV cell numbers NeuN and PV cell counts were performed on images of NeuN or PV immunostaining acquired using the tile function of a Zeiss LSM800 confocal microscope within a cortical region 500 µm to 2500 µm lateral to the midline. The confocal software Zen was used for cell counting. Sections (+0.2 AP) around the injection point with the most obvious stroke damage were used for the acquired images.

[0187] Analysis of local NeuN numbers 40x confocal images of NeuN immunostaining were used for quantification at 17 dpi. As shown in Figure 4A, three images were acquired in the virally infected area close to the stroke infarct (within 500 μm of the stroke core border). The Z-stack function was used with 10 sections spaced 1 μm apart. The confocal software Zen was used for cell counting. In the NeuroD1 group, cells positive for both GFP (NeuroD1-GFP) and NeuN (transformed neurons) or NeuN-positive GFP-negative (existing neurons) were counted. For the GFP control group, the total number of NeuN-positive cells was counted.

[0188] Electrophysiological analysis sEPSCs and sIPSCs were analyzed using the Mini Analysis Program (Synaptosoft, New Jersey, USA). To avoid multiple detection of large events, the analysis results were automatically detected and then visually checked. To compare sEPSC and sIPSC frequencies between different groups, more than 200 events or at least a 3-minute recording period were sampled for each cell before obtaining average values.

[0189] Blood vessel count analysis For quantification of Ly6C counts, single-layer 40x images were acquired using a Zeiss confocal microscope. Ly6C intensity was measured using ImageJ, and vessels with an intensity three times higher than background were identified as positive signals. The number of Ly6C+ vessels in three images of the peri-infarct region was quantified and averaged for each mouse.

[0190] Statistics and blind analysis Statistical analysis was performed using Prism 6 (GraphPad) software. All experiments shown were repeated in at least three animals, and representative data are shown. To determine significance between groups, comparisons were performed using a paired, two-tailed Student's t-test or repeated-measures ANOVA, as indicated. After initial screening for severe stroke before virus injection, animals were randomly assigned to groups matched for deficit level. All images for quantification were acquired by one researcher and quantified by another researcher who was unaware of the animal's condition and identity. As described above, mouse behavioral testing was performed in a blinded manner.

[0191] [Example 1] NeuroD1 converts stroke-induced reactive astrocytes into neurons In this example, we investigated whether in vivo cell conversion can achieve functional brain repair after ischemic stroke. We used a focal stroke model induced by the vasoconstrictor peptide endothelin-1 (ET-1), which produces consistent focal ischemic damage in rodents (Fuxe et al., 1997; Hughes et al., 2003; Roome et al., 2014; Windle et al., 2006).

[0192] When comparing two different ET-1 peptides, one with 21 amino acids (ET-1(1-21)) and another with 31 amino acids (ET-1(1-31)), we found that ET-1(1-31) caused more severe and prolonged brain damage than ET-1(1-21) (Figure 2A). Mouse strains with an FVB background produced more severe stroke damage than the commonly used B6 / C57 mice (Figure 2A). Notably, injection of ET-1(1-31) into the motor cortex of FVB mice induced significant cortical tissue loss (Figures 1A and 1B), establishing a severe focal stroke model with consistent tissue damage over a 2-month time course.

[0193] To determine the appropriate time window for NeuroD1 viral injection after stroke, we investigated the stroke area at different time points to determine the time point at which astrocytes became reactive, since NeuroD1 can convert reactive astrocytes into neurons.

[0194] At 5 days post-stroke (dps), as expected, there was a significant loss of the neuronal signal NeuN (see Figure 1C). Interestingly, GFAP signal, a commonly used reactive astrocyte marker, was also very low, suggesting that astrocytes were not activated at this early stage. However, at 10 dps, GFAP signal was significantly upregulated (see Figure 1D), suggesting that astrocytes had become reactive during this period.

[0195] We next determined whether these reactive astrocytes induced by ischemic stroke could be converted into neurons. Reactive glial cells infected with NeuroD1 retrovirus successfully converted into NeuN-positive neurons (Figure 1E; see 17 days after virus injection and 27 days after stroke).

[0196] Retroviruses have the advantage of primarily targeting dividing reactive glial cells after injury, but because the number of dividing glial cells is rather limited, they cannot generate a large number of neurons for neural repair. To generate a sufficient number of neurons for functional repair after stroke, adeno-associated viruses (AAVs) were used to infect both dividing and non-dividing glial cells. AAVs have the advantages of high infectivity and low pathogenicity in humans and have been approved by the FDA for clinical trials in the treatment of CNS disorders.

[0197] To specifically infect astrocytes, we constructed an AAV vector (recombinant serotype AAV9) expressing NeuroD1 under the control of the human GFAP promoter (hGFAP::NeuroD1-P2A-GFP). Two weeks after infection, many NeuroD1-infected cells were also converted into NeuN-positive neurons (see Figure 1F). However, one caveat of GFAP promoter-driven expression is that the GFAP promoter is silenced after astrocyte-to-neuron conversion, making it difficult to distinguish between unconverted and converted neurons.

[0198] To overcome this limitation, the GFAP promoter and NeuroD1 were separated into two different AAV9 vectors using the Cre-FLEX (flip-excision) homologous recombination system (see Atasoy et al., 2008). The human GFAP promoter was then used to drive the expression of Cre recombinase (hGFAP::Cre), which could then act on two pairs of heterologous antiparallel loxP recombination sites flanking the inverted sequences of NeuroD1-P2A-GFP (or NeuroD1-P2A-mCherry) under the control of the CAG promoter in another vector (CAG::FLEX-NeuroD1-P2A-GFP / mCherry) (see Figure 2B). The advantage of this system is that after Cre-mediated inversion, NeuroD1 expression is driven by the strong promoter CAG, resulting in a significant increase in its expression level. This was confirmed by the observation that 4 days after virus injection (AAV9 hGFAP::Cre and CAG::FLEX-NeuroD1-P2A-mCherry), many infected astrocytes already expressed high levels of NeuroD1 (see Figure 1G, top panel).

[0199] Interestingly, some GFAP-positive astrocytes were clearly visible in the transitional stage to NeuN-positive neurons (see Figure 1G, bottom). Two weeks after virus injection, many NeuroD1-infected cells lost GFAP signals and became NeuN-positive neurons (see Figure 1I, bottom). However, most control AAV FLEX-GFP-infected cells retained their glial morphology (see Figure 1I, top). Thus, using the Cre-FLEX system, we demonstrate that NeuroD1 can convert many reactive astrocytes into neurons after ischemic stroke.

[0200] We then evaluated the efficiency of neurotransmission and the type of neurons generated in the stroke area. To test the efficiency of NeuroD1-mediated neurotransmission, we examined the number of astrocytes and neurons among AAV-infected cells in the stroke area at 4, 7, and 17 days after virus injection (Figures 3A-D). GFAP immunostaining was used to identify astrocytes, and we confirmed that the majority of control virus-infected mCherry / GFP-infected cells colocalized with GFAP between 4 and 17 days after virus injection (Figure 3A, top).

[0201] However, in AAV-NeuroD1-mCherry-infected cells, GFAP signaling showed a dramatic reduction from 4 to 17 dpi (see Figure 3A, bottom panel), suggesting that many AAV-NeuroD1-infected cells lost their astrocytic identity. In contrast to GFAP, NeuN staining showed very few neurons in the mCherry-infected control group, although NeuroD1-mCherry-expressing cells gradually colocalized with NeuN (see Figure 3B).

[0202] Quantitative analysis revealed that approximately 70% of mCherry-infected cells maintained their astrocytic identity from 4 to 17 dpi (see Fig. 3C ), whereas >70% of NeuroD1-infected cells had acquired a neuronal identity by 17 dpi (see Fig. 3D ).

[0203] To further investigate the conversion efficiency and specificity, a Tri-AAV tracking system was developed to track astrocyte-converted neurons. This system included a virus expressing GFP in astrocytes (AAV9 hGFAP::GFP) along with the Cre-FLEX system of the present invention (see Figures 4A-4C). In the mCherry control group, only 10% of GFP+ cells showed NeuN+ signals by 60 dpi, whereas in the NeuroD1-mCherry group, as many as 95% of GFP+ cells were NeuN+ at 60 dpi (quantified in Figure 4B), further confirming the high conversion efficiency of NeuroD1 when expressed in astrocytes. These results suggest that the NeuroD1 AAV of the present invention can efficiently convert astrocytes into neurons in the stroke area.

[0204] We then assessed the identity of astrocyte-converted neurons in the stroke area. Astrocyte-converted neurons in the motor cortex were confirmed to be immunopositive for cortical pyramidal neuron markers, including Emx1, Tbr1, and Satb2 (Figures 3E and 3F). Only approximately 10% were immunopositive for GABAergic neuron markers, including parvalbumin and GABA (Figure 3F). Thus, after ectopic expression of NeuroD1 in the stroke area, astrocyte-converted neurons displayed a neuronal identity similar to that of their neighboring cortical neurons.

[0205] [Example 2] Reducing neuroinflammation after cell conversion Concomitant with the astrocyte-to-neuron conversion following NeuroD1 infection in the stroke area, we observed a significant reduction in GFAP signal in the stroke area (see Figure 5A). This is expected, as >70% of NeuroD1-infected reactive astrocytes are converted to neurons, with a corresponding decrease in GFAP signal. However, this also raises the question of whether astrocytes are depleted in the converted area. To answer this question, we performed a NeuroD1 AAV-heavily infected pro-infarct stroke area ( 17 days post-virus injection (dpi; 27 dps)), when astrocyte-to-neuron conversion is nearly complete. * ) was tested.

[0206] Although astrocytes were reduced in the NeuroD1-infected area, a significant number of astrocytes still remained (see Figure 5B). Surprisingly, not only was the number of astrocytes reduced, but the morphology of astrocytes also changed significantly in the NeuroD1-converted area. In the control group, astrocytes were enlarged and densely intermingled in the area surrounding the injury core (see Figure 5B, top panel). In the NeuroD1 group, most astrocytes had elongated processes, and only a few exhibited a hypertrophic morphology (see Figure 5B, bottom panel; and Figures 6A–6C). This suggests that the remaining astrocytes were less reactive in the NeuroD1-converted area.

[0207] Because GFAP labeled only the primary processes of astrocytes, we used GFAP-GFP transgenic mice to clarify the overall morphology of astrocytes. Similar to GFAP immunostaining, the use of GFAP-GFP mice also revealed fewer hypertrophic astrocytes in the NeuroD1 group (see Figure 5C). Notably, a large number of GFP-labeled astrocytes were observed in the NeuroD1-mCherry-expressing region (see Figure 5C, bottom panel), suggesting that astrocytes were not depleted after conversion. This is in stark contrast to methods that kill astrocytes. The persistence of astrocytes during in vivo cell conversion is likely due to the fact that astrocytes have the inherent ability to divide and regenerate themselves.

[0208] If 70-95% of NeuroD1-infected reactive astrocytes were converted into neurons at the stroke site, such a significant reduction in reactive astrocytes would be expected to have a significant impact on the local environment due to the close relationship between astrocytes and neurons as well as between astrocytes and microglia. Indeed, microglial cell-mediated neuroinflammation is closely related to reactive astrocytes after stroke injury.

[0209] In the control group, a large number of microglia (marked by Iba1) were observed in the injury core at 17 dpi (see Figure 5D, upper panel). However, in the NeuroD1 group, microglia showed a significant reduction (see Figure 5D, lower panel), supporting the interaction between microglia and astrocytes. Morphologically, microglia in the control group were mostly amoeboid (see 5E, upper panel), whereas in the NeuroD1 group, microglia mainly exhibited a branched shape with obvious processes (Figure 5E, lower panel; quantified in Figure 6D; also see Figures 6A-B for 7 dpi and 40 dpi signals, respectively).

[0210] Concomitant with these morphological changes in glia, glial secretory proteins, such as chondroitin sulfate proteoglycans (CSPGs) and lipocalin-2 (LCN2), were significantly reduced in the NeuroD1 group compared with the control group (see Figures 5F and 5G, quantified in Figures 6E-F). CSPGs are key players in the formation of the glial scar, which inhibits axon regeneration, and LCN2 is highly expressed in reactive glial cells and is associated with neuroinflammation.

[0211] To confirm the immunostaining results, we performed RT-PCR experiments at 17 dpi to quantitatively analyze glial markers and cytokines in control or NeuroD1-infected cortical tissues. First, we confirmed that NeuroD1 was indeed highly expressed in the NeuroD1-injected samples (see Figure 5H). GFAP transcription levels increased approximately 50-fold after stroke but significantly decreased after NeuroD1 treatment (see Figure 5H). The neuroinflammation-related factor LCN2 dramatically increased approximately 1600-fold after stroke but decreased only 15-fold in the NeuroD1-treated group (see Figure 5G).

[0212] Similarly, inflammatory factors, including interferon-γ, TNF-α, interleukin-1β (IL-β), and interleukin-6 (IL-6), all showed increases after stroke but were downregulated after NeuroD1 treatment (see Figure 5I). The unexpected reduction in neuroinflammation after NeuroD1 treatment suggests that the conversion of reactive astrocytes into neurons has far broader effects than simply generating new neurons.

[0213] [Example 3] Neuroregeneration leads to neuroprotection Consistent with the reduction in neuroinflammation, the number of NeuN-positive neurons appeared to be significantly increased in the NeuroD1-infected area (see Figure 7A). Surprisingly, in the stroke area close to the injury core, not only were NeuroD1-GFP-labeled neurons detected, but also a large number of non-transformed neurons that were neither GFP-positive (see arrows in Figure 7A) nor NeuroD1-positive (see arrows in Figure 7B).

[0214] Quantitative analysis revealed that approximately 40% of NeuN+ neurons in the peri-infarct region were NeuroD1 positive, and approximately 60% of neurons were NeuroD1 negative (see Figure 7C ; control group NeuN+, 27.1 ± 8.1 / 0.1 mm 2、 NeuroD1 group: NeuroD1- / NeuN+, 64.9, ±7.9 / 0.1mm 2, NeuroD1+ / NeuN+, 39.9, ±2.4 / 0.1mm 2 , n = 3 mice per group).

[0215] Similar results were obtained when quantifying GFP / NeuN neurons in NeuroD1-NeuroD1-GFP-infected neurons (see Figure 8A). Furthermore, non-converted neurons in NeuroD1-treated areas were more than twice as numerous as those in control GFP-infected areas (see Figure 7C), suggesting that NeuroD1-mediated glial-to-neuronal conversion significantly enhances neuronal survival.

[0216] Overall, the number of NeuN+ neurons in the NeuroD1-infected area was more than three times higher than that in the control group. In line with the significant increase in neuron number after NeuroD1 treatment, immunostaining for neuronal dendritic markers, including microtubule-associated protein 2 (Map2) and SMI32, revealed distinct dendritic morphologies in the NeuroD1 group but not in the GFP control group (Figures 7D and 7E; see Figure 8C for Map2 quantification and Figure 8B for Map2 staining at 40 dpi).

[0217] Similarly, using the axon markers SMI312 and NF200 and the myelination marker MBP, more axons were observed in the stroke area in the NeuroD1 group (see Figure 7F, quantified in Figure 8D), which was associated with higher levels of myelination (see Figure 7G, quantified in Figure 8E). To confirm the immunostaining results, RT-PCR experiments were performed to examine the expression levels of neuronal genes. Neuronal genes, including NeuN, Robo2, and Syn1, all showed significant decreases after stroke but were rescued by NeuroD1 treatment (see Figure 7H).

[0218] Finally, we quantified the total number of NeuN-positive neurons within the stroke-damaged cortical region (500 µm–2500 µm from the midline, 1500 µm from the midline) from 2 weeks to 2 months after viral infection. The total number of NeuN-positive neurons in the post-stroke control group was only approximately 20% of that in non-stroke brains, indicating 80% neuronal loss in a severe focal stroke model. However, the number of neurons in the NeuroD1 group increased significantly over 2 months, with approximately 70% of the total neurons in the stroke region being rescued (see Figure 7I).

[0219] This significant increase in the number of cortical neurons after in vivo cell conversion, including both converted and protected neurons, forms a solid basis for functional recovery.

[0220] [Example 4] Reconstruction of functional neural circuits after stroke One of the crucial questions regarding in vivo cell conversion is whether glial-converted neurons can form functional neural circuits. To answer this question, we examined the morphological structure in the stroke area after NeuroD1-mediated cell conversion. After stroke and viral injection, we examined the ischemic-injured motor cortex at 7, 17, 40, and 60 dpi in both control and NeuroD1-treated mice (see Figure 9A).

[0221] Over a 2-month time course, gradual loss of cortical tissue was observed in the group injected with the GFP control virus, whereas the NeuroD1-treated group showed significant tissue preservation (see Figure 9A). Analysis of motor cortex tissue revealed that the control group lost 70% of the damaged tissue over the 2-month period, whereas the NeuroD1-treated group preserved most of the damaged tissue, with only 20% tissue loss (see Figure 9B; also see Figure 10A for serial sections).

[0222] Notably, 60 days after NeuroD1 infection, clear cortical layer structures were observed in all treated animals (see Figure 9A , 60 dpi, NeuroD1 image), suggesting that NeuroD1 treatment could not only generate new neurons but also reconstruct / preserve cortical layers.

[0223] H&E (hematoxylin and eosin) staining (see Figure 10B) was also performed at 60 dpi. In the control group, in addition to tissue damage, many neurons had eosinophilic cytoplasm and pyknotic nuclei and appeared shrunken, whereas in the NeuroD1 group, neurons showed a very healthy morphology. Furthermore, in coronal sections, striatal axon bundles derived from NeuroD1-converted neurons in the stroke area were detected. To trace the projection locations of NeuroD1-infected neurons in the stroke area after in vivo cell conversion, we performed sagittal section analysis and determined that NeuroD1-converted cortical neurons could send axons ipsilaterally to the striatum, thalamus, and hypothalamus (see Figure 9C). On the contralateral side, NeuroD1-converted neurons projected their axons across the midline via the corpus callosum to the contralateral cortical area (see Figure 10C). Thus, NeuroD1-converted neurons can be integrated into global brain circuits.

[0224] We also investigated neuronal function in the stroke area after NeuroD1-mediated cell transformation. Whole-cell patch-clamp recordings were performed on AAV-NeuroD1-GFP-infected neurons in cortical slices 2 months after viral injection (see Figure 9D). Injection of depolarizing current into neurons evoked repetitive action potentials (see Figure 9D).

[0225] We recorded robust spontaneous synaptic events (both excitatory and inhibitory) in NeuroD1-expressing neurons after stroke (see Figure 9F), suggesting that these neurons were integrated into brain circuits through synaptic connections with other neurons. Quantitative comparison between the control and NeuroD1 groups demonstrated that the frequencies of both EPSCs and IPSCs in the NeuroD1 group were significantly higher than those in the control group (see Figure 9G).

[0226] To confirm that the recorded neurons were NeuroD1-converted rather than pre-existing neurons, biocytin was included in the recording pipette and immunostaining was performed after electrophysiological recording. Figure 9E demonstrates that the recorded neurons (biocytin-positive) were indeed NeuroD1-positive. Biocytin immunostaining also revealed the complex dendritic structure of NeuroD1-converted neurons within the motor cortex (see Figure 9E).

[0227] Distinct dendritic spines were observed in biocytin-labeled neurons transformed with NeuroD1 in the stroke area (Figure 12A), which was confirmed by Golgi staining (Figure 12B). Further immunostaining with the glutamatergic synaptic marker VGluT1 (vesicular glutamate transporter 1) revealed a significant increase in synaptic density in the NeuroD1 group compared with the control group (Figures 12C-D).

[0228] Furthermore, GABAergic neurons were observed by immunostaining for parvalbumin (PV, a specific marker for the GABAergic neuron subtype). Although ischemic stroke caused a significant loss of PV neurons (see Figure 12F), in the NeuroD1-treated group, numerous PV neurons were detected in the stroke area (see Figure 12E, quantified in Figure 12F). Although PV neurons typically did not colocalize with NeuroD1-GFP cells, they were intermingled with NeuroD1-GFP-expressing neurons (see Figure 12E), suggesting that GABAergic neurons may have been protected by NeuroD1 treatment.

[0229] Taken together, the data demonstrate that NeuroD1-mediated cellular transformation can reconstitute functional neural circuits in the stroke area through both the regeneration and preservation of excitatory and inhibitory neurons.

[0230] [Example 5] Rescue of the vascular and blood-brain barriers after cell conversion If NeuroD1 treatment can rescue brain tissue loss and form neural circuits in the stroke area, could it restore blood vessels and the blood-brain barrier after cell conversion? To answer this question, we examined blood vessels and astrocyte endfeet, which contact blood vessels and form the blood-brain barrier (BBB) ​​in the stroke area (see Figures 11A-11H). In normal brains without stroke, aquaporin 4 (AQP4, a water channel), a marker of astrocyte endfeet wrapping around blood vessels (labeled by Ly6C, an endothelial cell and monocyte marker), indicated an intact BBB (see Figure 11A, top). Conversely, 1 week after ET-1(1-31)-induced focal stroke, significant disruption of both blood vessels and astrocyte endfeet was observed (see Figure 11A, bottom).

[0231] AQP4-labeled astrocyte endfeet were detached from blood vessels and colocalized with GFAP (Figure 11A, bottom panel), suggesting mislocalization of AQP4 from endfeet to other regions of astrocytes. After NeuroD1 treatment, reassociation of AQP4-labeled astrocyte endfeet was observed in the stroke area, along with significant recovery of blood vessels (Figures 11B-C, 17 dpi and 27 dps).

[0232] Co-immunostaining for AQP4 and GFAP revealed that in the control group, AQP4 was diffusely distributed throughout astrocytic processes, whereas in the NeuroD1-treated group, AQP4 was largely concentrated in the endfeet surrounding blood vessels (Fig. 11D). Rearrangement of astrocytic endfeet around blood vessels in the NeuroD1 group was also clearly demonstrated by co-immunostaining for CD31 (vascular) and GFAP (Fig. 11C-E).

[0233] Quantitative analysis revealed a significant decrease in both AQP4 intensity (see Figure ​ Figure6F)11F and AQP4 coverage area (see Figure ​ Figure6G11G)11 in the NeuroD1-treated group compared with the GFP control group. Even in the NeuroD1 group, AQP4 signals appeared to be more associated with blood vessels in areas where NeuroD1 was highly expressed than in areas with low NeuroD1 signals (see Figure ​ Figure6G12G).

[0234] Blood vessels showed a significant increase in the NeuroD1 group compared with the control group (see Figure 11H). To examine the integrity of the BBB (blood-brain barrier), a biotin perfusion test was used to detect BBB leakage. In the peri-infarct area, biotin showed a strong signal outside of blood vessels (labeled with Ly6C) in the control group, indicating BBB leakage (see Figure 12H).

[0235] In the NeuroD1 group, biotin signals remained primarily within the blood vessels (see Figure 12H). Even two months after virus injection, the differences in AQP4 and Ly6C signals were still highly significant between the control and NeuroD1 groups (see Figure 12I). Taken together, this suggests that NeuroD1-mediated cell transformation promotes angiogenesis in the stroke area and restores the BBB after stroke injury.

[0236] [Example 6] Functional rescue of motor and memory disorders Considering the significant level of neuronal regeneration in the stroke area after NeuroD1 treatment, we conducted experiments to determine whether this treatment could rescue the functional impairment caused by stroke. After preliminary testing on various animal behavioral tests, detailed analysis of mouse forelimb function was performed using three tests: the food pellet retrieval test, the grid walking test, and the cylinder test (see Figure 13A, behavioral test paradigm).

[0237] For all behavioral tests, following the food pellet retrieval test, ET-1(1-31) was injected into two points on one side of the cortex (one in the forelimb motor cortex and one in the forelimb sensory cortex) to produce a severe unilateral stroke on the dominant side. To test food pellet retrieval, mice were fasted before testing to enhance their motivation for food. Before the stroke, normal animals were trained to retrieve an average of 5–6 pellets out of a total of 8 in 5 min (see Figure 14A for the pellet retrieval apparatus).

[0238] Nine days after the stroke, their pellet retrieval ability was significantly impaired (reduced to an average of approximately one pellet per 5 minutes) (see Figure 13B). The stroked animals were then randomly divided into two groups with similar deficits for injection with AAV9 expressing either GFP alone or NeuroD1-GFP, and their functional recovery was monitored over a two-month period. Ten days after the virus injection (day 20 after the stroke), there was no significant difference between the two groups; however, 20 days after the virus injection, the NeuroD1 group began to show improvement. By day 60 after the virus injection, the NeuroD1 group reached approximately four pellets per 5 minutes, while the GFP control group remained at less than two pellets per 5 minutes (see Figure 13B).

[0239] Similarly, in the grid walking test, normal animals had a low pre-stroke limb disability rate, typically about 5% within 5 min of free walking on the grid, but this increased to >10% after stroke (see Figure 13C). After viral injection, the NeuroD1 group consistently improved at 20 and 40 days after treatment, with the limb disability rate decreasing to about 7%, whereas the GFP control group maintained a high limb disability rate of >9% (see Figure 13C).

[0240] We also performed a cylinder test to assess forelimb function, in which animals reared and touched the side wall of the cylinder. Typically, normal animals use both forelimbs to steadily touch and push against the side wall, with a normal touch rate of approximately 85%. After a unilateral stroke in the forelimb motor cortex, the function of the contralateral forelimb was significantly weakened, and the affected limb often either failed to touch the side wall or briefly touched it before being dragged along the wall (see Figure 13D, where the normal touch rate was reduced to approximately 35%).

[0241] Forty to sixty days after viral infection, the NeuroD1 group showed significant recovery (approximately 65%) in touching the lateral wall with both forelimbs, whereas the majority of the GFP control mice remained weak and unable to use their injured forelimbs (see Figure 13D). In all three tests, injection of PBS as a sham control for ET-1(1-31) did not result in any behavioral impairment, and injection of ET-1(1-31) without virus resulted in impairments similar to those of the GFP control virus.

[0242] In the grid walking test and cylinder test, the non-stroke-affected forelimb showed no significant impairment compared to control animals without stroke. In the food pellet retrieval test, only the forelimb preferentially used to retrieve food pellets during the training session was damaged by stroke and tested. After completing the behavioral tests approximately 2 months after virus injection, mice were sacrificed and subjected to anatomical and immunocytochemical analyses. Consistent with the behavioral impairment, the brains of the GFP control group showed significant cavities in the stroke area, whereas the NeuroD1 group showed minimal damage at the stroke site (see Figure 14B).

[0243] Immunostaining results after behavioral testing also showed significant tissue loss in the control group, but better preserved cortex in the NeuroD1 group (see Figure 14C, quantified in Figure 14D). Thus, using three different mouse behavioral models, we demonstrate that NeuroD1 treatment not only regenerates new neurons and reduces brain tissue loss, but also rescues motor dysfunction induced by ischemic stroke.

[0244] Widespread effects of NeuroD1-mediated cellular transformation in vivo In the stroke area after NeuroD1-mediated glial-to-neuronal conversion in vivo, both glial morphology and function were significantly improved in the NeuroD1-infected area. The significant number of astrocytes after neuronal conversion is consistent with the proliferative capacity of reactive astrocytes. The improvement from hypertrophy to normal branched astroglial morphology in the NeuroD1-expressing area suggests that reactive glial cells are exposed to fewer injury signals, that newly generated astrocytes are less reactive, or both.

[0245] The change in microglial morphology from an amoeboid to a more branched shape in the stroke area after NeuroD1 treatment was consistent with a reduction in neuroinflammation, confirmed by a reduction in inflammatory factors, TNFα and IL-1β. Previous studies have reported a significant upregulation of CSPGs and LCN2 in the stroke area. As demonstrated herein, NeuroD1 treatment significantly reduced the levels of CSPGs and LCN2 in the stroke area, consistent with a significant reduction in reactive glial cells after conversion.

[0246] Following nerve regeneration, NeuroD1 treatment results in vascular repair and restoration of the BBB in the stroke area. This type of repair following nerve regeneration is consistent with neurovascular interactions during brain development and nerve repair. Notably, NeuroD1-mediated in vivo cellular transformation has widespread effects on local neuron-glia circuits, including the generation of large numbers of new neurons, a corresponding reduction in reactive astrocytes, reduced neuroinflammation, vascular repair, and restoration of the BBB. Such changes in the behavior of the entire neuron-glia circuit are unprecedented.

[0247] Structural support for functional rescue In vivo glia-to-neuron conversion can also functionally rescue motor deficits caused by ischemic stroke in rodent models. This functional rescue is based on structural changes, including both cellular conversion and circuit reorganization, after NeuroD1 treatment. A mixture of NeuroD1-GFP-labeled and unlabeled neurons was observed in the stroke area after NeuroD1 treatment. This suggests that the restored brain circuitry is an integration of both newly generated neurons and older, mature neurons.

[0248] Although NeuroD1-converted neurons were primarily glutamatergic, a significant number of GABAergic neurons not labeled by NeuroD1-GFP were observed. This suggests that GABAergic neurons are present among the neurons rescued by NeuroD1 treatment. The preservation of GABAergic neurons suggests that NeuroD1 treatment can maintain the excitation-inhibition balance after cell conversion in vivo, which is important for achieving normal brain function. Functional recovery is also achieved because NeuroD1 treatment can rescue over 70% of neurons in the stroke-damaged motor cortex.

[0249] item Item 1. A method of treating the effects of disruption of normal blood flow in the CNS of an individual subject in need thereof, comprising administering a therapeutically effective dose of exogenous NeuroD1 to the area where normal blood flow is disrupted.

[0250] Item 2. The method of item 1, wherein administering exogenous NeuroD1 comprises delivering to the region an expression vector comprising a nucleic acid encoding NeuroD1.

[0251] Item 3. The method of items 1 or 2, wherein administering exogenous NeuroD1 comprises delivering to the region a recombinant viral expression vector comprising a nucleic acid encoding NeuroD1.

[0252] Item 4. The method of any one of items 1 to 3, wherein expressing exogenous NeuroD1 comprises delivering to the region a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding NeuroD1.

[0253] Item 5. The method of any of items 1 to 4, wherein expressing exogenous NeuroD1 comprises delivering to the region an effective "flip-excision" recombinant expression vector combination of 1) a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding NeuroD1, and 2) a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a site-specific recombinase.

[0254] Item 6. The method of any of items 1 to 5, wherein NeuroD1 is the only exogenously expressed transcription factor delivered to the region.

[0255] Item 7. The method of any of items 1 to 6, wherein the exogenous NeuroD1 is administered once after normal blood flow in the CNS is disrupted if reactive astrocytes are present.

[0256] Item 8. The method of any of items 1 to 7, wherein the exogenous NeuroD1 is administered once after normal blood flow in the CNS is disrupted when a glial scar is present.

[0257] Item 9. The method of any of items 1 to 8, wherein the disruption of normal blood flow in the CNS is due to a disorder selected from the group consisting of ischemia, thrombosis, embolism, hemorrhage, chronic disease-mediated restriction of blood vessels, and a combination of any two or more thereof.

[0258] Item 10. The method of any of items 1 to 9, wherein the disruption of normal blood flow in the CNS is due to a disorder selected from the group consisting of ischemic stroke; hemorrhagic stroke; cerebral aneurysm; concussion, tumor, infection, inflammation, traumatic brain injury, traumatic spinal cord injury; ischemic or hemorrhagic myelopathy (spinal cord infarction); global cerebral ischemia caused by cardiac arrest or severe low blood pressure (shock); hypoxic-ischemic encephalopathy caused by hypoxia, hypoglycemia, or anemia; CNS embolism caused by infective endocarditis or atrial myxoma; fibrocartilaginous embolic myelopathy; CNS thrombosis caused by childhood leukemia; cerebral venous sinus thrombosis caused by nephrotic syndrome (kidney disease), chronic inflammatory disease, pregnancy, use of estrogen-based contraceptives, meningitis, dehydration, or a combination of any two or more thereof.

[0259] Item 11. The method of any one of items 1 to 10, wherein administering a therapeutically effective dose of NeuroD1 comprises administering a recombinant expression vector comprising a nucleic acid sequence encoding a NeuroD1 protein, wherein the nucleic acid sequence encoding the NeuroD1 protein comprises a nucleic acid sequence selected from the group consisting of a nucleic acid sequence encoding SEQ ID NO:2 or a functional fragment thereof, a nucleic acid sequence encoding SEQ ID NO:4 or a functional fragment thereof, SEQ ID NO:1 or a functional fragment thereof, SEQ ID NO:3 or a functional fragment thereof, and a nucleic acid sequence encoding a protein or functional fragment thereof having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:2 or SEQ ID NO:4.

[0260] Item 12. The method of any of items 1 to 11, wherein administering a therapeutically effective dose of NeuroD1 comprises stereotactic injection at or near the site of injury.

[0261] Item 13. The method of any one of items 1 to 12, wherein the nucleic acid sequence encoding NeuroD1 is operably linked to a ubiquitous promoter.

[0262] Item 14. The method of any of items 5 to 12, wherein the nucleic acid sequence encoding the site-specific recombinase is operably linked to a glial cell-specific promoter.

[0263] Item 15. The method of item 14, wherein the glial cell-specific promoter is a GFAP promoter.

[0264] Item 16. The method of item 15, wherein the GFAP promoter is a human GFAP promoter.

[0265] Item 17. The method of any of items 1 to 16, further comprising evaluating the effectiveness of the treatment in the subject.

[0266] Item 18. The method of item 17, wherein assessing the effectiveness of treatment in the subject comprises an assay selected from an electrophysiological assay, a blood flow assay, a tissue structure assay, a functional assay, and a combination of any two or more thereof.

[0267] Item 19. The method of item 17, wherein assessing the effectiveness of treatment in the subject comprises electroencephalography.

[0268] Item 20. The method of item 17, wherein assessing the effectiveness of treatment in the subject comprises an assay of blood flow selected from the group consisting of near-infrared spectroscopy and fMRI.

[0269] Item 21. The method of item 17, wherein assessing the effectiveness of treatment in the subject comprises a histological assay selected from the group consisting of MRI, PET scan, CAT scan, and ultrasound.

[0270] Item 22. The method of item 17, wherein assessing the effectiveness of the treatment in the subject comprises a behavioral assay.

[0271] Item 23. The method of item 17, wherein assessing the effectiveness of the treatment in the subject comprises an assay performed before administering a therapeutically effective dose of exogenous NeuroD1.

[0272] Item 24. In areas where normal blood flow has been disrupted, 10 adeno-associated virus particles containing nucleic acid encoding NeuroD1 are administered. 10 ~10 14 24. The method of any one of items 1 to 23, comprising administering to a subject 1 to 500 μl of a pharmaceutically acceptable carrier containing the adeno-associated virus particles at a concentration of 1 to 500 μl of the adeno-associated virus particles / 1 ml of the carrier at a controlled flow rate of 0.1 to 5 μl / min.

[0273] Item 25. The method of any one of items 1 to 24, further comprising a treatment selected from the group consisting of removing a blood clot, promoting blood flow, administering an anti-inflammatory agent, administering an antioxidant, reducing excitotoxicity, and two or more thereof.

[0274] Item 26. A composition comprising: 1) a recombinant adeno-associated adenovirus expression vector comprising a glial cell-specific promoter operably linked to a nucleic acid encoding a site-specific recombinase; and 2) a recombinant adeno-associated adenovirus expression vector comprising a ubiquitous promoter operably linked to a nucleic acid encoding NeuroD1, wherein the nucleic acid encoding NeuroD1 is inverted and flanked by two sets of site-specific recombinase recognition sites, whereby action of the recombinase irreversibly inverts the nucleic acid encoding NeuroD1 such that NeuroD1 is expressed in mammalian cells.

[0275] Item 27. A method of treating the effects of disruption of normal blood flow in the CNS of an individual subject in need thereof, substantially as described herein.

[0276] Item 28. A flip-excision expression vector combination substantially as described herein.

[0277] array SEQ ID NO:1 - Human NeuroD1 nucleic acid sequence encoding human NeuroD1 protein - 1071 nucleotides including the stop codon TIFF2025183293000002.tif121165

[0278] SEQ ID NO:2 - Human NeuroD1 amino acid sequence - 356 amino acids - encoded by SEQ ID NO:1 TIFF2025183293000003.tif42165

[0279] SEQ ID NO:3 - Mouse NeuroD1 nucleic acid sequence encoding mouse NeuroD1 protein - 1074 nucleotides including the stop codon TIFF2025183293000004.tif120165

[0280] SEQ ID NO:4 - Mouse NeuroD1 amino acid sequence - 357 amino acids - encoded by SEQ ID NO:3 TIFF2025183293000005.tif42165

[0281] Mouse LCN2 promoter - SEQ ID NO:5 TIFF2025183293000006.tif120164

[0282] Human GFAP promoter - SEQ ID NO: 6 TIFF2025183293000007.tif194164

[0283] Mouse Aldh1L1 promoter - SEQ ID NO: 7 TIFF2025183293000008.tif186165

[0284] Human NG2 promoter - SEQ ID NO:8 TIFF2025183293000009.tif158164

[0285] CAG::NeuroD1-IRES-GFP-SEQ ID NO:9 TIFF2025183293000010.tif244164TIFF2025183293000011.tif252161TIFF2025183293 000012.tif252162TIFF2025183293000013.tif254162TIFF2025183293000014.tif20165

[0286] Any patent or publication mentioned in this specification is herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. The compositions and methods described herein are presently representative of preferred embodiments and are not intended as limitations on the scope of the invention. Modifications and other uses will occur to those skilled in the art. Such modifications and other uses can be made without departing from the scope of the invention, as set forth in the claims.

Claims

1. A method for treating the effects of disruption of normal blood flow in the CNS of an individual subject in need thereof, comprising administering a therapeutically effective dose of exogenous NeuroD1 to the area where normal blood flow is disrupted.

2. 2. The method of claim 1, wherein administering exogenous NeuroD1 comprises delivering to the region an expression vector comprising a nucleic acid encoding NeuroD1.

3. 3. The method of claim 1 or 2, wherein administering exogenous NeuroD1 comprises delivering to the region a recombinant viral expression vector comprising a nucleic acid encoding NeuroD1.

4. 4. The method of any one of claims 1 to 3, wherein expressing exogenous NeuroD1 comprises delivering to the region a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding NeuroD1.

5. 5. The method of any one of claims 1 to 4, wherein expressing exogenous NeuroD1 comprises delivering to the region an effective "flip-excision" recombinant expression vector combination of 1) a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding NeuroD1, and 2) a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a site-specific recombinase.

6. 6. The method of any one of claims 1 to 5, wherein NeuroD1 is the only exogenously expressed transcription factor delivered to the region.

7. 7. The method of any one of claims 1 to 6, wherein the exogenous NeuroD1 is administered once after normal blood flow in the CNS has been disrupted if reactive astrocytes are present.

8. 8. The method of any one of claims 1 to 7, wherein exogenous NeuroD1 is administered once after normal blood flow in the CNS is disrupted when glial cells are reactive.

9. 9. The method of any one of claims 1 to 8, wherein the disruption of normal blood flow in the CNS is due to a disorder selected from the group consisting of ischemia, thrombosis, embolism, hemorrhage, concussion, blast, brain invasion, tumor, inflammation, infection, chronic disease-mediated restriction of blood vessels, and a combination of any two or more thereof.

10. 10. The method of any one of claims 1 to 9, wherein the disruption of normal blood flow in the CNS is due to a disorder selected from the group consisting of ischemic stroke; hemorrhagic stroke; cerebral aneurysm; traumatic brain injury; concussion; blast; brain invasion; tumor; inflammation; infection; traumatic spinal cord injury; ischemic or hemorrhagic myelopathy (spinal cord infarction); global cerebral ischemia caused by cardiac arrest or severe low blood pressure (shock); hypoxic-ischemic encephalopathy caused by hypoxia, hypoglycemia, or anemia; CNS embolism caused by infective endocarditis or atrial myxoma; fibrocartilaginous embolic myelopathy; CNS thrombosis caused by childhood leukemia; cerebral venous sinus thrombosis caused by nephrotic syndrome (kidney disease), chronic inflammatory disease, pregnancy, use of estrogen-based contraceptives, meningitis, dehydration; or a combination of any two or more thereof.

11. 11. The method of claim 1, wherein administering a therapeutically effective dose of NeuroD1 comprises administering a recombinant expression vector comprising a nucleic acid sequence encoding a NeuroD1 protein, wherein the nucleic acid sequence encoding the NeuroD1 protein comprises a nucleic acid sequence selected from the group consisting of a nucleic acid sequence encoding SEQ ID NO:2 or a functional fragment thereof, a nucleic acid sequence encoding SEQ ID NO:4 or a functional fragment thereof, SEQ ID NO:1 or a functional fragment thereof, SEQ ID NO:3 or a functional fragment thereof, and a nucleic acid sequence encoding a protein or functional fragment thereof having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:2 or SEQ ID NO:

4.

12. 12. The method of any one of claims 1 to 11, wherein administering a therapeutically effective dose of NeuroD1 comprises stereotactic injection in or near the area of ​​injury.

13. The method of any one of claims 1 to 12, wherein the nucleic acid sequence encoding NeuroD1 is operably linked to a ubiquitous promoter.

14. 13. The method of any one of claims 5 to 12, wherein the nucleic acid sequence encoding the site-specific recombinase is operably linked to a glial cell-specific promoter.

15. The method of claim 14, wherein the glial cell-specific promoter is a GFAP promoter.

16. The method of claim 15, wherein the GFAP promoter is a human GFAP promoter.

17. 17. The method of any one of claims 1 to 16, further comprising assessing the effectiveness of the treatment in the subject.

18. 18. The method of claim 17, wherein assessing the effectiveness of treatment in a subject comprises an assay selected from an electrophysiological assay, a blood flow assay, a tissue structure assay, a functional assay, and a combination of any two or more thereof.

19. 18. The method of claim 17, wherein assessing the effectiveness of treatment in the subject comprises electroencephalography.

20. 18. The method of claim 17, wherein assessing the effectiveness of treatment in the subject comprises an assay of blood flow selected from the group consisting of near-infrared spectroscopy and fMRI.

21. 18. The method of claim 17, wherein assessing the effectiveness of treatment in a subject comprises a histological assay selected from the group consisting of MRI, PET scan, CAT scan, and ultrasound.

22. 18. The method of claim 17, wherein assessing the effectiveness of the treatment in the subject comprises a behavioral assay.

23. 18. The method of claim 17, wherein assessing the effectiveness of treatment in the subject comprises an assay performed before administering a therapeutically effective dose of exogenous NeuroD1.

24. Adeno-associated virus particles containing nucleic acid encoding NeuroD1 were 10 ~10 14 24. The method of any one of claims 1 to 23, wherein 1 to 500 μl of a pharmaceutically acceptable carrier containing a concentration of adeno-associated virus particles / ml of carrier is injected into the subject at a controlled flow rate of 0.1 to 5 μl / min in the area where normal blood flow has been disrupted.

25. 25. The method of any one of claims 1 to 24, further comprising a treatment selected from the group consisting of removing a blood clot, promoting blood flow, administering an anti-inflammatory agent, administering an antioxidant, reducing excitotoxicity, and two or more thereof.

26. A composition comprising: 1) a recombinant adeno-associated adenovirus expression vector comprising a glial cell-specific promoter operably linked to a nucleic acid encoding a site-specific recombinase; and 2) a recombinant adeno-associated adenovirus expression vector comprising a ubiquitous promoter operably linked to a nucleic acid encoding NeuroD1, wherein the nucleic acid encoding NeuroD1 is inverted and flanked by two sets of site-specific recombinase recognition sites, such that the action of the recombinase irreversibly inverts the nucleic acid encoding NeuroD1 such that NeuroD1 is expressed in mammalian cells.

27. A method of treating the effects of disruption of normal blood flow in the CNS of an individual subject in need thereof substantially as described herein.

28. A flip-excision expression vector combination substantially as described herein.

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