Regenerating functional neurons for treatment of hemorrhagic stroke

Abstract

To provide methods and materials involved in treating mammals having had a hemorrhagic stroke.SOLUTION: For example, methods and materials for administering a composition containing exogenous nucleic acid encoding a NeuroD1 polypeptide and exogenous nucleic acid encoding a Dlx2 polypeptide to a mammal having had a hemorrhagic stroke are provided.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Patent Application No. 62 / 916,706, filed October 17, 2019. The disclosure of the prior application is considered part of (and is incorporated by reference into) the disclosure of this application.

[0002] The present specification relates to methods and materials for treating a mammal that has suffered a hemorrhagic stroke. For example, the present specification provides methods and materials for administering a composition containing an exogenous nucleic acid encoding a NeuroD1 polypeptide (or a biologically active fragment thereof) and a nucleic acid encoding a Dlx2 polypeptide (or a biologically active fragment thereof) to a mammal that has suffered a hemorrhagic stroke. [Background technology]

[0003] Stroke is a disease affecting the arteries leading to and within the brain. It is the fifth leading cause of death and a leading cause of disability in the United States. Stroke occurs when blood vessels carrying oxygen and nutrients to the brain are blocked, either by a blood clot or a rupture (Bonnard et al., Stroke, 50:1318-1324 (2019)). This prevents parts of the brain from receiving the blood (and oxygen) they need, leading to the death of brain cells. Strokes can be caused either by a blood clot blocking blood flow to the brain (called an ischemic stroke) or by a blood vessel rupturing and obstructing blood flow to the brain (called a hemorrhagic stroke). TIAs (transient ischemic attacks), or "mini-strokes," are caused by temporary blood clots. Recent advances in neuroimaging, organized stroke care, dedicated neuro-ICUs, and medical and surgical management have improved the management of hemorrhagic strokes. However, there remains a significant unmet need for treatment of patients who have suffered a hemorrhagic stroke. Summary of the Invention

[0004] The present specification provides methods and materials involved in treating a mammal that has suffered a hemorrhagic stroke. For example, the present specification provides methods and materials for administering a composition containing an exogenous nucleic acid encoding a NeuroD1 polypeptide (or a biologically active fragment thereof) and a nucleic acid encoding a Dlx2 polypeptide (or a biologically active fragment thereof) to a mammal that has suffered a hemorrhagic stroke.

[0005] In general, one aspect of the present disclosure features a method for (1), (2), (3), or (4) in a mammal that has suffered a hemorrhagic stroke and is in need of (1) generating new glutamatergic neurons, (2) increasing the survival of GABAergic neurons, (3) generating new non-reactive astrocytes, or (4) reducing the number of reactive astrocytes. The method comprises (or consists essentially of, or consists of) administering to the mammal a composition comprising an exogenous nucleic acid encoding a neurogenic differentiation 1 (NeuroD1) polypeptide or a biologically active fragment thereof, and an exogenous nucleic acid encoding a distal-less homeobox 2 (Dlx2) polypeptide or a biologically active fragment thereof. The mammal may be a human. The hemorrhagic stroke may be caused by a condition selected from the group consisting of ischemic stroke, physical injury, tumor, inflammation, infection, global ischemia caused by cardiac arrest or severe hypotension (shock), hypoxic-ischemic encephalopathy caused by hypoxia, hypoglycemia, or anemia, meningitis, and dehydration, or a combination of any two or more thereof. The administering step may include delivering an expression vector containing a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof and an expression vector containing a nucleic acid encoding a Dlx2 polypeptide or a biologically active fragment thereof to the location of the hemorrhagic stroke in the brain. The administering step may include delivering a recombinant viral expression vector containing a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof and a recombinant viral expression vector containing a nucleic acid encoding a Dlx2 polypeptide or a biologically active fragment thereof to the location of the hemorrhagic stroke in the brain. The administering step may include delivering a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof, and a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a Dlx2 polypeptide or a biologically active fragment thereof, to the location of the hemorrhagic stroke in the brain. The administering step may include stereotactic intracranial injection into the location of the hemorrhagic stroke in the brain.The administering step may further comprise administering an exogenous nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof and an exogenous nucleic acid encoding a Dlx2 polypeptide or a biologically active fragment thereof in a single expression vector, a single recombinant viral expression vector, or a single recombinant adeno-associated viral expression vector. The composition comprises a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof and a nucleic acid encoding a Dlx2 polypeptide or a biologically active fragment thereof. 10 ~10 14 The composition may comprise about 1 μL to about 500 μL of a pharmaceutically acceptable carrier containing an adeno-associated virus at a concentration of adeno-associated virus particles / mL of carrier. The composition may be injected into the mammalian brain at a controlled flow rate of about 0.1 μL / min to about 5 μL / min.

[0006] In another aspect, the document features a method for (1), (2), (3), or (4) in a mammal that has suffered a hemorrhagic stroke and is in need of (1) generating new GABAergic and glutamatergic neurons, (2) increasing the survival of GABAergic and glutamatergic neurons, (3) generating new non-reactive astrocytes, or (4) reducing the number of reactive astrocytes. The method comprises (or consists essentially of, or consists of) administering to the mammal, within three days of the hemorrhagic stroke, a composition comprising an exogenous nucleic acid encoding a neurogenic differentiation 1 (NeuroD1) polypeptide or a biologically active fragment thereof, and an exogenous nucleic acid encoding a distal-less homeobox 2 (Dlx2) polypeptide or a biologically active fragment thereof. The mammal can be a human. Hemorrhagic stroke may be caused by bleeding in the brain; aneurysm; intracranial hematoma; subarachnoid hemorrhage; brain trauma; high blood pressure; weak blood vessels; vascular malformations; ischemic stroke; physical injury; tumor; inflammation; infection; generalized ischemia caused by cardiac arrest or severe hypotension (shock); hypoxic-ischemic encephalopathy caused by hypoxia, hypoglycemia, or anemia; meningitis; and dehydration; or any combination of two or more thereof. The administering step may include delivering an expression vector containing a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof and an expression vector containing a nucleic acid encoding a Dlx2 polypeptide or a biologically active fragment thereof to the location of the hemorrhagic stroke in the brain. The administering step may include delivering a recombinant viral expression vector containing a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof and a recombinant viral expression vector containing a nucleic acid encoding a Dlx2 polypeptide or a biologically active fragment thereof to the location of the hemorrhagic stroke in the brain.The administering step may include delivering a recombinant adeno-associated virus expression vector containing a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof and a recombinant adeno-associated virus expression vector containing a nucleic acid encoding a Dlx2 polypeptide or a biologically active fragment thereof to the site of hemorrhagic stroke in the brain. The administering step may include stereotactic intracranial injection to the site of hemorrhagic stroke in the brain. The administering step may further include administering an exogenous nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof and an exogenous nucleic acid encoding a Dlx2 polypeptide or a biologically active fragment thereof in one expression vector, one recombinant virus expression vector, or one recombinant adeno-associated virus expression vector. The composition includes a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof and a nucleic acid encoding a Dlx2 polypeptide or a biologically active fragment thereof. 10 ~10 14 The composition may comprise about 1 μL to about 500 μL of a pharmaceutically acceptable carrier containing an adeno-associated virus at a concentration of adeno-associated virus particles / mL of carrier. The composition may be injected into the mammalian brain at a controlled flow rate of about 0.1 μL / min to about 5 μL / min.

[0007] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains.Although the present invention can be practiced or tested using methods and materials similar or equivalent to those described herein, suitable methods and materials are described below.All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.In case of conflict, the present specification, including definitions, will control.In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0008] Other features and advantages of the invention will become apparent from the following detailed description and claims. [Brief explanation of the drawings]

[0009] [Figure 1A] Iron evolution in a collagenase-induced intracerebral hemorrhage (ICH) model. (Figure 1A) At 1 and 2 days poststroke (dps), very low levels of ferric iron were detected via iron staining, and microglia began to migrate into the hematoma as determined by DAB staining. (Figure 1B) At 8 and 29 dps, high levels of iron were detected via iron staining in the injury core, intermingled with microglia, and astrocytes formed a glial scar around the injury core as determined by DAB staining. These results suggest that therapy within 2 days poststroke may be preferable. [Figure 1B] Same as above. [Figure 2A]Conversion of astrocytes into neurons. Figure 2A is a schematic diagram showing the in vivo conversion of astrocytes into functional neurons in a collagenase-induced ICH model. Figure 2B shows the experimental design used to confirm the in vivo conversion of reactive astrocytes into neurons in an ICH model (intracerebral hemorrhage). ICH was induced by intrastriatal injection of 0.2 μL of collagenase. The control virus was AAV5-GFAP-Cre (3 × 10 11 , 1 μL) + AAV5-CAG-flex-GFP (3.4 × 10 11 , 1 μL), and the treatment viruses were AAV5-GFAP-Cre (3 × 10 11 , 1 μL) + AAV5-CAG-flex-ND1-GFP (4.55 × 10 11 , 1 μL) + AAV5-CAG-flex-Dlx2-GFP (2.36 × 10 12 , 1 μL). Figure 2C shows immunofluorescence staining for GFP, GFAP, and NeuN 21 days after infection (days postinfection, dpi) with ND1 and Dlx2 viruses injected at 0 dps. Mild ICH was observed. GFAP signals were downregulated in the lesion. Most GFP+ cells exhibited neuronal morphology. Figure 2D shows immunofluorescence staining for GFP, GFAP, and NeuN 21 days after infection with viruses engineered to express ND1 and Dlx2 injected at 0 dps. Numbers 1, 2, and 3 refer to three adjacent regions around the injury core. Most GFP+ cells expressed NeuN. (Figure 2E) 19 days after induction with control or treatment viruses at 2 dps, many GFP+ cells exhibited neuronal morphology on the treated side. Figure 2F shows immunofluorescence staining for GFP, GFAP, and NeuN 19 days after induction with viruses engineered to express ND1 and Dlx2 at 2 dps. Numbers 1, 2, and 3 refer to three nearby regions around the injury core. Many of the GFP+ cells expressed NeuN. (Figure 2G) At 17 days after induction with control or treatment viruses at 4 dps, fewer GFP+ cells displayed neuronal morphology in the treated side. Figure 2H shows immunofluorescence staining for GFP, GFAP, and NeuN at 17 days after induction with viruses designed to express ND1 and Dlx2 at 4 dps.Numbers 1, 2, and 3 refer to three adjacent regions surrounding the injury core. Some GFP+ neurons exhibited neuronal morphology, while others were astrocytes. (Figure 2I) 14 days after induction with control or treatment viruses at 7 dps, few GFP+ cells with neuronal morphology were observed. Figure 2J shows immunofluorescence staining for GFP, GFAP, and NeuN 14 days after induction with viruses designed to express ND1 and Dlx2 at 7 dps. Numbers 1, 2, and 3 refer to three adjacent regions surrounding the injury core. Almost all GFP+ cells remained in astrocyte morphology. Figure 2K shows immunofluorescence staining for GFP, GFAP, and NeuN in normal control, virus control, and treated mice treated with viruses designed to express ND1 and Dlx2 at 0, 2, 4, or 7 dps. Fewer GFP+ neurons, less neuronal density, and more reactive astrocytes were observed with delayed injection time points. The optimal time point should not exceed 2 dps. Figure 2L shows the disappearance of GFAP observed in both the treated and control groups. Figure 2M shows the disappearance of GFAP and NeuN signals 21 days after induction with the control virus. Figure 2N shows that S100b signals were present in the GFAP-deficient region in treated mice, but were absent in the same region in control mice. (Figure 2O) Nineteen days after induction with the control or treated virus at 2 dps, S100b signals appeared to be downregulated. Figure 2P shows the downregulation of S100b in the treated group, but the S100b signals still showed the morphology of reactive astrocytes in the control group. [Figure 2B] Same as above. [Figure 2C] Same as above. [Figure 2D] Same as above. [Figure 2E] Same as above. [Figure 2F] Same as above. [Figure 2G] Same as above. [Figure 2H] Same as above. [Figure 2I] Same as above. [Figure 2J] Same as above. [Figure 2K-1] Same as above. [Figure 2K-2] Same as above. [Figure 2L] Same as above. [Figure 2M] Same as above. [Figure 2N] Same as above. [Figure 2O] Same as above. [Figure 2P] Same as above. [Figure 3A]Long-term in vivo conversion of reactive astrocytes into neurons in ICH. Figure 3A shows the experimental design used to confirm the long-term conversion of reactive astrocytes into neurons in ICH. ICH was induced by intrastriatal injection of 0.35 μL of collagenase. The control virus was AAV5-GFAP-Cre (3 × 10 11 , 1 μL) + AAV5-CAG-flex-GFP (3.4 × 10 11 , 1 μL), and the treatment viruses were AAV5-GFAP-Cre (3 × 10 11 , 1 μL) + AAV5-CAG-flex-ND1-GFP (4.55 × 10 11 , 1 μL) + AAV5-CAG-flex-Dlx2-GFP (2.36 × 10 12 , 1 μL). Figure 3B shows immunofluorescence staining for GFP, GFAP, and NeuN 2 months after induction for mice treated with a virus designed to express ND1 and Dlx2 at 0 dps. Mild ICH was observed. Most GFP+ cells are neuronal. Figure 3C shows immunofluorescence staining for GFP, GFAP, and NeuN 2 months after induction for mice treated with a virus designed to express ND1 and Dlx2 at 0 dps. Almost all GFP+ cells expressed NeuN. Figure 3D shows immunofluorescence staining for GFP, GFAP, and NeuN 2 months after induction for mice treated with a virus designed to express ND1 and Dlx2 at 2 dps. Viral infection was not widespread, possibly due to infection being too close to the ventricles. Figure 3E shows immunofluorescence staining for GFP, GFAP, and NeuN 2 months after induction for mice treated with a virus designed to express ND1 and Dlx2 at 7 dps. Mild ICH was observed. Many GFP+ neuron-like cells were observed. Figure 3F shows immunofluorescence staining for GFP, GFAP, and NeuN 2 months after induction for mice treated with a virus designed to express ND1 and Dlx2 at 7 dps. A lower infection rate was observed than for mice treated with a control virus at 0 dps. Figure 3G shows immunofluorescence staining for GFP, GFAP, and NeuN 2 months after induction for mice treated with a control virus at 0 dps.Although many GFP+ cells remained astrocytes, some GFP+ neurons were observed. Figure 3H contains graphs plotting the conversion (or leakage) rate (%) (left graph) and neuronal density (cell count × 104 / mm3) (right graph) for mice treated as indicated. 2dps-2M data were excluded due to inefficient viral infection. 0dps-2M achieved the highest conversion rate (86%) and the highest neuronal density (147,000 / mm3). [Figure 3B] Same as above. [Figure 3C] Same as above. [Figure 3D] Same as above. [Figure 3E] Same as above. [Figure 3F] Same as above. [Figure 3G] Same as above. [Figure 3H] Same as above. [Figure 4A]AAV9-non-enriched 1.6kb-GFAP-cre / flex system. Figure 4A shows RFP staining 19 days after induction with control virus (AAV9-non-enriched 1.6kb-GFAP-Cre + AAV9-flex-mCherry, left) or treatment virus (AAV9-non-enriched 1.6kb-GFAP-Cre + AAV9-flex-ND1-mCherry + AAV9-flex-Dlx2-mCherry, right) at 2 dps. Stroke was induced with 0.2 μL (0.03 units) of collagenase in each case. Figure 4B shows immunofluorescence staining for NeuN, ND1, and RFP 19 days after induction with viruses designed to express ND1 and Dlx2 at 2 dps. Although not many neurons overexpressed ND1, ND1 signals were still detectable. Figure 4C shows immunofluorescence staining for NeuN, Dlx2, and RFP 19 days after induction with a virus designed to express ND1 and Dlx2 at 2 dps. Most neurons expressed Dlx2, while some showed no RFP signal. Figure 4D shows immunofluorescence staining for GFAP, RFP, and NeuN 19 days after induction with control or treated viruses at 2 dps. RFP signal was reduced in the treated group. High leakage was still present in the AAV9 non-concentrated Cre. Figure 4E shows immunofluorescence staining for Iba1 and RFP 19 days after induction with control or treated viruses at 2 dps. Microglia in the treated group appeared more reactive than those in the control group. Figure 4F shows immunofluorescence staining for AQP4 (aquaporin 4) and RFP 19 days after induction with control or treated viruses at 2 dps. No significant differences were observed in AQP4 staining between the control and treated groups. [Figure 4B] Same as above. [Figure 4C] Same as above. [Figure 4D] Same as above. [Figure 4E] Same as above. [Figure 4F] Same as above. [Figure 5A]AAV5-1.6kb-GFAP-cre / flex system. Figure 5A shows GFP staining 19 days after induction with control virus (AAV5-1.6kb-GFAP-Cre + AAV5-flex-GFP, left) or treatment virus (AAV5-1.6kb-GFAP-Cre + AAV5-flex-ND1-GFP + AAV5-flex-Dlx2-GFP, right) at 2 dps. In each case, stroke was induced with 0.2 μL (0.03 units) of collagenase. Figure 5B shows immunofluorescence staining for NeuN, GFP, ND1, and Dlx2 19 days after induction with viruses designed to express ND1 and Dlx2 at 2 dps. ND1 signal was not detected. Many neurons overexpressed Dlx2. Generally, the signal was weaker than that observed with AAV9. Figure 5C shows immunofluorescence staining for GFAP, GFP, and NeuN 19 days after induction with control or treated virus at 2 dps. Astrocytes in the treatment group appeared more reactive throughout the striatum. Astrocytes in the control group appeared more reactive only around the injury core. Figure 5D shows immunofluorescence staining for Iba1 and GFP 19 days after induction with control or treated virus at 2 dps. In the control group, reactive microglia were densely distributed in the injury core, whereas reactive microglia in the treatment group were also observed around the injury periphery. Figure 5E shows immunofluorescence staining for AQP4 and RFP 19 days after induction with control or treated virus at 2 dps. The AQP4 signal in the treatment group was potentially slightly stronger than that observed in the control group. [Figure 5B] Same as above. [Figure 5C] Same as above. [Figure 5D] Same as above. [Figure 5E] Same as above. [Figure 6A]Figure 6A shows GFP, GFAP, and NeuN staining 14 days after induction with control virus (AAV5-1.6kb-GFAP-Cre-5-flex-GFP) at 2 dps, induced with 0.5 μL (0.075 units) of collagenase. Figure 6B shows GFP, GFAP, and NeuN staining in mild stroke mice 14 days after induction with treatment virus (AAV5-1.6kb-GFAP-Cre-5-flex-ND1-GFP-5-flex-Dlx2-GFP) at 2 dps, induced with 0.5 μL (0.075 units) of collagenase. Figure 6C shows GFP, GFAP, and NeuN staining for a severe stroke 14 days after induction with treatment virus (AAV5-1.6kb-GFAP-Cre-5-flex-ND1-GFP-5-flex-Dlx2-GFP) at 2 dps, induced with 0.5 μL (0.075 units) of collagenase. Figure 6D shows GFP, GFAP, and NeuN staining for a mild stroke 2 months after induction with treatment virus (AAV5-0.6kb-GFAP-Cre+AAV5-flex-ND1-GFP+AAV5-flex-Dlx2-GFP) at 2 dps, induced with 0.5 μL (0.075 units) of collagenase. MRI images were performed at 1 dps. Figure 6E shows GFP, GFAP, and NeuN staining for a severe stroke 2 months after induction with treatment virus (AAV5-0.6kb-GFAP-Cre + AAV5-flex-ND1-GFP + AAV5-flex-Dlx2-GFP) induced with 0.5 μL (0.075 units) of collagenase at 2 dps. MRI images were performed at 1 dps. [Figure 6B] Same as above. [Figure 6C] Same as above. [Figure 6D] Same as above. [Figure 6E] Same as above. [Figure 7]The hematoma does not dissolve until 7 dps. RFP staining 4 days after induction with control virus (AAV9 non-concentrated GFAP-Cre + AAV9-flex-mCherry) at 2 dps, induced with 0.2 µL (0.03 units) of collagenase. Virus enters the hematoma when injected in situ before 7 dps. The presence of a hematoma may prevent the virus from targeting astrocytes. [Figure 8] Peak proliferation of reactive astrocytes after ICH occurs approximately 7 dps. Astrocytes become reactive at 4 dps and begin to form a glial scar before 8 dps. See also Sukumari-Ramesh, et al., J. Neurotrauma, 29(18):2798-28044 (2012). [Figure 9] In addition to the time point of virus injection, various injury conditions may also affect the rate of astrocyte-to-neuron conversion. GFP staining was measured 19, 17, or 14 days after induction with the treatment virus (AAV5-0.6kb-GFAP-Cre + AAV5-flex-ND1-GFP + AAV5-flex-Dlx2-GFP) at 2, 4, or 7 dps, respectively, using 0.2 μL (0.03 units) of collagenase. [Figure 10A]Comparison of astrocyte-to-neuron conversion rates under comparable injury conditions. Mouse #1 received treatment virus (AAV5-0.6kb-GFAP-Cre + AAV5-flex-ND1-GFP + AAV5-flex-Dlx2-GFP) at 2 dps, induced with 0.325 μL (0.05 units) of collagenase. Mouse #2 received control virus (AAV5-0.6kb-GFAP-mCherry-Cre + AAV5-flex-GFP) in the left hemisphere at 7 dps and treatment virus (AAV5-0.6kb-GFAP-Cre + AAV5-flex-ND1-GFP + AAV5-flex-Dlx2-GFP) in the right hemisphere at 7 dps, induced with 0.2 μL (0.03 units) of collagenase on each side. Figure 10A shows MRI scans of mouse #1 at 1 dpz (top) and mouse #2 at 3 dpz (bottom). Figure 10B shows GFP, GFAP, and NeuN staining of mouse #1 and mouse #2 14 days after induction. Figure 10C shows MRI images of hematoma size in these two mice. Figure 10D shows better recovery of the striatum on the treated side. MRI showed comparable hematomas on both sides at 3 dpz, but a smaller injury core and smaller ventricles were observed 14 days after treatment on the right side. This suggests that treatment can alleviate striatal shrinkage after ICH. MRI scans were obtained at 3 dpz. [Figure 10B] Same as above. [Figure 10C] Same as above. [Figure 10D] Same as above. [Figure 11] FIG. 1 illustrates the processes involved in ICH. DETAILED DESCRIPTION OF THE INVENTION

[0010] The present specification provides methods and materials involved in treating a mammal that has suffered a hemorrhagic stroke. For example, the present specification provides methods and materials for administering a composition containing an exogenous nucleic acid encoding a NeuroD1 polypeptide and a nucleic acid encoding a Dlx2 polypeptide to a mammal that has been identified as having suffered a hemorrhagic stroke.

[0011] Any suitable mammal can be identified as having suffered a hemorrhagic stroke, for example, humans and other primates, such as monkeys, can be identified as having suffered a hemorrhagic stroke.

[0012] Any suitable type of hemorrhagic stroke (e.g., intracranial hemorrhage) can be treated as described herein. For example, intraaxial (intracerebral) hemorrhagic stroke, such as intracerebral hemorrhage, can be treated as described herein. In some cases, extraaxial (extracerebral) hemorrhage, such as epidural hemorrhage (e.g., caused by trauma), subdural hemorrhage (e.g., caused by trauma), or subarachnoid hemorrhage (e.g., caused by trauma or aneurysm), can be treated as described herein. Approximately 10 to 20 percent of all strokes may involve intracerebral hemorrhage, which can have a high mortality rate of 40 percent within one month and 54 percent within one year. Causes of intracerebral hemorrhage include hypertension and secondary effects of other diseases, such as amyloid angiopathy (e.g., Alzheimer's disease) or brain tumors. A common location of intracerebral hemorrhage is in the striatum (e.g., approximately 50 percent). Three models of intracerebral hemorrhage are autologous blood (or lysed blood cell) injection, striatal balloon inflation, and collagenase injection. For autologous blood (or lysed blood cell) injection, approximately 50–100 μL of whole blood, lysed RBCs, or RBC-plus cell fractions are injected into the striatum. A characteristic feature is blood-borne toxicity without lesion expansion. For striatal balloon inflation, an embolization balloon is inserted into the striatum and slowly inflated with saline. The balloon can be left in place or withdrawn for the desired imitation. A characteristic feature is the isolated mechanical effects of a massive hematoma. For collagenase injection, approximately 0.075–0.4 units of bacterial collagenase are injected into the striatum to induce basal lamina breakdown and ICH. A characteristic feature is an expanding hematoma resulting from in situ rupture, which best mimics ICH in humans.

[0013] Intracerebral hemorrhage can result in primary and secondary damage to the brain. For example, intracerebral hemorrhage can result in primary damage caused by the physical pressure induced by the hematoma, and toxicity from blood components, such as ferric iron (Fe 3+ This can result in ferroptosis induced by NeuroD1 and subsequent secondary damage caused by oxidative stress and inflammation. The methods and materials provided herein (e.g., administration of a nucleic acid encoding a NeuroD1 polypeptide (or a biologically active fragment thereof) and a nucleic acid encoding a Dlx2 polypeptide (or a biologically active fragment thereof)) can be used to reduce the severity of one or more primary or secondary injuries to the brain of a mammal (e.g., a human) that has suffered an intracerebral hemorrhage.

[0014] In some cases, the hemorrhagic stroke results from a condition selected from the group consisting of ruptured blood vessels, high blood pressure, an aneurysm, an ischemic stroke, physical injury, a tumor, inflammation, an infection, generalized ischemia, hypoxic-ischemic encephalopathy, meningitis, and dehydration.

[0015] In some cases, the hemorrhagic stroke results from a condition selected from the group consisting of bleeding in the brain, an aneurysm, an intracranial hematoma, a subarachnoid hemorrhage, brain trauma, high blood pressure, a weak blood vessel, a vascular malformation, an ischemic stroke, a physical injury, a tumor, inflammation, an infection, generalized ischemia, hypoxic-ischemic encephalopathy, meningitis, and dehydration.

[0016] In some cases, global ischemia is caused by cardiac arrest or severe hypotension (shock). In some cases, hypoxic-ischemic encephalopathy is caused by hypoxia, hypoglycemia, or anemia.

[0017] In some cases, hemorrhagic stroke is caused by bleeding in the brain. In some cases, hemorrhagic stroke is caused by an aneurysm. In some cases, hemorrhagic stroke is caused by an intracranial hematoma. In some cases, hemorrhagic stroke is caused by a subarachnoid hemorrhage. In some cases, hemorrhagic stroke is caused by brain trauma. In some cases, hemorrhagic stroke is caused by high blood pressure. In some cases, hemorrhagic stroke is caused by weak blood vessels. In some cases, hemorrhagic stroke is caused by vascular malformations. In some cases, hemorrhagic stroke is caused by ischemic stroke. In some cases, hemorrhagic stroke is caused by physical injury. In some cases, hemorrhagic stroke is caused by a tumor. In some cases, hemorrhagic stroke is caused by inflammation. In some cases, hemorrhagic stroke is caused by infection. In some cases, hemorrhagic stroke is caused by generalized ischemia. In some cases, hemorrhagic stroke is caused by hypoxic-ischemic encephalopathy. In some cases, hemorrhagic stroke is caused by meningitis. In some cases, hemorrhagic stroke is caused by dehydration.

[0018] In some cases, administering a therapeutically effective amount of an exogenous nucleic acid encoding a NeuroD1 polypeptide (or a biologically active fragment thereof) and a nucleic acid encoding a Dlx2 polypeptide (or a biologically active fragment thereof) to a subject affected by hemorrhagic stroke mediates the generation of new glutamatergic neurons by converting reactive astrocytes into glutamatergic neurons, a reduction in the number of reactive astrocytes, survival of damaged neurons, including GABAergic and glutamatergic neurons, the generation of new non-reactive astrocytes, a reduction in the reactivity of unconverted reactive astrocytes, and reconnection of blood vessels into the damaged area.

[0019] In some cases, the methods or compositions provided herein generate new glutamatergic neurons and increase the number of glutamatergic neurons by about 1% to 500% from baseline levels after administration of the compositions provided herein. In some cases, the methods or compositions provided herein generate new glutamatergic neurons and increase the number of glutamatergic neurons by about 1% to 50%, about 1% to 100%, about 1% to 150%, about 50% to 100%, about 50% to 150%, about 50% to 200%, about 100% to 150%, about 100% to 200%, about 100% to 250%, about 150% to 2 Increase by approximately 00%, approximately 150% to 250%, approximately 150% to 300%, 200% to 250%, 200% to 300%, 200% to 350%, 250% to 300%, 250% to 350%, approximately 250% to 400%, approximately 300% to 350%, approximately 300% to 400%, approximately 300% to 450%, approximately 350% to 400%, approximately 350% to 450%, approximately 350% to 500%, approximately 400% to 450%, approximately 400% to 500%, or approximately 450% to 500%.

[0020] In some cases, the methods or compositions provided herein reduce the number of reactive astrocytes by about 1% to about 100% after administration of the compositions provided herein. In some cases, the methods or compositions provided herein reduce the number of reactive astrocytes by about 1% to about 10%, 1% to about 20%, 1% to about 30%, 10% to about 20%, 10% to about 30%, about 10% to about 40%, about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 30% to about 40%, about 30% to about 50%, about 30% to about 50%, about 30% to about 60%, about 30% to about 60%, about 30% to about 70%, about 30% to about 70%, about 30% to about 80%, about 30% to about 80%, about 30% to about 90%, about 30% to about 90%, about 30% to about 100%, about 30% to about 120%, about 30% to about 130%, about 30% to about 140%, about 30% to about 150%, about 30% to about 160%, about 30% to about 170%, about 30% to about 180%, about 30% to about 190%, about 30% to about 210%, about 30% to about 220%, about 30% to about 230%, about 30% to about 240%, about 30% to about 250%, about 30% to about 260%, about 30% to about 270%, about 30% to about 280%, about 30% to about 320%, about 32% to about 330%, about 33% to about 340 Reduced by about 60%, about 40% to about 50%, about 40% to about 60%, about 40% to about 70%, about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 60% to about 70%, about 60% to about 80%, about 60% to about 90%, about 70% to about 80%, about 70% to about 90%, about 70% to about 100%, about 80% to about 90%, about 80% to about 100%, or about 90% to about 100%.

[0021] In some cases, the methods or compositions provided herein increase GABAergic neuron survival by about 1% to 100% after administration of a composition provided herein compared to no administration. In some cases, the methods or compositions provided herein increase GABAergic neuron survival by about 1% to about 10%, 1% to about 20%, 1% to about 30%, 10% to about 20%, 10% to about 30%, about 10% to about 40%, about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 30% to about 40%, about 30% to about Increased survival of GABAergic neurons can be measured by any appropriate method. For example, immunostaining for γ-aminobutyric acid (GABA), the GABA-synthesizing enzyme glutamic acid decarboxylase 67 (GAD67), and / or parvalbumin (PV) can be performed to measure the number of GABAergic neurons. A decrease in the number of GABAergic neurons may indicate GABAergic neuron loss. A stable number may indicate survival of GABAergic neurons. An increase in the number of GABAergic neurons may indicate the occurrence of GABAergic regeneration.

[0022] In some cases, the methods or compositions provided herein increase glutamatergic neuron survival by about 1% to 100% after administration of a composition provided herein compared to no administration. In some cases, the methods or compositions provided herein increase glutamatergic neuron survival by about 1% to about 10%, 1% to about 20%, 1% to about 30%, 10% to about 20%, 10% to about 30%, about 10% to about 40%, about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 30% to about 40%, about 30% to about 50%, about 5 ... Increased survival of glutamatergic neurons can be measured by approximately 50%, approximately 30% to approximately 60%, approximately 40% to approximately 50%, approximately 40% to approximately 60%, approximately 40% to approximately 70%, approximately 50% to approximately 60%, approximately 50% to approximately 70%, approximately 50% to approximately 80%, approximately 60% to approximately 70%, approximately 60% to approximately 80%, approximately 60% to approximately 90%, approximately 70% to approximately 80%, approximately 70% to approximately 90%, approximately 70% to approximately 100%, approximately 80% to approximately 90%, approximately 80% to approximately 100%, or approximately 90% to approximately 100%. Increased survival of glutamatergic neurons can be assessed using any appropriate method. For example, immunostaining using a glutamatergic neuron marker can be performed to measure the number of glutamatergic neurons. A decrease in the number of glutamatergic neurons can indicate glutamatergic neuron loss. If the number remains unchanged, it may indicate that the glutamatergic neurons are surviving. An increase in the number of glutamatergic neurons may indicate the occurrence of glutamatergic regeneration.

[0023] In some cases, the methods or compositions provided herein generate new non-reactive astrocytes, and after administration of the compositions provided herein, the number of new non-reactive astrocytes increases from baseline levels by about 1% to about 100%. In some cases, the methods or compositions provided herein generate new non-reactive astrocytes, and after administration of the compositions provided herein, the number of new non-reactive astrocytes increases from baseline levels by about 1% to about 10%, 1% to about 20%, 1% to about 30%, 10% to about 20%, 10% to about 30%, about 10% to about 40%, about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, or about 30% to about 40%. , about 30% to about 50%, about 30% to about 60%, about 40% to about 50%, about 40% to about 60%, about 40% to about 70%, about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 60% to about 70%, about 60% to about 80%, about 60% to about 90%, about 70% to about 80%, about 70% to about 90%, about 70% to about 100%, about 80% to about 90%, about 80% to about 100%, or about 90% to about 100%.

[0024] In some cases, the methods or compositions provided herein reduce the reactivity of unconverted reactive astrocytes by about 1% to 100% from baseline levels after administration of the compositions provided herein. In some cases, the methods or compositions provided herein reduce the reactivity of unconverted reactive astrocytes by about 1% to about 10%, 1% to about 20%, 1% to about 30%, 10% to about 20%, 10% to about 30%, about 10% to about 40%, about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 30% to about 40%, about 30% to about 50%, about 30% to about 60%, about 40% to about 50%, about 40% to about 60%, about 40% to about 70%, about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 60% to about 70%, about 60% to about 80%, about 60% to about 90%, about 70% to about 80%, about 70% to about 90%, about 70% to about 100%, about 80% to about 90%, about 80% to about 100%, or about 90% to about 100%.

[0025] In some cases, administering a therapeutically effective amount of an exogenous nucleic acid encoding a NeuroD1 polypeptide (or a biologically active fragment thereof) and a nucleic acid encoding a Dlx2 polypeptide (or a biologically active fragment thereof) to a subject affected by hemorrhagic stroke mediates a reduction in inflammation at the injury site, a reduction in neuronal inhibition at the injury site, a re-establishment of normal microglial morphology at the injury site, a re-establishment of neural circuitry at the injury site, an increase in blood vessels at the injury site, a re-establishment of the blood-brain barrier at the injury site, a re-establishment of normal tissue architecture at the injury site, and an improvement in motor deficits due to disruption of normal blood flow.

[0026] In some cases, administering therapeutically effective amounts of an exogenous nucleic acid encoding a NeuroD1 polypeptide (or a biologically active fragment thereof) and a nucleic acid encoding a Dlx2 polypeptide (or a biologically active fragment thereof) to ameliorate the effects of ICH in an individual subject in need thereof has a greater beneficial effect when administered to reactive astrocytes than when administered to quiescent astrocytes.

[0027] Treatment with exogenous nucleic acids encoding NeuroD1 polypeptides (or biologically active fragments thereof) and Dlx2 polypeptides (or biologically active fragments thereof) can be administered to areas of injury diagnosed by magnetic resonance imaging (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 point of injury can be non-invasively assessed via near-infrared spectroscopy and fMRI. Blood flow within the area can either increase, as seen in aneurysms, or decrease, as seen in ischemia. Damage to the CNS caused by blood flow disruption further causes short- and long-term changes in tissue structure that can be used to diagnose the point of injury. In the short term, injury causes local swelling. In the long term, cell death causes tissue loss. Non-invasive methods for assaying the structural changes caused by tissue death include MRI, position emission tomography (PET) scans, computerized axial tomography (CAT) scans, or ultrasound. These methods can be used alone or in any combination to pinpoint the location of foci of damage.

[0028] As mentioned above, non-invasive methods for assaying structural changes caused by tissue death include MRI, CAT scan, or ultrasound. Functional assays may include EEG recording.

[0029] In some embodiments of the methods for treating a mammal that has suffered a hemorrhagic stroke as described herein, the exogenous NeuroD1 polypeptide (or a biologically active fragment thereof) and Dlx2 polypeptide (or a biologically active fragment thereof) are administered as expression vectors containing nucleic acid sequences encoding NeuroD1 and Dlx2.

[0030] In some embodiments of the method for treating neurological disorders described herein, viral vectors (e.g., AAV) comprising nucleic acids encoding NeuroD1 polypeptide and Dlx2 polypeptide are delivered by injection into the brain of a subject, such as stereotactic intracranial injection or retroorbital injection.In some cases, the composition containing adeno-associated virus encoding NeuroD1 polypeptide and Dlx2 polypeptide is administered to the brain using two or more intracranial injections at the same location in the brain.In some cases, the composition containing adeno-associated virus encoding NeuroD1 polypeptide and Dlx2 polypeptide is administered to the brain using two or more intracranial injections at two or more different locations in the brain.In some cases, the composition containing adeno-associated virus encoding NeuroD1 polypeptide and Dlx2 polypeptide is administered to the brain using one or more extracranial injections.

[0031] The term "expression vector" refers to a recombinant vehicle for introducing a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof and a nucleic acid encoding a Dlx2 polypeptide or a biologically active fragment thereof into a host cell in vitro or in vivo, where the nucleic acid is expressed to produce the NeuroD1 polypeptide and the Dlx2 polypeptide. In certain embodiments, an expression vector comprising SEQ ID NO: 1 or 3, or a substantially identical nucleic acid sequence, is expressed to produce NeuroD1 in cells containing the expression vector. In certain embodiments, an expression vector comprising SEQ ID NO: 10 or 12, or a substantially identical nucleic acid sequence, is expressed to produce Dlx2 in cells containing the expression vector.

[0032] The term "recombinant" is used to refer to a nucleic acid construct in which two or more nucleic acids are linked and are not found linked together in nature. Expression vectors include, but are not limited to, plasmids, viruses, BACs and YACs. Specific viral expression vectors include, for example, those derived from adenoviruses, adeno-associated viruses, retroviruses and lentiviruses.

[0033] The present disclosure provides materials and methods for treating symptoms of hemorrhagic stroke in a subject in need thereof, including providing a viral vector containing a nucleic acid encoding NeuroD1 and Dlx2, and delivering the viral vector to the subject's brain, whereby the viral vector infects glial cells in the central nervous system, respectively, producing infected glial cells, whereby an exogenous nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof and a nucleic acid encoding a Dlx2 polypeptide or a biologically active fragment thereof are expressed at therapeutically effective levels in the infected glial cells. Expression of the NeuroD1 polypeptide and the Dlx2 polypeptide in the infected cells results in a higher number of neurons in the subject compared to untreated subjects with the same neurological condition, thereby treating the neurological disorder. In addition to the generation of new neurons, the number of reactive glial cells is also reduced, resulting in less neuroinhibitory factors being released, less neuroinflammation, and / or more uniformly distributed blood vessels, making the local environment more permissive for neuronal growth or axonal penetration, thus alleviating the neurological condition.

[0034] In some cases, adeno-associated vectors can be used in the methods described herein to infect 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. Any of a variety of recombinant adeno-associated viruses, such as serotypes 1-9, can be used as described herein. In some cases, AAV-PHP.eb is used to administer exogenous NeuroD1 and Dlx2.

[0035] The "FLEX" switch approach is used to express NeuroD1 and Dlx2 in infected cells according to some embodiments described herein. The terms "FLEX" and "Flip-excision" are used interchangeably to refer to a method in which two pairs of heterotypic, antiparallel loxP-type recombination sites are placed on either side of an inverted NeuroD1 or Dlx2 coding sequence, which first undergoes inversion of the coding sequence, followed by excision of the two sites, so that one of each orthogonal recombination site is oriented in the opposite direction and cannot undergo 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 and Dlx2 are also expressed in glial cells, including reactive astrocytes. Then, when the stop codon before NeuroD1 or Dlx2 is removed by recombination, a constitutive or neuron-specific promoter drives high expression of NeuroD1 and Dlx2, enabling the conversion of reactive astrocytes into functional neurons.

[0036] According to certain embodiments, exogenous nucleic acids encoding a NeuroD1 polypeptide or a biologically active fragment thereof, and nucleic acids encoding a Dlx2 polypeptide or a biologically active fragment thereof, are 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 DNA sequences encoding NeuroD1 polypeptide and Dlx2 polypeptide under the transcriptional control of a ubiquitous (constitutive) promoter or a neuron-specific promoter, wherein the DNA sequences encoding NeuroD1 and Dlx2 are reversed and in the wrong orientation for expression of NeuroD1 and Dlx2 until the site-specific recombinase reverses the reverse DNA sequences encoding NeuroD1 and Dlx2, thereby enabling expression of NeuroD1 and Dlx2.

[0037] Examples of site-specific recombinases and their recognition sites include Cre recombinase with the recognition sites loxP and lox2272, or FLP-FRT recombination, or a combination thereof.

[0038] A composition comprising an exogenous nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof and a nucleic acid encoding a Dlx2 polypeptide or a biologically active fragment thereof (e.g., an AAV encoding a NeuroD1 polypeptide and a Dlx2 polypeptide) can be formulated into a pharmaceutical composition for administration into a mammal. For example, a therapeutically effective amount of a composition comprising an exogenous nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof and an exogenous nucleic acid encoding a Dlx2 polypeptide or a biologically active fragment thereof can be formulated with one or more pharmaceutically acceptable carriers (additives) and / or diluents. A pharmaceutical composition comprising an exogenous nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof and an exogenous nucleic acid encoding a Dlx2 polypeptide or a biologically active fragment thereof (e.g., an AAV encoding a NeuroD1 polypeptide and a Dlx2 polypeptide) can be formulated for oral administration via various routes, such as capsules, liquids, etc. In some cases, the exogenous nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof and the viral vector (e.g., AAV) carrying the exogenous nucleic acid encoding a Dlx2 polypeptide or a biologically active fragment thereof are administered parenterally, preferably by intravenous injection or infusion. Administration can be, for example, by intravenous infusion over a period of, for example, 60 minutes, 30 minutes, or 15 minutes. In some cases, the intravenous infusion can last for 1 minute to 60 minutes. In some cases, the intravenous infusion can last for 1 minute to 5 minutes, 1 minute to 10 minutes, 1 minute to 15 minutes, 5 minutes to 10 minutes, 5 minutes to 15 minutes, 5 minutes to 20 minutes, 10 minutes to 15 minutes, 10 minutes to 20 minutes, 10 minutes to 25 minutes, 15 minutes to 20 minutes, 15 minutes to 25 minutes, 15 minutes to 30 minutes, 20 minutes to 25 minutes, 20 minutes to 30 minutes, 20 minutes to 35 minutes, 25 minutes to 30 minutes, 25 minutes to 25 minutes, or 25 minutes. It can be up to 35 minutes, 25 to 40 minutes, 30 to 35 minutes, 30 to 40 minutes, 30 to 45 minutes, 35 to 40 minutes, 35 to 45 minutes, 35 to 50 minutes, 40 to 45 minutes, 40 to 50 minutes, 40 to 55 minutes, 45 to 50 minutes, 45 to 60 minutes, 50 to 55 minutes, 50 to 60 minutes, or 55 to 60 minutes.

[0039] In some cases, the administration can be provided to the mammal 1 day to 60 days after the hemorrhagic stroke. In some cases, the administration can be provided 1 day to 5 days, 1 day to 10 days, 1 day to 15 days, 5 days to 10 days, 5 days to 15 days, 5 days to 20 days, 10 days to 15 days, 10 days to 20 days, 10 days to 25 days, 15 days to 20 days, 15 days to 25 days, 15 days to 30 days, 20 days to 25 days, 20 days to 30 days, 20 days to 35 days, 25 days to 30 ... It may be provided to the mammal after 5 days, 25 to 40 days, 30 to 35 days, 30 to 40 days, 30 to 45 days, 35 to 40 days, 35 to 45 days, 35 to 50 days, 40 to 45 days, 40 to 50 days, 40 to 55 days, 45 to 50 days, 45 to 55 days, 45 to 60 days, 50 to 55 days, 50 to 60 days, or 55 to 60 days.

[0040] In some cases, the administration may be provided to the mammal at the time of the hemorrhagic stroke. In some cases, the administration may be provided to the mammal one day after the hemorrhagic stroke. In some cases, the administration may be provided to the mammal two days after the hemorrhagic stroke. In some cases, the administration may be provided to the mammal three days after the hemorrhagic stroke. In some cases, the administration may be provided to the mammal four days after the hemorrhagic stroke. In some cases, the administration may be provided to the mammal five days after the hemorrhagic stroke. In some cases, the administration may be provided to the mammal six days after the hemorrhagic stroke. In some cases, the administration may be provided to the mammal seven days after the hemorrhagic stroke. In some cases, the administration may be provided to the mammal one week after the hemorrhagic stroke. In some cases, the administration may be provided to the mammal two weeks after the hemorrhagic stroke. In some cases, the administration may be provided to the mammal three weeks after the hemorrhagic stroke. In some cases, the administration may be provided to the mammal four weeks after the hemorrhagic stroke. In some cases, the administration may be provided to the mammal 5 weeks after the hemorrhagic stroke. In some cases, the administration may be provided to the mammal 6 weeks after the hemorrhagic stroke. In some cases, the administration may be provided to the mammal 7 weeks after the hemorrhagic stroke. In some cases, the administration may be provided to the mammal 8 weeks after the hemorrhagic stroke.

[0041] In some cases, viral vectors (e.g., AAVs encoding NeuroD1 and Dlx2 polypeptides) are administered locally via injection into the brain during surgery. Compositions suitable for administration by injection and / or infusion include solutions and dispersions, as well as powders from which corresponding solutions and dispersions can be prepared. Such compositions comprise a viral vector and at least one suitable pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers for intravenous administration include, but are not limited to, bacterial static water, Ringer's solution, physiological saline, phosphate-buffered saline (PBS), and Cremophor EL™. Sterile compositions for injection and / or infusion can be prepared by incorporating the required amount of viral vectors (e.g., AAVs encoding NeuroD1 and Dlx2 polypeptides) into a suitable carrier and then sterilizing by filtration. Compositions for administration by injection or infusion must remain stable under storage conditions for extended periods after their preparation. The compositions may contain a preservative for this purpose. Suitable preservatives include chlorobutanol, phenol, ascorbic acid, and thimerosal.

[0042] In some embodiments, the gene delivery vector can be an AAV vector.For example, the AAV vector can be selected from the group consisting of AAV2 vector, AAV5 vector, AAV8 vector, AAV1 vector, AAV7 vector, AAV9 vector, AAV3 vector, AAV6 vector, AAV10 vector and AAV11 vector.

[0043] Pharmaceutical compositions can be formulated for administration in solid or liquid form, including, but not limited to, sterile solutions, suspensions, sustained-release formulations, tablets, capsules, pills, powders, and granules.The formulations can be presented in unit-dose or multi-dose containers, such as sealed ampoules and vials, and can be stored in a freeze-dried (lyophilized) state, requiring only the addition of a sterile liquid carrier, such as water for injection, immediately before use.Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets.

[0044] Additional pharmaceutically acceptable carriers, fillers, and vehicles that may be used in the pharmaceutical compositions described herein include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, saturated vegetable fatty acids, partial glyceride mixtures of water, salts, or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic materials, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol, and wool fat.

[0045] As used herein, the term "adeno-associated viral particle" refers to the packaged capsid form of the AAV virus that delivers its nucleic acid genome to a cell.

[0046] An effective amount of a composition containing exogenous NeuroD1 and Dlx2 can be any amount that ameliorates symptoms of a neurological disorder in a mammal (e.g., a human) and does not produce severe toxicity to the mammal. For example, an effective amount of an adeno-associated virus encoding a NeuroD1 polypeptide and a Dlx2 polypeptide can be about 10 10 ~10 14The concentration of the adeno-associated virus particles / mL can be 10 ... 10 adeno-associated virus particles / mL ~ 10 11 adeno-associated virus particles / mL, 10 10 adeno-associated virus particles / mL ~ 10 12 adeno-associated virus particles / mL, 10 10 adeno-associated virus particles / mL ~ 10 13 adeno-associated virus particles / mL, 10 11 adeno-associated virus particles / mL ~ 10 12 adeno-associated virus particles / mL, 10 11 adeno-associated virus particles / mL ~ 10 13 adeno-associated virus particles / mL, 10 11 adeno-associated virus particles / mL ~ 10 14 adeno-associated virus particles / mL, 10 12 adeno-associated virus particles / mL ~ 10 13 adeno-associated virus particles / mL, 10 12 adeno-associated virus particles / mL ~ 10 14 adeno-associated virus particles / mL, or 10 13 adeno-associated virus particles / mL ~ 10 14The amount of viral vector (e.g., AAV encoding NeuroD1 and Dlx2 polypeptides) administered may be in the range of 1000 to 10000 adeno-associated virus particles / mL. Factors related to the amount of viral vector (e.g., AAV encoding NeuroD1 and Dlx2 polypeptides) administered include, for example, the route of administration of the viral vector, the nature and severity of the disease, the patient's disease history, and the age, weight, height, and health of the patient. In some cases, the level of transgene expression required to achieve therapeutic efficacy, the patient's immune response, and the stability of the gene product are related to the amount administered. In some cases, the viral vector (e.g., AAV encoding exogenous NeuroD1 and Dlx2) is administered in an amount that results in a complete or substantially complete cure of brain dysfunction or disease.

[0047] In some cases, a composition containing an effective amount of exogenous NeuroD1 and Dlx2 can be optionally administered at a controlled flow rate of about 0.1 μL / min to about 5 μL / min.

[0048] In some cases, the controlled flow rate is between 0.1 μL / min and 0.2 μL / min, between 0.1 μL / min and 0.3 μL / min, between 0.1 μL / min and 0.4 μL / min, between 0.2 μL / min and 0.3 μL / min, between 0.2 μL / min and 0.4 μL / min, between 0.2 μL / min and 0.5 μL / min, between 0.3 μL / min and 0.4 μL / min, between 0.3 μL / min and 0.5 μL / min, between 0.3 μL / min and 0.6 μL / min, between 0.4 μL / min and 0.5 μL / min, between 0.4 μL / min and 0.6 μL / min, between 0.4 μL / min and 0.7 μL / min, between 0.5 μL / min and 0.6 μL / min, between 0.5 μL / min and 0.7 μL / min, between 0.5 μL / min and 0.5 μL / min. min~0.8μL / min, 0.6μL / min~0.7μL / min, 0.6μL / min~0.8μL / min, 0.6μL / min~0.9μL / min, 0.7μ L / min~0.8μL / min, 0.7μL / min~0.9μL / min, 0.7μL / min~1.0μL / min, 0.8μL / min~0.9μL / min, 0. 8μL / min~1.0μL / min, 0.8μL / min~1.1μL / min, 0.9μL / min~1.0μL / min, 0.9μL / min~1.1μL / min, 0.9μL / min~1.2μL / min, 1.0μL / min~1.1μL / min, 1.0μL / min~1.2μL / min, 1.0μL / min~1.3μL / min , 1.1μL / min~1.2μL / min, 1.1μL / min~1.3μL / min, 1.1μL / min~1.4μL / min, 1.2μL / min~1.3μL / min, 1.2μL / min~1.4μL / min, 1.2μL / min~1.5μL / min, 1.3μL / min~1.4μL / min, 1.3μL / min~1.5 μL / min, 1.3μL / min~1.6μL / min, 1.4μL / min~1.5μL / min, 1.4μL / min~1.6μL / min, 1.4μL / min~1 .7μL / min, 1.5μL / min~1.6μL / min, 1.5μL / min~1.7μL / min, 1.5μL / min~1.8μL / min, 1.6μL / min~ 1.7μL / min, 1.6μL / min~1.8μL / min, 1.6μL / min~1.9μL / min, 1.7μL / min~1.8μL / min, 1.7μL / min min~1.9μL / min, 1.7μL / min~2.0μL / min, 1.8μL / min~1.9μL / min, 1.8μL / min~2.0μL / min, 1.8μL / min~2.1μL / min, 1.9μL / min~2.0μL / min, 1.9μL / min~2.1μL / min, 1.9μL / min~2.2μL / min, 2.0 μL / min~2.1μL / min, 2.0μL / min~2.2μL / min, 2.0μL / min~2.3μL / min, 2.1μL / min~2.2μL / min, 2.1 μL / min ~ 2.3 μL / min, 2.1 μL / min ~ 2.4 μL / min, 2.2 μL / min ~ 2.3 μL / min, 2.2 μL / min ~ 2.4 μL / min, 2.2 μL / min ~ 2.5 μL / min, 2.3 μL / min ~ 2.4 μL / min, 2.3 μL / min ~ 2.5 μL / min, 2.3 μL / min ~ 2.6 μL / min 2.4 μL / min ~ 2.5 μL / min, 2.4 μL / min ~ 2.6 μL / min, 2.4 μL / min ~ 2.7 μL / min, 2.5 μL / min ~ 2.6 μL / min, 2.5 μL / min ~ 2.7 μL / min, 2.5 μL / min ~ 2.8 μL / min, 2.6 μL / min ~ 2.7 μL / min, 2.6 μL / min ~ 2.8 μL / min L / min, 2.6μL / min~2.9μL / min, 2.7μL / min~2.8μL / min, 2.7μL / min~2.9μL / min, 2.7μL / min~3.0μL / min, 2.8μL / min~2.9μL / min, 2.8μL / min~3.0μL / min, 2.8μL / min~3.1μL / min, 2.9μL / min~3 0.0 μL / min, 2.9 μL / min~3.1 μL / min, 2.9 μL / min~3.2 μL / min, 3.0 μL / min~3.1 μL / min, 3.0 μL / min~3.2 μL / min, 3.0 μL / min~3.3 μL / min, 3.1 μL / min~3.2 μL / min, 3.1 μL / min~3.3 μL / min, 3.1 μL / min 3.4 μL / min, 3.2 μL / min to 3.3 μL / min, 3.2 μL / min to 3.4 μL / min, 3.2 μL / min to 3.5 μL / min, 3.3 μL / min to 3.4 μL / min, 3.3 μL / min to 3.5 μL / min, 3.3 μL / min to 3.6 μL / min, 3.4 μL / min to 3.5 μL / min, 3.4 μL / min μL / min ~ 3.6 μL / min, 3.4 μL / min ~ 3.7 μL / min, 3.5 μL / min ~ 3.6 μL / min, 3.5 μL / min ~ 3.7 μL / min, 3.5 μL / min ~ 3.8 μL / min, 3.6 μL / min ~ 3.7 μL / min, 3.6 μL / min ~ 3.8 μL / min, 3.6 μL / min ~ 3.9 μL / min 3.7 μL / min ~ 3.8 μL / min, 3.7 μL / min ~ 3.9 μL / min, 3.7 μL / min ~ 4.0 μL / min, 3.8 μL / min ~ 3.9 μL / min, 3.8 μL / min ~ 4.0 μL / min, 3.8 μL / min ~ 4.1 μL / min, 3.9 μL / min ~ 4.0 μL / min, 3.9 μL / min ~ 4.1 μL / min, 3.9μL / min~4.2μL / min, 4.0μL / min~4.1μL / min, 4.0μL / min~4.2μL / min, 4.0μL / min~4.3μL / min, 4.1μL / min~4.2μL / min, 4.1μL / min~4.3μL / min, 4.1μL / min~4.4μL / min, 4.2μL / min~4.3μL / min, 4.2μL / min~4.4μL / min, 4.2μL / min~4.5μL / min, 4.3μL / min~4.4μL / min, 4.3μL / min~4.5μL / min, 4.3μL / min~4.6μL / min, 4.4μL / min~4.5μL / min, 4.4μL / min~4.6μL / min, 4.4μL / min~4.7μL / min, 4.5μL / min~4.6μL / min, 4.5μL / min~4.7μL / min, 4.5 μL / min to 4.8 μL / min, 4.6 μL / min to 4.7 μL / min, 4.6 μL / min to 4.8 μL / min, 4.6 μL / min to 4.9 μL / min, 4.7 μL / min to 4.8 μL / min, 4.7 μL / min to 4.9 μL / min, 4.7 μL / min to 5.0 μL / min, 4.8 μL / min to 4.9 μL / min, 4.8 μL / min to 5.0 μL / min, or 4.9 μL / min to 5.0 μL / min.

[0049] A viral vector (e.g., an AAV containing a nucleic acid encoding a NeuroD1 polypeptide and a nucleic acid encoding a Dlx2 polypeptide) can be expressed in an amount of approximately 1.0×10 10 ~Approx. 1.0×10 14 In some cases, the viral vector (e.g., an AAV containing a nucleic acid encoding a NeuroD1 polypeptide and a nucleic acid encoding a Dlx2 polypeptide) can be administered in an amount corresponding to a viral dose in the range of about 1.0×10 11 ~Approx. 1.0×10 12 vg / kg, which corresponds to a viral dose in the range of about 5.0 x 10 11 ~Approx. 5.0×10 12 vg / kg range, or approximately 1.0 × 10 12 ~Approx. 5.0×10 11 In some cases, a viral vector (e.g., an AAV containing a nucleic acid encoding a NeuroD1 polypeptide and a nucleic acid encoding a Dlx2 polypeptide) is administered in a range of about 2.5 x 10 12The effective amount of a viral vector (e.g., an AAV containing a nucleic acid encoding a NeuroD1 polypeptide and a nucleic acid encoding a Dlx2 polypeptide) may be administered in an amount corresponding to a dose of vg / kg (viral genomes per kg body weight) described herein, in a volume of about 1 μL to about 500 μL, corresponding to the dose volume of vg / kg (viral genomes per kg body weight) described herein. In some cases, the amount of a viral vector (e.g., an AAV containing a nucleic acid encoding a NeuroD1 polypeptide and a nucleic acid encoding a Dlx2 polypeptide) administered is adjusted according to the intensity of expression of one or more exogenous nucleic acids encoding polypeptides (e.g., NeuroD1 and Dlx2).

[0050] In some cases, the effective administration volume of the viral vector is between 1 μL and 25 μL, between 1 μL and 50 μL, between 1 μL and 75 μL, between 25 μL and 50 μL, between 25 μL and 75 μL, between 25 μL and 100 μL, between 50 μL and 75 μL, between 50 μL and 100 μL, between 50 μL and 125 μL, between 75 μL and 100 μL, between 75 μL and 125 μL, between 75 μL and 150 μL, between 100 μL and 125 μL, between 100 μL and 150 μL, 100μL~175μL, 125μL~150μL, 125μL~175μL, 125μL~200μL, 150μL~175μL, 150μL~200μL, 150μL~225μL, 175 μL~200μL, 175μL~225μL, 175μL~250μL, 200μL~225μL, 200μL~250μL, 200μL~275μL, 225μL~250μL, 225μL~ 275μL, 225μL~300μL, 250μL~275μL, 250μL~300μL, 250μL~325μL, 275μL~300μL, 275μL~325μL, 275μL~350 μL, 300μL~325μL, 300μL~350μL, 300μL~375μL, 325μL~350μL, 325μL~375μL, 325μL~400μL, 350μL~375μL, 350μL to 400μL, 350μL to 425μL, 375μL to 400μL, 375μL to 425μL, 375μL to 450μL, 400μL to 425μL, 400μL to 450μL, 400μL to 475μL, 425μL to 450μL, 425μL to 475μL, 425μL to 500μL, 450μL to 475μL, 450μL to 500μL, or 475μL to 500μL.

[0051] In some cases, an adeno-associated virus vector comprising nucleic acids encoding NeuroD1 and Dlx2 polypeptides under the transcriptional control of a ubiquitous (constitutive) promoter or a neuron-specific promoter, wherein the nucleic acid sequences encoding NeuroD1 and Dlx2 are inverted and in the wrong orientation relative to expression of NeuroD1 and Dlx2 until a site-specific recombinase reverses the reverse nucleic acid sequences encoding NeuroD1 and Dlx2 to enable expression of the NeuroD1 and Dlx2 polypeptides, 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.

[0052] In some cases, an adeno-associated virus vector comprising a nucleic acid encoding a NeuroD1 polypeptide and a Dlx2 polypeptide under the transcriptional control of a ubiquitous (constitutive) promoter or a neuron-specific promoter, wherein the nucleic acid sequences encoding the NeuroD1 polypeptide and the Dlx2 polypeptide are inverted and in the wrong orientation relative to expression of NeuroD1 and Dlx2 until a site-specific recombinase reverses the reverse nucleic acid sequences encoding NeuroD1 and Dlx2 to enable expression of the NeuroD1 polypeptide and the Dlx2 polypeptide, and further comprising a site for recombinase activity by the site-specific recombinase, is delivered by stereotactic injection into the brain of a subject in a region or at a site of interest, together with an adeno-associated virus encoding the site-specific recombinase.

[0053] In some cases, the site-specific recombinase is Cre recombinase and the sites for recombinase activity are recognition sites loxP and lox2272 sites.

[0054] In some cases, treatment with the subject's exogenous nucleic acid encoding NeuroD1 and Dlx2 polypeptides is monitored during or after treatment to monitor the progress and / or final outcome of treatment. Successful post-treatment restoration of neuronal cell integration and the tissue microenvironment can be diagnosed by restoration or near-recovery of normal electrophysiology, blood flow, tissue structure, and function. Non-invasive methods for assessing neuronal function include EEG. Blood flow can be non-invasively assessed via near-infrared spectroscopy and fMRI. Non-invasive methods for assessing tissue structure include MRI, CAT scans, PET scans, or ultrasound. Behavioral assays can be used to non-invasively assess the recovery of brain function. Behavioral assays should be consistent with the loss of function caused by the original brain injury. For example, if the injury caused paralysis, the patient's mobility and limb dexterity should be tested. If the injury caused speech loss or delay, the patient's ability to communicate through spoken language should be assessed. Restoration of normal behavior after treatment with exogenous nucleic acids encoding NeuroD1 and Dlx2 polypeptides indicates successful creation and integration of effective neuronal circuits. These methods can be used alone or in any combination to assay for neural function and tissue health. Assays to evaluate treatment can be performed at any time point, such as 1 day, 2 days, 3 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, 1 year, or later, after NeuroD1 and Dlx2 treatment. Such assays can be performed before NeuroD1 and Dlx2 treatment to establish a baseline comparison, if necessary.

[0055] Scientific and technical terms used herein are intended to have meanings that are commonly understood by those of ordinary skill in the art.Such terms are used, illustratively, in J. Sambrook and DWRussell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 3rd Ed., 2001, FMAsubel, Ed., Short Protocols in Molecular Biology, Current Protocols; 5th Ed., 2002, B. Alberts et al., Molecular Biology of the Cell, 4th Ed., Garland, 2002, DL Nelson and MMCox, Lehninger Principles of Biochemistry, 4th Ed., WH Freeman & Company, 2004, Engelke, DR, 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. Behringer, Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 3rd Ed.; 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.

[0056] As used herein, the singular forms "a," "an," and "the" are not intended to be limiting and include plural referents unless clearly indicated otherwise or the context clearly dictates otherwise.

[0057] As used herein, the term "NeuroD1 protein" refers to a bHLH proneural transcription factor involved in fetal brain development and adult neurogenesis, see Cho et al., Mol. Neurobiol., 30:35-47 (2004), Kuwabara et al., Nature Neurosci., 12:1097-1105 (2009), and Gao et al., Nature Neurosci., 12:1090-1092 (2009). NeuroD1 is primarily expressed in the nervous system during late development and is involved in neuronal differentiation, maturation, and survival.

[0058] The term "NeuroD1 protein" or "exogenous NeuroD1" 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, that may be included in the methods described herein. As used herein, the term "variant" refers to naturally occurring genetic variations and recombinantly prepared variations, each of which contains one or more changes in its amino acid sequence compared to a reference NeuroD1 protein, such as SEQ ID NO:2 or SEQ ID NO:4. Such variations include those in which one or more amino acid residues are modified by amino acid substitution, addition, or deletion. The term "variant" encompasses human NeuroD1 orthologs, including mammalian and avian NeuroD1, such as, but not limited to, NeuroD1 orthologs from non-human primates, cats, dogs, sheep, goats, horses, cattle, pigs, birds, poultry, and rodents, including, but not limited to, mice and rats. In a non-limiting example, mouse NeuroD1, exemplified herein as amino acid sequence SEQ ID NO: 4, is an ortholog of human NeuroD1.

[0059] In some cases, 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.

[0060] 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 changing the functional properties of NeuroD1 protein.For example, one or more amino acid substitutions, additions, or deletions can be made without changing the functional properties of the NeuroD1 protein of SEQ ID NO: 2 or 4.

[0061] Conservative amino acid substitution can be made in NeuroD1 protein to produce NeuroD1 protein variant.Conservative amino acid substitution is the technically recognized substitution of one amino acid with another amino acid with similar characteristics.For example, each amino acid can be described as having one or more of the following characteristics: positive charge, negative charge, aliphatic, aromatic, polar, hydrophobic and hydrophilic.Conservative substitution is the substitution of one amino acid with specific structural or functional characteristics with another amino acid with the same characteristics. 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 may also be described in terms of their relative size, with alanine, cysteine, aspartate, glycine, asparagine, proline, threonine, serine, and valine, all of which are typically considered small.

[0062] NeuroD1 variants may include synthetic amino acid analogs, amino acid derivatives, and / or non-standard amino acids, including, but not limited to, α-aminobutyric acid, citrulline, canavanine, cyanoalanine, diaminobutyric acid, diaminopimelic acid, dihydroxy-phenylalanine, dienkolinic acid, homoarginine, hydroxyproline, norleucine, norvaline, 3-phosphoserine, homoserine, 5-hydroxytryptophan, 1-methylhistidine, 3-methylhistidine, and ornithine.

[0063] 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 sequence of the first amino acid or nucleic acid sequence for optimal alignment with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. If 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 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.

[0064] The determination of percent identity between two sequences can also be achieved using mathematical algorithms.A preferred, non-limiting example of mathematical algorithms used to compare two sequences is the algorithm of Karlin and Altschul, PNAS, 87:2264-2268 (1990), modified as in Karlin and Altschul, PNAS, 90:5873-5877 (1993).Such algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., J. Mol. Biol., 215:403 (1990).To obtain the nucleotide sequence homologous to the nucleic acid molecules described herein, for example, BLAST nucleotide search is performed using the NBLAST nucleotide program parameters set as score=100 and word length=12.

[0065] To obtain amino acid sequences homologous to the protein molecules described herein, BLAST protein searches are performed, for example, using the XBLAST program parameters set to score=50 and word length=3. To obtain gapped alignments for comparison purposes, gapped BLAST is used as described in Altschul et al., Nucleic Acids Res., 25:3389-3402 (1997). Alternatively, PSI BLAST is used to perform an iterated search that detects distant relationships between molecules. When using BLAST, Gapped BLAST, and PSI BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) are used (see, for example, the NCBI website).

[0066] Another preferred, non-limiting example of a mathematical algorithm utilized for comparing sequences is the algorithm of Myers and Miller, CABIOS, 4:11-17 (1988). 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.

[0067] The percent identity between two sequences is determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[0068] The term "NeuroD1 protein" encompasses fragments of NeuroD1 protein, such as fragments of SEQ ID NOs: 2 and 4, and variants thereof, that are operable in the methods or compositions described herein.

[0069] 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 express NeuroD1. Alternatively, NeuroD1 proteins or nucleic acids can be recombinantly produced in vitro or in vivo, such as by expression using an expression construct. NeuroD1 proteins and nucleic acids can also be synthesized by well-known methods.

[0070] The NeuroD1 included in the methods or compositions described herein can be produced using recombinant nucleic acid technology. Producing recombinant NeuroD1 involves introducing a recombinant expression vector containing a DNA sequence encoding NeuroD1 into a host cell.

[0071] In some cases, 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.

[0072] In some cases, a 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. In some cases, a 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. In some cases, a nucleic acid sequence identified herein as SEQ ID NO: 10 encodes SEQ ID NO: 11 and is included in an expression vector and expressed to produce Dlx2. In some cases, a nucleic acid sequence identified herein as SEQ ID NO: 12 encodes SEQ ID NO: 13 and is included in an expression vector and expressed to produce Dlx2.

[0073] It is 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 such alternative nucleic acids can be included in expression vectors and expressed to produce NeuroD1 and variants of NeuroD1. One of skill in the art will understand that fragments of nucleic acids encoding NeuroD1 protein can be used to produce fragments of NeuroD1 protein.

[0074] As used herein, the term "Dlx2" refers to distal-less homeobox 2, which functions as a transcriptional activator and plays a role in the terminal differentiation of interneurons, such as amacrine and bipolar cells, in the developing retina. Dlx2 plays a regulatory role in ventral forebrain development and may play a role in craniofacial patterning and morphogenesis. The term "Dlx2 protein" or "exogenous Dlx2" encompasses the human Dlx2 protein identified herein as SEQ ID NO: 11 and the mouse Dlx2 protein identified herein as SEQ ID NO: 13. In addition to the Dlx2 proteins of SEQ ID NO: 11 and SEQ ID NO: 13, the term "Dlx2 protein" encompasses variants of Dlx2 proteins, such as variants of SEQ ID NO: 11 and SEQ ID NO: 13, that may be included in the methods described herein.

[0075] 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 linkage 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 controls some aspect of the expression of an operably linked nucleic acid. Exemplary regulatory elements include enhancers, introns, origins of replication, polyadenylation signals (pA), promoters, transcription termination sequences, and upstream regulatory domains, such as, but not limited to, the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), internal ribosome entry site (IRES), or 2A domain, which contribute to the replication, transcription, and posttranscriptional 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 with no more than routine experimentation.

[0076] As used herein, the term "promoter" refers to a DNA sequence operably linked to a nucleic acid sequence to be transcribed, such as a nucleic acid sequence encoding NeuroD1 and / or a nucleic acid sequence encoding Dlx2. A promoter is generally positioned upstream of a nucleic acid sequence to be transcribed and provides a site for specific binding by RNA polymerase and other transcription factors. In specific embodiments, a promoter is generally positioned upstream of a 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.

[0077] 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 necessary to drive expression of an operably linked nucleic acid is used. Assays for determining 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.

[0078] The specific promoters used to drive expression of NeuroD1 and / or Dlx2 according to the methods described herein are "ubiquitous" or "constitutive" promoters, which drive 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 and / or Dlx2 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 using routine methodologies or obtained from commercial sources. The promoter may be derived entirely from a single gene, or may be chimeric, having portions derived from more than one gene.

[0079] A combination of regulatory sequences can be included in an expression vector and used to drive expression of NeuroD1 and / or Dlx2. A non-limiting example of an expression vector for driving expression of NeuroD1 and / or Dlx2 is the CAG promoter in combination with the cytomegalovirus CMV early enhancer element and the chicken beta actin promoter.

[0080] Particular promoters used to drive expression of NeuroD1 and / or Dlx2 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.

[0081] 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.

[0082] 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.

[0083] Particular promoters used to drive expression of NeuroD1 and / or Dlx2 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.

[0084] 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.

[0085] Homologs and variants of ubiquitous and cell type-specific promoters can be used for expression of NeuroD1 and / or Dlx2.

[0086] In some cases, promoter homologs and promoter variants can be included in expression vectors for expressing NeuroD1 and / or Dlx2. The terms "promoter homolog" and "promoter variant" refer to promoters that have substantially similar functional properties to those disclosed herein to confer a desired type of expression, such as cell-type-specific expression of NeuroD1 (and / or Dlx2) on an operably linked nucleic acid encoding NeuroD1 (and / or Dlx2), or ubiquitous expression of NeuroD1 (and / or Dlx2). For example, promoter homologs or variants have substantially similar functional properties to GFAP, S100b, Aldh1L1, NG2, lcn2, and CAG promoters to confer cell-type-specific expression on an operably linked nucleic acid encoding NeuroD1 (and / or Dlx2).

[0087] 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 produce promoter variants.As used herein, the term "promoter variant" refers to either isolated naturally occurring or recombinantly prepared variants of reference promoters, such as but not limited to GFAP, S100b, Aldh1L1, NG2, lcn2, and pCAG promoters.

[0088] It is known in the art that promoters from other species are functional, for example, the mouse Aldh1L1 promoter is functional in human cells.Homologues and homologous promoters from other species can be identified using bioinformatics tools known in the art, for example, see Xuan et al., Genome Biol., 6:R72 (2005), Zhao et al., Nucl.Acid Res., 33:D103-107 (2005), and Halees et al., Nucl.Acid Res., 31:3554-3559 (2003).

[0089] Structurally, homologs and variants of a cell type-specific promoter or / and ubiquitous promoter 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.

[0090] A nucleic acid sequence that is substantially identical to SEQ ID NO:1 or SEQ ID NO:3 is characterized as having a complementary nucleic acid sequence capable of hybridizing to SEQ ID NO:1 or SEQ ID NO:3 under highly stringent hybridization conditions.

[0091] In addition to one or more nucleic acids encoding NeuroD1, one or more nucleic acid sequences encoding additional proteins can be included in the expression vector, including, for example, non-NeuroD1 proteins such as reporters, including, but not limited to, beta-galactosidase, green fluorescent protein, and antibiotic resistance reporters.

[0092] 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.

[0093] 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.

[0094] 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 and the nucleic acid encoding EGFP. SEQ ID NO: 9 is inserted into an expression vector for the expression of NeuroD1 and the reporter gene EGFP. Optionally, the IRES and the nucleic acid encoding EGFP are removed, and the remaining operably linked nucleic acid encoding the CAG promoter and NeuroD1 is inserted into an expression vector for the expression of NeuroD1. A WPRE or another enhancer is optionally included.

[0095] Optionally, a reporter gene is included in the recombinant expression vector encoding NeuroD1 (and / or Dlx2). The reporter gene may be included to produce a peptide or protein that serves as a surrogate marker for expression of NeuroD1 (and / or Dlx2) from the recombinant expression vector. As used herein, the term "reporter gene" refers to a gene that, when expressed, is readily detectable, 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).

[0096] The process of introducing genetic material into recipient host cells, such as the transient or stable expression of desired protein encoded by genetic material in host cells, 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 adenovirus, adeno-associated virus and lentivirus.

[0097] 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 / or Dlx2), and then returned to the subject.

[0098] Introduction of a recombinant expression vector containing a nucleic acid encoding NeuroD1 or a functional fragment thereof, and / or a nucleic acid encoding Dlx2 or a functional fragment thereof, into host glial cells in vitro or in vivo to express exogenous NeuroD1 and / or Dlx2 in the host glial cells and convert the glial cells into neurons can be achieved by any of a variety of transfection methodologies.

[0099] Expression of exogenous NeuroD1 and / or Dlx2 in host glial cells to convert the glial cells into neurons is optionally achieved by introducing mRNA encoding NeuroD1 or a functional fragment thereof, and / or mRNA encoding Dlx2 or a fragment thereof into the host glial cells in vitro or in vivo.

[0100] Expression of exogenous NeuroD1 and / or Dlx2 in host glial cells to convert glial cells into neurons is optionally achieved by introducing NeuroD1 protein and / or Dlx2 protein into host glial cells in vitro or in vivo. Details of these and other techniques are known in the art, for example, as described in J. Sambrook and DW Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 3rd Ed., 2001; FMA Russell, 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.

[0101] The nucleic acid encoding NeuroD1 or a functional fragment thereof, and / or Dlx2 or a functional fragment thereof, the mRNA encoding NeuroD1 or a functional fragment thereof, and / or the mRNA encoding Dlx2 or a functional fragment thereof, and / or the expression vector containing NeuroD1 protein and / or Dlx2 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.

[0102] In certain embodiments, the carrier is a particulate carrier, such as a lipid particle, including a liposome, a micelle, a unilamellar, or a multilamellar vesicle, a polymer particle, such as a hydrogel particle, a polyglycolic acid particle, or a polylactic acid particle, an inorganic particle, such as calcium phosphate particles, such as those described elsewhere (e.g., U.S. Pat. No. 5,648,097), and an inorganic / organic particulate carrier, such as those described elsewhere (e.g., U.S. Pat. No. 6,630,486).

[0103] The particulate carriers can be selected from lipid particles, polymer particles, inorganic particles, and inorganic / organic particles. Mixtures of particle types can also be included as particulate pharmaceutically acceptable carriers.

[0104] 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.

[0105] Further description of liposomes and methods related to their preparation and use can be found in Liposomes: A Practical Approach (The Practical Approach Series, 264), V.P. Torchilin and V. Weissig (Eds.), Oxford University Press; 2nd ed., 2003. Further aspects of nanoparticles are described in S.M. Moghimi et al., FASEB J., 19:311-30 (2005).

[0106] Expression of NeuroD1 and / or Dlx2 using a recombinant expression vector is achieved by introducing the expression vector into a eukaryotic or prokaryotic host cell expression system, such as an art-recognized insect cell, mammalian cell, yeast cell, bacterial cell, or any other single- or multicellular organism. The host cell is optionally a primary cell or an immortalized derivative cell. An immortalized cell is one that can be maintained in vitro for at least five replicative passages.

[0107] Host cells containing the recombinant expression vector are maintained under conditions in which NeuroD1 and / or Dlx2 are 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 regard to specific nutrients, oxygen, tension, carbon dioxide, and reduced serum levels, can be selected and optimized by those skilled in the art.

[0108] In some cases, a recombinant expression vector containing a nucleic acid encoding NeuroD1 and / or Dlx2 is introduced into glial cells of a subject. Expression of exogenous NeuroD1 and / or Dlx2 in glial cells "converts" the glial cells into neurons.

[0109] In some cases, a recombinant expression vector containing a nucleic acid encoding NeuroD1 and / or Dlx2, or a functional fragment thereof, is introduced into astrocytes of a subject. Expression of exogenous NeuroD1 and / or exogenous Dlx2 in glial cells "converts" the astrocytes into neurons.

[0110] In some cases, a recombinant expression vector containing a nucleic acid encoding NeuroD1 and / or a nucleic acid encoding Dlx2, or a functional fragment thereof, is introduced into reactive astrocytes of a subject. Expression of exogenous NeuroD1 and / or exogenous Dlx2, or a functional fragment thereof, in reactive astrocytes "converts" the reactive astrocytes into neurons.

[0111] In some cases, a recombinant expression vector containing a nucleic acid encoding NeuroD1 and / or a nucleic acid encoding Dlx2, or a functional fragment thereof, is introduced into a subject's NG2 cells. Expression of exogenous NeuroD1 and / or exogenous Dlx2, or a functional fragment thereof, in the NG2 cells "converts" the NG2 cells into neurons.

[0112] Detection of expression of exogenous NeuroD1 and / or exogenous Dlx2 following introduction of a recombinant expression vector containing a nucleic acid encoding exogenous NeuroD1 and / or a nucleic acid encoding exogenous Dlx2, or a functional fragment thereof, is accomplished using any of a variety of standard methodologies, including, but not limited to, immunoassays for detecting NeuroD1 and / or Dlx2, nucleic acid assays for detecting NeuroD1 and / or Dlx2 nucleic acids, and detection of a reporter gene co-expressed with exogenous NeuroD1 and / or exogenous Dlx2.

[0113] The terms "transforming" and "transformed" are used herein to describe the effect of expression of NeuroD1 or a functional fragment thereof, and / or Dlx2 or a functional fragment thereof, resulting in a change in the phenotype of glial cells, astrocytes, or reactive astrocytes to a neuronal phenotype. Similarly, the phrases "NeuroD1-transformed neuron," "Dlx2-transformed neuron," "NeuroD1- and Dlx2-transformed neuron," and "transformed neuron" are used herein to designate cells containing exogenous NeuroD1 protein or a functional fragment thereof that have a resulting neuronal phenotype.

[0114] The term "phenotype" refers to the well-known detectable characteristics of cells referred to herein.Neuronal phenotype can be, but is not limited to, one or more of neuronal morphology, the expression of one or more neuronal markers, neuronal electrophysiological characteristics, synapse formation, and neurotransmitter release.For example, neuronal phenotype includes, but is not limited to, the characteristic morphological aspects of neurons, such as the presence of dendrites, axons, and dendritic spines, the presence of synaptic proteins in synaptic puncta, the expression and distribution of characteristic neuronal proteins, such as the presence of MAP2 in dendrites, and characteristic electrophysiological signs, such as spontaneous and evoked synaptic events.

[0115] In further examples, glial phenotypes, such as astrocytic and reactive astrocyte phenotypes, generally include, but are not limited to, characteristic morphological aspects of astrocytes and reactive astrocytes, such as a "star-shaped" morphology, and characteristic astrocyte and reactive astrocyte protein expression, such as the presence of glial fibrillary acidic protein (GFAP).

[0116] The term "nucleic acid" refers to an RNA or DNA molecule having two or more nucleotides in any form, including single-stranded, double-stranded, oligonucleotide, or polynucleotide. The term "nucleotide sequence" refers to the order of nucleotides in an oligonucleotide or polynucleotide in the single-stranded form of a nucleic acid.

[0117] The term "NeuroD1 nucleic acid" refers to an isolated NeuroD1 nucleic acid molecule, and includes an isolated NeuroD1 nucleic acid having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the DNA sequence set forth in SEQ ID NO:1 or SEQ ID NO:3, or its complement, or a fragment thereof, or an isolated DNA molecule having a sequence that hybridizes to the nucleic acid set forth in SEQ ID NO:1 or SEQ ID NO:3, or its complement, or a fragment thereof, under highly stringent hybridization conditions.

[0118] The nucleic acid of SEQ ID NO: 3 is an example of an isolated DNA molecule having a sequence that hybridizes under highly stringent 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 the embodiments described herein, including NeuroD1 nucleic acids.

[0119] 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. The nucleic acid probe can be an oligonucleotide of at least 10, 15, 30, 50, or 100 nucleotides in length, sufficient to specifically hybridize to NeuroD1 mRNA or cDNA or its complementary sequence under stringent conditions. The nucleic acid probe can be an oligonucleotide of at least 10, 15, or 20 nucleotides in length, sufficient to specifically hybridize to mRNA or cDNA or its complementary sequence under stringent conditions.

[0120] The term "Dlx2 nucleic acid" refers to an isolated Dlx2 nucleic acid molecule, and includes an isolated Dlx2 nucleic acid having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the DNA sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 12, or its complement, or a fragment thereof, or an isolated DNA molecule having a sequence that hybridizes to the nucleic acid set forth in SEQ ID NO: 10 or SEQ ID NO: 12, or its complement, or a fragment thereof under highly stringent hybridization conditions.

[0121] The nucleic acid of SEQ ID NO: 12 is an example of an isolated DNA molecule having a sequence that hybridizes under highly stringent hybridization conditions to the nucleic acid set forth in SEQ ID NO: 10. A fragment of a Dlx2 nucleic acid is any fragment of a Dlx2 nucleic acid that is operable in the embodiments described herein, including Dlx2 nucleic acids.

[0122] Nucleic acid probes or primers capable of hybridizing to target Dlx2 mRNA or cDNA can be used to detect and / or quantify mRNA or cDNA encoding Dlx2 protein. The nucleic acid probe can be an oligonucleotide of at least 10, 15, 30, 50, or 100 nucleotides in length, sufficient to specifically hybridize to NeuroD1 mRNA or cDNA, or its complementary sequence, under stringent conditions. The nucleic acid probe can be an oligonucleotide of at least 10, 15, or 20 nucleotides in length, sufficient to specifically hybridize to mRNA or cDNA, or its complementary sequence, under stringent conditions.

[0123] The terms "complement" and "complementary" refer to Watson-Crick base pairing between nucleotides, specifically nucleotides hydrogen-bonded to each other with a thymine or uracil residue linked to an adenine residue by two hydrogen bonds, and cytosine and guanine residues linked by three hydrogen bonds. Generally, nucleic acids 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 the region of the nucleotide sequence 5'-TTAGCTGG-3'.

[0124] The terms "hybridization" and "hybridizing" refer to the pairing and binding of complementary nucleic acids.As is well known in the art, hybridization occurs to various degrees between two nucleic acids, depending on factors such as the degree of complementarity of nucleic acids, the melting temperature, Tm, and the stringency of hybridization conditions.The term "stringency of hybridization conditions" refers to the temperature, ionic strength, and composition conditions of the hybridization medium, with respect to certain common additives such as formamide and Denhardt's solution.

[0125] Determining specific hybridization conditions for 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.A. Russell, Ed., Short Protocols in Molecular Biology, Current Protocols; 5th Ed., 2002. Highly stringent hybridization conditions are conditions that allow only hybridization of substantially complementary nucleic acids. Typically, nucleic acids with about 85-100% complementarity are considered highly complementary and hybridize under highly stringent conditions. Intermediate stringency conditions are exemplified by conditions under which nucleic acids with intermediate complementarity, about 50-84%, and high complementarity, hybridize. In contrast, low stringency hybridization conditions are conditions under which nucleic acids with a low degree of complementarity will hybridize.

[0126] 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.

[0127] As known to those skilled in the art, the stringency of hybridization and washing conditions depends on several factors, including the Tm of probe and target and the ionic strength of hybridization and washing conditions.The hybridization and conditions for achieving desired stringency of hybridization 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.

[0128] An example of a highly stringent hybridization condition is hybridization of a nucleic acid of approximately 100 nucleotides in length in a solution containing 6xSSC, 5xDenhardt's solution, 30% formamide, and 100 micrograms / mL denatured salmon sperm at 37°C overnight, followed by washing in a solution of 0.1xSSC and 0.1% SDS at 60°C for 15 minutes. SSC is 0.15M NaCl / 0.015M sodium citrate. Denhardt's solution is 0.02% bovine serum albumin / 0.02% Ficoll / 0.02% polyvinylpyrrolidone. Under highly stringent conditions, SEQ ID NO: 1 and SEQ ID NO: 3 hybridize to the complement of a substantially identical target and do not hybridize to unrelated sequences.

[0129] Provided according to some embodiments described herein are methods for treating a neurological condition in a subject in need thereof, comprising delivering a therapeutically effective amount of NeuroD1 and / or Dlx2 to glial cells in the central or peripheral nervous system of the subject, wherein the therapeutically effective amount of NeuroD1 and / or Dlx2 in the glial cells results in a higher number of neurons in the subject compared to an untreated subject with the same neurological condition, thereby treating the neurological condition.

[0130] Additionally, the conversion of reactive glial cells into neurons reduces neuroinflammatory and neuroinhibitory factors associated with reactive glial cells, thereby making the glial scar tissue more permissive for neuronal growth such that the neurological condition is alleviated.

[0131] As used herein, the term "neurological condition" or "neurological disorder" refers to any condition of a subject's central nervous system that is alleviated, ameliorated, or prevented by additional neurons. Injuries or diseases that result in the loss or inhibition of neurons and / or the loss or inhibition of neuronal function are neurological conditions for treatment by the methods described herein.

[0132] Injuries or diseases that result in the loss or inhibition of glutamatergic neurons and / or the loss or inhibition of glutamatergic neuron function are neurological conditions that can be treated as described herein. Loss or inhibition of other types of neurons, such as GABAergic, cholinergic, dopaminergic, norepinephrine, or serotonergic neurons, can be treated in a similar manner.

[0133] As used herein, the term "therapeutically effective amount" 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 with signs and / or symptoms of a neurological condition.

[0134] As used herein, the terms "treat," "treatment," "treating," "NeuroD1 treatment," "Dlx2 treatment," and "NeuroD1 and Dlx2 treatment," or grammatical equivalents, refer to 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, including, but not limited to, therapeutic treatment and / or prophylactic treatment.

[0135] The signs and symptoms of neurological conditions are well known in the art, along with methods for detecting and assessing such signs and symptoms.

[0136] In some cases, a combination of therapies for a subject's neurological condition may be administered.

[0137] According to certain embodiments, additional agents or therapeutic treatments administered to a subject to treat the effects of disruption of normal blood flow in the CNS in an individual subject in need thereof include, 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.

[0138] The term "subject" refers to humans, and also to non-human mammals such as, but not limited to, non-human primates, cats, dogs, sheep, goats, horses, cows, pigs, and rodents, including, but not limited to, mice and rats, and non-mammalian animals such as, but not limited to, birds, poultry, reptiles, and amphibians.

[0139] Embodiments of the compositions and methods of the present invention are illustrated 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. [Example]

[0140] Example 1 - Histology of intracerebral hemorrhage 0.2 μL of collagenase was injected into the mouse striatum. Data were collected 1, 2, 8, and 29 days later, and DAB and iron staining was performed. Figures 1A and 1B show DAB staining of Iba1 and S100b accompanied by iron staining 1 to 29 days after ICH induction.

[0141] These results demonstrated the morphological changes of astrocytes and microglia after ICH, as well as the process of ferric iron accumulation. These results provided a reference for selecting the time point to intervene to treat ICH.

[0142] Example 2 - In vivo conversion of reactive astrocytes into neurons in a mouse model of intracerebral hemorrhage (short term) A series of experiments were performed to evaluate the in vivo conversion of reactive astrocytes into neurons after treatment with AAV5 viruses encoding NeuroD1 and Dlx2. ICH induction on day 0 was performed by intrastriatal injection of 0.2 μL of collagenase. Mice were treated with 1 μL of AAV5-GFA104-cre:3 × 10 11 , 1 μL AAV5-CAG-flex-GFP: 3.4 × 10 11 , 1 μL AAV5-CAG-flex-ND1-GFP: 4.55 × 10 11 , or 1 μL of AAV5-CAG-flex-Dlx2-GFP: 2.36 × 10 12 were injected 2, 4, and 7 days after ICH induction. Data on astrocyte conversion were collected on day 21.

[0143] Figures 2A and 2B show a schematic diagram of the short-term in vivo conversion experiment. Various virus injection times (immediately after, 2 dps, 4 dps, and 7 dps) were performed to find the optimal time window for recovering from ICH. Figures 2C-2P reveal corresponding immunostaining for GFP, GFAP, and NeuN. The results consistently showed a decrease in conversion, a decrease in neuronal density, and an increase in reactive astrocytes around the injury core with a delay in the virus injection time point.

[0144] These results demonstrate that earlier viral injection has a better therapeutic effect: Injecting the virus immediately after or within two days after a stroke can achieve a higher conversion rate, resulting in less reactive astrocytes.

[0145] Example 3 - In vivo conversion of reactive astrocytes into neurons in a mouse model of intracerebral hemorrhage (long-term) A series of experiments were performed to evaluate the in vivo conversion of reactive astrocytes into neurons after treatment with AAV5 viruses encoding NeuroD1 and Dlx2. ICH induction on day 0 was performed by intrastriatal injection of 0.35 μL of collagenase. Mice were treated with 1 μL of AAV5-GFA104-cre:3 × 10 11 , 1 μL AAV5-CAG-flex-GFP: 3.4 × 10 11 , 1 μL AAV5-CAG-flex-ND1-GFP: 4.55 × 10 11 , or 1 μL of AAV5-CAG-flex-Dlx2-GFP: 2.36 × 10 12 The mice were injected 2 and 7 days after ICH induction. Two months after induction, the mice were harvested and data were collected.

[0146] Figure 3A shows the experimental design for the long-term recovery effect of ND1 and Dlx2 on ICH. Figures 3B-3G show immunostaining for GFP, GFAP, and NeuN. Figures 3B and 3C show that when the virus was injected immediately after ICH, almost all GFP-positive cells had a neuronal morphology and expressed NeuN two months after viral infection. Figure 3D shows the results two months after viral infection when the virus was injected two days after ICH. Infection was not widespread, which may be due to the virus injection point being too close to the ventricle. Figures 3E and 3F show immunostaining two months after viral infection after injection 7 days after ICH. The conversion rate was lower than that after viral injection immediately after ICH. Figure 3H shows a comparison of the conversion rate and neuronal density for various viral injection time points (2 dps was excluded due to low infection). This indicates that immediate viral injection may be an ideal time point for treating ICH.

[0147] These results demonstrate that early viral injection after ICH may result in better recovery outcomes, higher conversion rates, and higher neuronal densities.

[0148] Example 4 - Evaluation of viral vectors in in vivo transduction after ICH: AAV9-1.6kb-GFAP-cre-flex system To achieve higher infection and higher expression of ND1 and Dlx2, we developed the following viral system: AAV9-1.6kb-GFAP-cre with flex-ND1-mCherry and flex-Dlx2-mCherry. The results in Figures 4A-4F suggest that AAV9 can achieve higher expression of ND1 and Dlx2, but with more leakage than AAV5. However, the treatment still showed a less dense glial scar reflected by GFAP and slightly better vascular morphology as indicated by AQP4. Iba1 signaling was stronger in the treatment than in the control, while the role of microglia in the transformation was unclear.

[0149] These results demonstrate that, despite leakage, the AAV9-1.6kb-GFAP-cre-flex system can be an effective alternative for in vivo astrocyte-to-neuron conversion after ICH.

[0150] Example 5 - Evaluation of viral vectors for in vivo transduction after ICH: AAV5-1.6kb-GFAP-cre-flex system and the effect of injury on transduction rate Figures 5A-5E show infection by the AAV5 system. Few neurons were GFP-positive, indicating that this system was relatively clean. Furthermore, the recovery effect was observed in various ways. Downregulation of GFAP signals around the injury core, increased neuronal density, and more AQP4 signals around blood vessels suggest restoration of the blood-brain barrier. This demonstrated that the AAV5 system is an effective system for converting astrocytes into neurons in vivo and treating ICH. Figures 6A-6E show the effect of injury on the conversion rate. The more severe the injury, the lower the conversion rate.

[0151] Example 6 - Deducing the ideal time point for therapeutic application for in vivo conversion after ICH Figure 7 shows viral infection over a 4-day period, 2 days after collagenase injection. A hematoma was observed, but there was no viral signal within the hematoma. Significant viral infection was observed in the area surrounding the hematoma. The presence of the hematoma may have hindered viral infection and recovery after ICH. To address this issue, one or more small molecules can be administered to inhibit hematoma growth, and / or one or more additional doses of virus can be administered after the hematoma has been absorbed to improve ND1 and Dlx2 expression.

[0152] Figure 8 reveals that it is beneficial to take action as soon as ICH occurs. Astrocytes begin to proliferate after ICH, peaking at approximately 5 dps. Figure 8 also reveals that a dense glial scar formed at 8 dps. The glial scar isolated the injury core and rendered the damage irreversible. Therefore, to avoid the formation of a glial scar, treatment can be applied as early as possible (e.g., less than 5 dps, less than 4 dps, less than 3 dps, less than 2 dps, less than 1 dps, within 12 hours after stroke, within 8 hours after stroke, or within 6 hours after stroke).

[0153] Example 7 - Other Materials Figure 9 shows that early virus injection can result in smaller injury cores and higher conversion rates. Figure 10 shows a rare situation in which virus injection at 7 days post-ICH may be superior to injection at 2 days post-ICH. However, the initial conditions were measured at different time points after ICH. Figure 11 shows a simplified diagram of the ICH process and the corresponding treatments for each step. This technique can be used for long-term recovery after ICH.

[0154] Example 8 - Additional Embodiments Embodiment 1. A method for (1), (2), (3), or (4) in a mammal that has suffered a hemorrhagic stroke and is in need of (1) generating new glutamatergic neurons, (2) increasing survival of GABAergic neurons, (3) generating new non-reactive astrocytes, or (4) reducing the number of reactive astrocytes, comprising administering to said mammal a composition comprising an exogenous nucleic acid encoding a neurogenic differentiation 1 (NeuroD1) polypeptide or a biologically active fragment thereof, and an exogenous nucleic acid encoding a distal-less homeobox 2 (Dlx2) polypeptide or a biologically active fragment thereof.

[0155] Embodiment 2. The method of embodiment 1, wherein said mammal is a human.

[0156] Embodiment 3. The method of embodiment 1, wherein the hemorrhagic stroke results from a condition selected from the group consisting of ischemic stroke; physical injury; tumor; inflammation; infection; generalized ischemia caused by cardiac arrest or severe low blood pressure (shock); hypoxic-ischemic encephalopathy caused by hypoxia, hypoglycemia, or anemia; meningitis; and dehydration; or a combination of any two or more thereof.

[0157] Embodiment 4. The method of embodiment 1, wherein the administering step comprises delivering an expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof, and an expression vector comprising a nucleic acid encoding a Dlx2 polypeptide or a biologically active fragment thereof to the location of the hemorrhagic stroke in the brain.

[0158] Embodiment 5. The method of embodiment 1 or 2, wherein the administering step comprises delivering a recombinant viral expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof, and a recombinant viral expression vector comprising a nucleic acid encoding a Dlx2 polypeptide or a biologically active fragment thereof to the location of the hemorrhagic stroke in the brain.

[0159] Embodiment 6. The method of any one of embodiments 1 to 3, wherein the administering step comprises delivering a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof, and a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a Dlx2 polypeptide or a biologically active fragment thereof to the location of the hemorrhagic stroke in the brain.

[0160] Embodiment 7. The method of any one of embodiments 1-6, wherein said administering comprises a stereotactic intracranial injection into the location of the hemorrhagic stroke in the brain.

[0161] Embodiment 8. The method of any one of embodiments 1 to 7, wherein the administering step further comprises administering an exogenous nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof, and an exogenous nucleic acid encoding a Dlx2 polypeptide or a biologically active fragment thereof, in an expression vector, a recombinant viral expression vector, or a recombinant adeno-associated viral expression vector.

[0162] Embodiment 9. A composition comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof, and a nucleic acid encoding a Dlx2 polypeptide or a biologically active fragment thereof. 10 ~10 14 2. The method of embodiment 1, comprising about 1 μL to about 500 μL of a pharmaceutically acceptable carrier containing adeno-associated virus at a concentration of adeno-associated virus particles / mL of carrier.

[0163] Embodiment 10. The method of embodiment 9, wherein the composition is injected into the brain of said mammal at a controlled flow rate of about 0.1 μL / min to about 5 μL / min.

[0164] Embodiment 11. A method for (1), (2), (3), or (4) in a mammal that has suffered a hemorrhagic stroke and is in need of (1) generating new GABAergic and glutamatergic neurons, (2) increasing survival of GABAergic and glutamatergic neurons, (3) generating new non-reactive astrocytes, or (4) reducing the number of reactive astrocytes, comprising administering to said mammal, within three days of said hemorrhagic stroke, a composition comprising an exogenous nucleic acid encoding a neurogenic differentiation 1 (NeuroD1) polypeptide or a biologically active fragment thereof, and an exogenous nucleic acid encoding a distal-less homeobox 2 (Dlx2) polypeptide or a biologically active fragment thereof.

[0165] Embodiment 12 The method of embodiment 11, wherein said mammal is a human.

[0166] Embodiment 13. The method of embodiment 11, wherein the hemorrhagic stroke is caused by a condition selected from the group consisting of bleeding in the brain; an aneurysm; an intracranial hematoma; a subarachnoid hemorrhage; a brain trauma; high blood pressure; a weak blood vessel; a vascular malformation; an ischemic stroke; a physical injury; a tumor; inflammation; an infection; systemic ischemia caused by cardiac arrest or severe low blood pressure (shock); hypoxic-ischemic encephalopathy caused by hypoxia, hypoglycemia, or anemia; meningitis; and dehydration; or a combination of any two or more thereof.

[0167] Embodiment 14. The method of embodiment 11, wherein the administering step comprises delivering an expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof, and an expression vector comprising a nucleic acid encoding a Dlx2 polypeptide or a biologically active fragment thereof to the location of the hemorrhagic stroke in the brain.

[0168] Embodiment 15. The method of embodiment 11 or 12, wherein the administering step comprises delivering a recombinant viral expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof, and a recombinant viral expression vector comprising a nucleic acid encoding a Dlx2 polypeptide or a biologically active fragment thereof to the location of the hemorrhagic stroke in the brain.

[0169] Embodiment 16. The method of any one of embodiments 11 to 13, wherein the administering step comprises delivering a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof, and a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a Dlx2 polypeptide or a biologically active fragment thereof to the location of the hemorrhagic stroke in the brain.

[0170] Embodiment 17 The method of any one of embodiments 11-16, wherein said administering step comprises a stereotactic intracranial injection into the location of the hemorrhagic stroke in the brain.

[0171] Embodiment 18. The method of any one of embodiments 11 to 17, wherein the administering step further comprises administering an exogenous nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof, and an exogenous nucleic acid encoding a Dlx2 polypeptide or a biologically active fragment thereof, in an expression vector, a recombinant viral expression vector, or a recombinant adeno-associated viral expression vector.

[0172] Embodiment 19. A composition comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof, and a nucleic acid encoding a Dlx2 polypeptide or a biologically active fragment thereof. 10 ~10 14 12. The method of embodiment 11, comprising about 1 μL to about 500 μL of a pharmaceutically acceptable carrier containing adeno-associated virus at a concentration of adeno-associated virus particles / mL of carrier.

[0173] Embodiment 20. The method of embodiment 19, wherein the composition is injected into the brain of said mammal at a controlled flow rate of about 0.1 μL / min to about 5 μL / min.

[0174] array SEQ ID NO:1 - Human NeuroD1 nucleic acid sequence encoding human NeuroD1 protein - 1071 nucleotides, including a stop codon SEQ ID NO:2 - Human NeuroD1 amino acid sequence encoded by SEQ ID NO:1 - 356 amino acids MTKSYSESGLMGEPQPQGPPSWTDECLSSQDEEHEADKKEDDLEAMNAEEDSLRNGGEEEDEDEDEDLEEEEEEEDDDQKPKRRGPKKKKMTKARLERFKLRRMKANARERNRMHGLNAALDNLRKVVPCYSKTQKLSKIETLRLAKNYIWALSEILRSGKSPDLVSFVQTLCKGLSQ PTTNLVAGCLQLNPRTFLPEQNQDMPPHLPTASASFPVHPYSYQSPGLPSPPYGTMDSSHVFHVKPPPHAYSAALEPFFESPLTDCTSPSFDGPLSPPLSINGNFSFKHEPSAEFEKNYAFTMHYPAATLAGAQSHGSIFSGTAAPRCEIPIDNIMSFDSHSHHERVMSAQLNAIFHD SEQ ID NO:3 - Mouse NeuroD1 nucleic acid sequence encoding mouse NeuroD1 protein - 1074 nucleotides, including a stop codon SEQ ID NO: 4: Mouse NeuroD1 amino acid sequence encoded by SEQ ID NO: 3 - 357 amino acids MTKSYSESGLMGEPQPQGPPSWTDECLSSQDEEHEADKKEDELEAMNAEEDSLRNGGEEEEEDEDLEEEEEEEEEEDQKPKRRGPKKKKMTKARLERFKLRRMKANARERNRMHGLNAALDNLRKVVPCYSKTQKLSKIETLRLAKNYIWALSEILRSGKSPDLVSFVQTLCKGLSQ PTTNLVAGCLQLNPRTFLPEQNPDMPPHLPTASASFPVHPYSYQSPGLPSPPYGTMDSSHVFHVKPPPHAYSAALEPFFESPLTDCTSPSFDGPLSPPLSINGNFSFKHEPSAEFEKNYAFTMHYPAATLAGPQSHGSIFSSGAAAPRCEIPIDNIMSFDSHSHHERVMSAQLNAIFHD Mouse LCN2 promoter - SEQ ID NO:5 Human GFAP promoter - SEQ ID NO: 6 Mouse Aldh1L1 promoter - SEQ ID NO: 7 Human NG2 promoter - SEQ ID NO:8 CAG::NeuroD1-IRES-GFP-SEQ ID NO:9 TATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAACACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCG GGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAAT SEQ ID NO: 10 - Human Dlx2 nucleic acid sequence encoding human Dlx2 protein ATGACTGGAGTCTTTGACAGTCTAGTGGCTGATATGCACTCGACCCAGATCGCCGCCTCCAGCACGTACCACCAGCACCAGCAGCCCCCGAGCGGCGGCGGCGCCGGCCCGGGTGGCAACAGCAGCAGCAGCAGCAGCCTCCACAAGCCCCAGGAGTCGCCCACCCTTCCGGTGTCCACCGCCACCGACAGCAGCTACTACACCAACCAGCAGCACCCGGCGGGCGGCGGCGGCGGCGGGGGCTCGCCCTACGCGCACATGGGTTCCTACCAGTACCAAGCCAGCGGCCTCAACAACGTCCCTTACTCCGCCAAGAGCAGCTATGACCTGGGCTACACCGCCGCCTACACCTCCTACGCTCCCTATGGAACCAGTTCGTCCCCAGCCAACAACGAGCCTGAGAAGGAGGACCTTGAGCCTGAAATTCGGATAGTGAACGGGAAGCCAAAGAAAGTCCGGAAACCCCGCACCATCTACTCCAGTTTCCAGCTGGCGGCTCTTCAGCGGCGTTTCCAAAAGACTCAATACTTGGCCTTGCCGGAGCGAGCCGAGCTGGCGGCCTCTCTGGGCCTCACCCAGACTCAGGTCAAAATCTGGTTCCAGAACCGCCGGTCCAAGTTCAAGAAGATGTGGAAAAGTGGTGAGATCCCCTCGGAGCAGCACCCTGGGGCCAGCGCTTCTCCACCTTGTGCTTCGCCGCCAGTCTCAGCGCCGGCCTCCTGGGACTTTGGTGTGCCGCAGCGGATGGCGGGCGGCGGTGGTCCGGGCAGTGGCGGCAGCGGCGCCGGCAGCTCGGGCTCCAGCCCGAGCAGCGCGGCCTCGGCTTTTCTGGGCAACTACCCCTGGTACCACCAGACCTCGGGATCCGCCTCACACCTGCAGGCCACGGCGCCGCTGCTGCACCCCACTCAGACCCCGCAGCCGCATCACCACCACCACCATCACGGCGGCGGGGGCGCCCCGGTGAGCGCGGGGACGATTTTCTAA SEQ ID NO:11 -- Human Dlx2 amino acid sequence encoded by SEQ ID NO:10 MTGVFDSLVADMHSTQIAASSTYHQHQQPPSGGGAGPGGNSSSSSSLHKPQESPTLPVSTATDSSYYTNQQHPAGGGGGGGSPYAHMGSYQYQASGLNNVPYSAKSSYDLGYTAAYTSYAPYGTSSSPANNEPEKEDLEPEIRIVNGKPKKVRKPRTIYSSFQL AALQRRFQKTQYLALPERAELAASLGLTQTQVKIWFQNRRSKFKKMWKSGEIPSEQHPGASASPPCASPPVSAPASWDFGVPQRMAGGGGPGSGGAGSSGSSPSSAASAFLGNYPWYHQTSGSASHLQATAPLLHPTQTPQPHHHHHHHGGGAPVSAGTIF SEQ ID NO: 12—Mouse Dlx2 nucleic acid sequence encoding mouse Dlx2 protein ATGACTGGAGTCTTTGACAGTCTGGTGGCTGATATGCACTCGACCCAGATCACCGCCTCCAGCACGTACCACCAGCACCAGCAGCCCCCGAGCGGTGCGGGCGCCGGCCCTGGCGGCAACAGCAACAGCAGCAGCAGCAACAGCAGCCTGCACAAGCCCCAGGAGTCGCCAACCCTCCCGGTGTCCACGGCTACGGACAGCAGCTACTACACCAACCAGCAGCACCCGGCGGGCGGCGGCGGCGGGGGGGCCTCGCCCTACGCGCACATGGGCTCCTACCAGTACCACGCCAGCGGCCTCAACAATGTCTCCTACTCCGCCAAAAGCAGCTACGACCTGGGCTACACCGCCGCGTACACCTCCTACGCGCCCTACGGCACCAGTTCGTCTCCGGTCAACAACGAGCCGGACAAGGAAGACCTTGAGCCTGAAATCCGAATAGTGAACGGGAAGCCAAAGAAAGTCCGGAAACCACGCACCATCTACTCCAGTTTCCAGCTGGCGGCCCTTCAACGACGCTTCCAGAAGACCCAGTATCTGGCCCTGCCAGAGCGAGCCGAGCTGGCGGCGTCCCTGGGCCTCACCCAAACTCAGGTCAAAATCTGGTTCCAGAACCGCCGATCCAAGTTCAAGAAGATGTGGAAAAGCGGCGAGATACCCACCGAGCAGCACCCTGGAGCCAGCGCTTCTCCTCCTTGTGCCTCCCCGCCGGTCTCGGCGCCAGCATCCTGGGACTTCGGCGCGCCGCAGCGGATGGCTGGCGGCGGCCCGGGCAGCGGAGGCGGCGGTGCGGGCAGCTCTGGCTCCAGCCCGAGCAGCGCCGCCTCGGCCTTTCTGGGAAACTACCCGTGGTACCACCAGGCTTCGGGCTCCGCTTCACACCTGCAGGCCACAGCGCCACTTCTGCATCCTTCGCAGACTCCGCAGGCGCACCATCACCACCATCACCACCACCACGCAGGCGGGGGCGCCCCGGTGAGCGCGGGGACGATTTTCTAA SEQ ID NO:13 - Mouse Dlx2 amino acid sequence encoded by SEQ ID NO:12 MTGVFDSLVADMHSTQITASSTYHQHQQPPSGAGAGPGGNSNSSSSNSSLHKPQESPTLPVSTATDSSYYTNQQHPAGGGGGASPYAHMGSYQYHASGLNNVSYSAKSSYDLGYTAAYTSYAPYGTSSSPVNNEPDKEDLEPEIRIVNGKPKKVRKPRTIYSSFQ LAALQRRFQKTQYLALPERAELAASLGLTQTQVKIWFQNRRSKFKKMWKSGEIPTEQHPGASASPPCASPPVSAPASWDFGAPQRMAGGGPGSGGGAGSSGSSPSSAASAFLGNYPWYHQASGSASHLQATAPLLHPSQTPQAHHHHHHHHHAGGGAPVSAGTIF

[0175] Other embodiments While the present invention has been described in conjunction with its detailed description, it should be understood that the foregoing description is illustrative of, but not intended to limit, the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. 1. A method for (1), (2), (3), or (4) in a mammal that has suffered a hemorrhagic stroke and is in need of (1) generating new glutamatergic neurons, (2) increasing survival of GABAergic neurons, (3) generating new non-reactive astrocytes, or (4) reducing the number of reactive astrocytes, comprising administering to the mammal a composition comprising an exogenous nucleic acid encoding a neurogenic differentiation 1 (NeuroD1) polypeptide or a biologically active fragment thereof, and an exogenous nucleic acid encoding a distal-less homeobox 2 (Dlx2) polypeptide or a biologically active fragment thereof.

2. The method of claim 1 , wherein the mammal is a human.

3. 2. The method of claim 1, wherein the hemorrhagic stroke is caused by a condition selected from the group consisting of ischemic stroke; physical injury; tumor; inflammation; infection; generalized ischemia caused by cardiac arrest or severe low blood pressure (shock); hypoxic-ischemic encephalopathy caused by hypoxia, hypoglycemia, or anemia; meningitis; and dehydration; or a combination of any two or more thereof.

4. The method of claim 1, wherein the administering step comprises delivering an expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof, and an expression vector comprising a nucleic acid encoding a Dlx2 polypeptide or a biologically active fragment thereof to the location of the hemorrhagic stroke in the brain.

5. The method of claim 1 or 2, wherein the administering step comprises delivering a recombinant viral expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof, and a recombinant viral expression vector comprising a nucleic acid encoding a Dlx2 polypeptide or a biologically active fragment thereof to the location of the hemorrhagic stroke in the brain.

6. 4. The method of claim 1, wherein the administering step comprises delivering a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof, and a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a Dlx2 polypeptide or a biologically active fragment thereof to the location of the hemorrhagic stroke in the brain.

7. 7. The method of any one of claims 1 to 6, wherein the administering step comprises stereotactic intracranial injection into the location of the hemorrhagic stroke in the brain.

8. 8. The method of claim 1, wherein the administering step further comprises administering the exogenous nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof and the exogenous nucleic acid encoding a Dlx2 polypeptide or a biologically active fragment thereof in an expression vector, a recombinant virus expression vector, or a recombinant adeno-associated virus expression vector.

9. 10. The composition comprises a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof, and a nucleic acid encoding a Dlx2 polypeptide or a biologically active fragment thereof. １０ ~10 １４ 10. The method of claim 1, comprising about 1 μL to about 500 μL of a pharmaceutically acceptable carrier containing adeno-associated virus at a concentration of adeno-associated virus particles / mL of carrier.

10. 10. The method of claim 9, wherein the composition is injected into the mammalian brain at a controlled flow rate of about 0.1 μL / min to about 5 μL / min.

11. 1. A method for (1), (2), (3), or (4) in a mammal that has suffered a hemorrhagic stroke and is in need of (1) generating new GABAergic and glutamatergic neurons, (2) increasing the survival of GABAergic and glutamatergic neurons, (3) generating new non-reactive astrocytes, or (4) reducing the number of reactive astrocytes, the method comprising administering to the mammal, within three days of the hemorrhagic stroke, a composition comprising an exogenous nucleic acid encoding a neurogenic differentiation 1 (NeuroD1) polypeptide or a biologically active fragment thereof, and an exogenous nucleic acid encoding a distal-less homeobox 2 (Dlx2) polypeptide or a biologically active fragment thereof.

12. The method of claim 11 , wherein the mammal is a human.

13. 12. The method of claim 11, wherein the hemorrhagic stroke is caused by a condition selected from the group consisting of bleeding in the brain; an aneurysm; an intracranial hematoma; a subarachnoid hemorrhage; a brain trauma; high blood pressure; a weak blood vessel; a vascular malformation; an ischemic stroke; a physical injury; a tumor; inflammation; an infection; generalized ischemia caused by cardiac arrest or severe low blood pressure (shock); hypoxic-ischemic encephalopathy caused by hypoxia, hypoglycemia, or anemia; meningitis; and dehydration; or a combination of any two or more thereof.

14. The method of claim 11, wherein the administering step comprises delivering an expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof, and an expression vector comprising a nucleic acid encoding a Dlx2 polypeptide or a biologically active fragment thereof to the location of the hemorrhagic stroke in the brain.

15. The method of claim 11 or 12, wherein the administering step comprises delivering a recombinant viral expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof, and a recombinant viral expression vector comprising a nucleic acid encoding a Dlx2 polypeptide or a biologically active fragment thereof to the location of the hemorrhagic stroke in the brain.

16. 14. The method of any one of claims 11 to 13, wherein the administering step comprises delivering a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof, and a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a Dlx2 polypeptide or a biologically active fragment thereof to the location of the hemorrhagic stroke in the brain.

17. 17. The method of any one of claims 11 to 16, wherein the administering step comprises stereotactic intracranial injection into the location of the hemorrhagic stroke in the brain.

18. 18. The method of any one of claims 11 to 17, wherein the administering step further comprises administering the exogenous nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof and the exogenous nucleic acid encoding a Dlx2 polypeptide or a biologically active fragment thereof in one expression vector, one recombinant virus expression vector, or one recombinant adeno-associated virus expression vector.

19. 10. The composition comprises a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof, and a nucleic acid encoding a Dlx2 polypeptide or a biologically active fragment thereof. １０ ~10 １４ 12. The method of claim 11, comprising about 1 μL to about 500 μL of a pharmaceutically acceptable carrier containing adeno-associated virus at a concentration of adeno-associated virus particles / mL of carrier.

20. 20. The method of claim 19, wherein the composition is injected into the mammalian brain at a controlled flow rate of about 0.1 μL / min to about 5 μL / min.

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