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  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4702—Regulators; Modulating activity
• • A—HUMAN NECESSITIES
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  • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
  • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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  • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
  • C12N2750/00011—Details
  • C12N2750/14011—Parvoviridae
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  • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
  • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

• Traumatic brain injury is one of the leading causes of death and disability all over the world.
• the CDC has reported that about 1.7 million people needed medical care for TBI each year in the US, at a cost of more than 77 billion dollars yearly.
• TBI Traumatic brain injury
• 50 million people are affected by TBI at a cost of 400 billion dollars annually (Maas et al., Lancet Neurol., 16(12):987-1048, 2017).
• TBI causes acute damage to the brain tissue, and also results in secondary injuries to the nervous system, leading to the consequences of chronic physical and/or mental deficits. TBI results in blood brain barrier breakdown, microgliosis, astrogliosis, and neuronal degeneration.
• the adult mammalian brain lacks the ability to regenerate neurons after injury and there is a lack of treatments capable of promoting neuronal regeneration following TBI. There is a continuing need for treatments promoting repair of the damaged brain after TBI.
• Methods of treating traumatic brain injury are provided according to aspects of the present disclosure including: converting reactive astrocytes to functional neurons by providing exogenous neurogenic differentiation 1 (NeuroDl, also called ND1 herein) to at least one reactive astrocyte in a damaged region of a subject’s brain.
• the TBI is a closed head injury.
• the damaged region of the brain includes non-functional neurons and reactive astrocytes due to the TBI.
• the non- functional neurons are selected from the group consisting of dead neurons, dying neurons, and a combination thereof.
• non- functional neurons present in the damaged region of the brain are detected by a functional MRI (fMRI).
• fMRI functional MRI
• the subject is human.
• Methods of treating TBI including: converting reactive astrocytes to functional neurons by providing exogenous NeuroDl to at least one reactive astrocyte in a damaged region of a subject’s brain wherein the damaged region of the brain includes non-functional neurons and reactive astrocytes due to the TBI.
• the presence of non-functional neurons and reactive astrocytes in the damaged region are not primarily due to bleeding in the damaged region.
• the presence of non-functional neurons and reactive astrocytes are not primarily due to ischemia in the damaged region.
• the TBI is a closed head injury.
• the non-functional neurons are dead neurons.
• the non- functional neurons are dying neurons.
• non-functional neurons present in the damaged region of the brain are detected by a functional MRI (fMRI).
• the subject is human.
• providing the exogenous NeuroDl includes providing exogenous NeuroDl to the at least one reactive astrocyte at a first treatment time in the range of about two days to about ten days after the traumatic brain injury.
• the traumatic brain injury causes a period of astrogliosis in the damaged region
• providing the exogenous NeuroDl includes providing exogenous NeuroDl to the at least one reactive astrocyte at a first treatment time during the period of astrogliosis or within four weeks after the period of astrogliosis.
• providing the exogenous NeuroDl includes providing exogenous NeuroDl to the at least one reactive astrocyte at a second treatment time after the first treatment time and during the period of astrogliosis or within four weeks after the period of astrogliosis.
• providing the exogenous NeuroDl includes providing exogenous NeuroDl to the at least one reactive astrocyte at a third treatment time after the second treatment time and during the period of astrogliosis or within four weeks after the period of astrogliosis.
• providing the exogenous NeuroDl includes administering a recombinant expression vector to the subject, wherein the recombinant expression vector includes a nucleic acid sequence encoding NeuroDl.
• providing the exogenous NeuroDl includes administering a recombinant expression vector to the subject, wherein the recombinant expression vector is a viral expression vector including a nucleic acid sequence encoding NeuroDl.
• providing the exogenous NeuroDl includes administering a recombinant expression vector to the subject, wherein the recombinant expression vector is a recombinant adeno-associated virus expression vector, and wherein the recombinant adeno-associated virus vector includes a nucleic acid sequence encoding NeuroDl.
• the nucleic acid sequence encoding NeuroDl is operably linked to a promoter.
• the promoter is a glial-cell specific promoter.
• the glial-cell specific promoter is a glial fibrillary acidic protein (GFAP) promoter.
• GFAP glial fibrillary acidic protein
• the GFAP promoter is a human GFAP (hGFP) promoter.
• no exogenous transcription factor other than NeuroDl is provided to the at least one reactive astrocyte.
• the NeuroDl includes an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, a functional fragment of SEQ ID NO: 2, a functional fragment of SEQ ID NO: 4, an amino acid sequence having at least 85% identity to SEQ ID NO: 2, and an amino acid sequence having at least 85% identity to SEQ ID NO: 4.
• the NeuroDl is encoded by a nucleic acid sequence including SEQ ID NO: 1, a nucleic acid sequence having at least 85% identity to SEQ ID NO: 1, a nucleic acid sequence including SEQ ID NO: 3, or a nucleic acid sequence having at least 85% identity to SEQ ID NO: 3.
• providing the exogenous NeuroDl includes injection into the damaged region of the brain.
• the nucleic acid sequence encoding NeuroDl is present in a virus particle.
• providing the exogenous NeuroDl includes administering about 10 7 to about 10 14 virus particles to the damaged brain region of the subject.
• a composition including NeuroDl are provided in the manufacture of a medicament for converting reactive astrocytes to functional neurons in a damaged region of a subject’s brain, wherein the damaged region of the brain includes non- functional neurons and reactive astrocytes, due to a TBI.
• the non-functional neurons are dead neurons.
• the non-functional neurons are dying neurons.
• the traumatic brain injury is a closed head injury.
• the NeuroDl is encoded by a nucleic acid sequence includes a nucleic acid sequence having at least 85% identity to SEQ ID NO: 1.
• the nucleic acid encoding NeuroDl includes a nucleic acid sequence having at least 85% identity to SEQ ID NO: 3.
• the NeuroDl includes an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, a functional fragment of SEQ ID NO: 2, a functional fragment of SEQ ID NO: 4, an amino acid sequence having at least 85% identity to SEQ ID NO: 2, and an amino acid sequence having at least 85% identity to SEQ ID NO: 4.
• the NeuroDl is encoded by a nucleic acid sequence included in a recombinant expression vector.
• the nucleic acid sequence encoding NeuroDl is operably linked to a promoter.
• the promoter is a glial-cell specific promoter.
• the glial-cell specific promoter is a GFAP promoter.
• the GFAP promoter is an hGFP promoter.
• the NeuroDl is encoded by a nucleic acid sequence included a viral expression vector.
• the NeuroDl is encoded by a nucleic acid sequence included a recombinant adeno-associated virus expression vector.
• Figure 1A shows aspects of establishment of a focal closed head injury model for study of treatment of traumatic brain injury; the model includes used of an electric-magnet controlled device to induce a focal closed head injury to the motor cortex shown diagrammatically on a mouse head;
• Figure IB is a schematic illustration of the timeline for injury induction and pathology investigation
• Figure 1C is a set of images showing results of immunostaining for a neuronal marker (NeuN) and an astrocytic marker (GFAP) which reflected the cell density of surviving neurons and reactive astrocytes in mouse brain from sham-traumatic brain injury mice (Sham-TBI group) or in mouse brain from traumatic brain injury mice (TBI group) at the indicated time points following traumatic brain injury;
• Neuronal marker Neuronal marker
• GFAP astrocytic marker
• Figure ID is a graph showing NeuN density significantly decreased in the injury core
• Figure IE is a graph showing NeuN density significantly decreased in the peri-injury area
• Figure IF is a graph showing that reactive astrocyte density significantly increased in the total injury area
• Figure 1G is a set of images showing results of co-immunostaining for microglia marker (Ibal), astrocytic marker (GFAP), and cell proliferation marker (Ki67) in mouse brain from sham-traumatic brain injury mice (Sham-TBI group) or in mouse brain from traumatic brain injury mice (TBI group) at the indicated time points following traumatic brain injury, showing the neuroinflammation process at these early time points after TBI;
• Ibal microglia marker
• GFAP astrocytic marker
• Ki67 cell proliferation marker
• Figure 1H is a graph showing that proliferation rate of microglia cells reached a peak around 1 day after TBI and proliferation rate of astrocytes reached a peak around 4 days after TBI;
• Figure 2A illustrates the definition of injury core and peri-injury area of mouse motor cortex in a CHI model
• FIG. 2B is a set of images showing results of immunostaining of damaged brain tissue at early time points, 6 hours and 4 days, after CHI.
• the immunostaining results showed that a cell apoptosis marker, TUNEL, colocalized with a neuronal marker, NeuN, which suggested that there would be death and loss of many neurons , especially in the superficial layer of the damaged motor cortex;
• Figure 2C is a set of images showing results of immunostaining of damaged brain tissue for myelin basic protein (MBP) and neurofilament protein (NF200) at 7 days after CHI.
• MBP myelin basic protein
• NF200 neurofilament protein
• Figure 3A shows diagrammatic Illustrations of the closed head injury in mouse motor cortex and administration of ND1 at or near the impact site after the CHI.
• Figure 3B diagrammatically shows an experimental scheme of CHI induction, NeuroDl -encoding virus injection and immunofluorescence experiments described in detail in Examples herein;
• Figure 3C is a set of representative images showing the injured cortex 7 days after injection of AAV-GFAP::GFP virus (control group, left panel) or injection of AAV-GFAP::ND1-GFP virus (NDl group, right panel);
• Figure 3D is a set of images showing GFP fluorescence and immunofluorescence of the indicated marker; as shown, under GFAP promotor control, GFP was mainly expressed in GFAP+ astrocytes, whereas very low GFP expression was found in other cortical cells of different subtypes at 7 days after AAV-GFAP::GFP virus injection in control group;
• Figure 3E is a set of “zoomed-in” images from Figure 2C illustrating that NeuroDl was highly expressed in GFP+ astrocytes in the NDl group 7 days after AAV- GFAP::ND1-GFP virus injection (lower panels) compared to control group (upper panels);
• Figure 3F is a set of images showing results of co-staining for GFAP, NeuN and ND1 which showed the astrocyte-to-neuron conversion process at different time points after AAV-GFAP::ND1-GFP virus injection;
• Figure 3G is a graph showing quantification of the percentage of different types among the total GFP-expressing cortical cells were shown in Figure 3D;
• Figure 3H is a graph showing quantification of the percentage of cells expressing a neuronal marker, NeuN, with GFP at different time points after AAV- GFAP::ND1-GFP virus injection;
• Figure 4A is a set of images from damaged brain at 4 days after CHI, illustrating that some GFP+ cells showed both GFAP and NeuN signal at the same time, which indicated that they were in the transitional stage from reactive astrocyte to neuron;
• Figure 4B is a set of images showing that, among the converted neurons, the variation trend of immature neuron marker (Tujl) and mature neuron marker (MAP2) implied that converted neurons became mature gradually;
• Figure 4C is a set of images showing GFP fluorescence, NeuN immunofluorescence, and GFAP immunofluorescence and showing that “astrocyte to neuron” (AtN) conversion by NeuroDl was confirmed using (retrovirus) CAG::ND1- GFP or (retrovirus) CAG::GFP expression constructs
• Figure 4D is a graph showing that “astrocyte to neuron” (AtN) conversion by NeuroDl was confirmed using (retrovirus) CAG::ND1-GFP or (retrovirus) CAG::GFP expression constructs and that retrovirus carrying NDl converted about half of GFP- expressing cells to NeuN+, while there was no conversion of astrocytes to neurons in the control group;
• Figure 5A is a set of images showing that most converted neurons showed FoxGl signal and many converted neurons showed Tbrl signal;
• Figure 5B is an image showing that, after NDl treatment, immunostaining with the superficial cortical marker (Cuxl) and deep layer marker (Ctip2) suggested that cortical layers were still well organized.
• Figure 5C is a set of images showing that some converted neurons were Cuxl+ or Ctip2+ in superficial layer or deep layer in mouse cortex;
• Figure 5D is a graph showing results of quantification of the percentage of converted neurons expressing cortical markers FoxGl, and/or Tbrl, or layer markers Cuxl, and/or Ctip2, with GFP and NeuN at 28 days after GFAP::ND1- GFP virus injection;
• Figure 6A is a set of images showing that, at 28 days after ND1 treatment, some converted neurons had both GABA and GAD67 signal inside cell soma, which indicated that they were GABAergic neurons;
• Figure 6B is a set of images showing that some converted neurons could be positive for markers of different subtypes of GABAergic neurons in mouse cortex, like Pavabulmin, Calretinin, Neuropeptide Y, Somatostatin;
• Figure 6C is a graph showing quantification of the percentage of cells expressing neuron subtype markers 28 days after AAV-GFAP::ND1-GFP virus injection;
• Figure 7A is a set of images showing morphology of converted neurons at a, b, and c, along with GFP fluorescence and NeuN immunofluorescence;
• Figure 7B is a set of three traces of action potential firing patterns obtained by whole cell patch recording representative of three different action potential firing patterns, I, II, and III;
• Figure 7C is a pie chart graph showing results of quantitation of converted neurons having either action potential firing pattern I, II, or III;
• Figure 7D is a trace showing that converted neurons fired sEPSCs of which the frequency and amplitude was higher than those from wild type control;
• Figure 7E is a trace showing that converted neurons fired sIPSCs of which the frequency and amplitude was higher than those from wild type control;
• Figure 7F is a set of graphs showing that converted neurons fired sEPSCs of which the frequency and amplitude was higher than those from wild type control;
• Figure 7G is a set of graphs showing that converted neurons fired sIPSCs of which the frequency and amplitude was higher than those from wild type control;
• Figure 8A is a graph demonstrating that the frequency of sEPSCs showed a trend of increase at early time points, and then decreased at later time points to the control level;
• Figure 8B is a graph demonstrating that the amplitude of sEPSCs increased significantly after the first week post-NeuroDl administration, then went down to the control level two months later;
• Figure 8C is a diagram showing an experimental scheme for showing neural innervation on converted neurons at an early time point (day 7) post-NeuroDl administration;
• Figure 8D is a set of images illustrating colocalization of a synaptic marker (VGAT) with GFP and NeuN in the cell soma of converted neurons at 7 days after NeuroDl virus injection and CTB-647 injection on the contralateral side; CTB signal from contralateral side was also observed on the cell soma;
• VGAT synaptic marker
• Figure 8E is a set of images illustrating colocalization of a synaptic vesicle marker (SV2) with GFP and NeuN in the cell soma of converted neurons at 7 days after NeuroDl virus injection and CTB-647 injection on the contralateral side; CTB signal from contralateral side was also observed on the cell soma;
• SV2 synaptic vesicle marker
• Figure 9A is a set of images showing that a glutamatergic synaptic marker (vGlutl), or a GABAergic synaptic marker (vGAT), colocalize with GPF on the cell soma of ND1 converted neurons;
• vGlutl glutamatergic synaptic marker
• vGAT GABAergic synaptic marker
• Figure 9B is a set of images showing that a synaptic terminal marker (synaptophysin, SP1), or a synaptic vesicle marker (SV2), colocalize with GPF around the cell boundary of ND1 converted neurons;
• a synaptic terminal marker synaptophysin, SP1
• SV2 synaptic vesicle marker
• Figure 9C is a set of images showing that ND1 converted neurons demonstrated comparable cFos expression with endogenous neurons in mouse motor cortex;
• Figures 9D-9F show that thalamus neurons were labeled by (AAV)Synapsin::Cre + CAG::Flex-mCherry in the NeuroDl group for anterograde tracing;
• Figure 9D is an image illustrating that, for anterograde tracing in mice to which the NDl-GFP expressing virus was administered, viruses AAV-synapsin::Cre + AAV-CAG::FlexmCherry (which express a red fluorescent protein, mCherry) were further injected into mouse thalamus, thereby labeling neurons to visualize their axon projections ontoNDl converted neurons expressing GFP;
• Figure 9E is a set of images showing an ND1 converted neuron which had GFP-containing synaptic boutons on the soma illustrating local innervation from other converted neurons;
• Figure 9F is a set of images showing an ND1 converted neuron which had mCherry-containing synaptic boutons on the soma illustrating innervation from remote thalamus neurons;
• Figure 9G is a set of images showing that CTB-467 was injected for retrograde tracing in the contralateral side to the NeuroDl -expressing virus injection site and CTB signal was found in some converted neurons;
• Figure 9H is a set of graphs showing that the average CTB signal inside converted neurons increased over time after the NeuroDl -expressing virus was injected as the conversion process proceeded; CTB was injected 7 days before the brain samples were acquired for all the indicated time points.
• the expression “A and/or B” is intended to mean either or both of A and B - i.e., A alone, B alone, or A and B in combination.
• the expression “A, B and/or C” is intended to mean A alone, B alone, C alone, A and B in combination, A and C in combination, B and C in combination, or A, B, and C in combination.
• range is understood to be inclusive of the edges of the range as well as any number between the defined edges of the range.
• “between 1 and 10” includes any number between 1 and 10, as well as the number 1 and the number 10.
• compositions and methods for treating traumatic brain injury (TBI) in a subject are provided according to aspects of the present disclosure.
• Methods effective to reverse the neuronal loss resulting from TBI are provided according to aspects of the present disclosure.
• expression of exogenous neurogenic differentiation 1 (NeuroDl) in glial cells, particularly astrocytes and/or reactive astrocytes treats TBI in a subject in need thereof.
• methods of treatment of TBI in a subject including administration of a therapeutically effective amount of NeuroDl to the subject.
• TBI traumatic brain injury
• NeuroDl refers to a bHLH proneural transcription factor, neurogenic differentiation 1, involved in embryonic brain development and in adult neurogenesis, see Cho, J.H. et al., Mol, Neurobiol., 30:35-47, 2004; Kuwabara, T. et al., Nature Neurosci., 12:1097-1105, 2009; and Gao, Z. et al ., Nature Neurosci., 12:1090- 1092, 2009. NeuroDl is expressed late in development, mainly in the nervous system and is involved in neuronal differentiation, maturation and survival.
• NeuroDl refers to NeuroDl present in a glial cell, particularly an astrocyte and/or reactive astrocyte, to be converted to a functional neuron by operation of a method of the present disclosure and is not naturally present in the glial cell.
• the term “functional” with respect to a neuron refers to a neuron exhibiting and/or a maintaining a capability to perform an action and/or task for which the neuron is specially fitted or exists to perform.
• treat refers to alleviating, inhibiting or ameliorating a TBI, symptoms or signs of a TBI, and preventing symptoms or signs of TBI, and include, but are not limited to therapeutic and/or prophylactic treatments.
• a therapeutically effective amount is intended to mean an amount of an inventive composition which is effective to alleviate, ameliorate or prevent a symptom or sign of a TBI to be treated. According to aspects of the present disclosure, a therapeutically effective amount is an amount which has a beneficial effect in a subject having signs and/or symptoms of TBI.
• administration of a therapeutically effective amount of NeuroDl to a subject affected by a TBI provides the generation of new functional neurons by conversion of reactive astrocytes to functional neurons; reduction of the number of reactive astrocytes; the generation of new non-reactive astrocytes; and integration of the new functional neurons into the neuronal network both in the injured region and in non-injured regions of the brain of the subject.
• TBI traumatic brain injury
• CHI closed head injury
• penetrating head injury is an object piercing the skull and entering the brain.
• a TBI is a CHI.
• a TBI is a penetrating head injury. TBI can result from direct impact to the head from any of various sources such as, but not limited to, a fall, car accidents, sports accidents, being struck with an object, or an indirect impact such as shock waves from an explosion.
• a non-limiting example of an explosion is a battlefield explosion.
• a TBI results from a fall.
• a TBI results from a car accident.
• a TBI results from a sports accident.
• a TBI results from being struck by an object.
• a TBI results from an indirect impact such as shock waves from an explosion.
• a non-limiting example of a TBI is a brain injury resulting from an impact to the head of a subject which is alleviated, ameliorated or prevented by additional functional neurons.
• CHI closed head injury
• the TBI is “focal” such that primary damage to the brain is localized to an area of the brain adjacent to the impact site. Secondary damage to the brain may be present in other regions of the brain resulting from the primary damage.
• the term “primary damage” refers to presence of non-functional neurons, such as dead and/or dying neurons, and reactive astrocytes, in the area adjacent the impact site, wherein presence of the non-functional neurons, such as dead and/or dying neurons, and reactive astrocytes in the damaged region are not primarily due to bleeding and/or ischemia in the damaged region.
• dead or dying neurons are measured by apoptotic assays and functional assay.
• Non- limiting examples of apoptotic assays include electron microscopy, TUNEL assay, flow cytometry, the DNA ladder assay, detection of cytochrome c, detection of annexin V, and caspase activity assays.
• Non-limiting examples of functional assays include functional magnetic resonance imaging (fMRI).
• TBI may result from two or more impacts and that each of the two or more impacts is associated with an impact site such that primary damage to the brain is associated to each of the two or more impact sites.
• the therapeutically effective amount of NeuroDl in the glial cells treats at least one sign and/or symptom of TBI in the subject, whereby the TBI is treated.
• Signs and symptoms of TBI are well-known in the art along with methods of detection and assessment of such signs and symptoms. Signs and symptoms of TBI in a subject include loss of consciousness, confusion, disorientation, headache, fatigue, speech problems, sleep problems, dizziness, balance problems, sensory problems, sensitivity to light, loss of sight or changes in vision, loss or alterations in the sense of smell, loss or alterations in the sense of taste, tinnitus, loss or alterations in the sense of hearing, memory problems, concentration problems, depression, anxiety, agitation, mood swings, seizures, loss or diminishment of coordination, motor issues, cognitive issues including difficulty learning, negative changes in reasoning ability, negative changes in judgement, and negative changes in attention or concentration.
• Signs and/or symptoms of TBI in a subject include presence of non- functional neurons, such as dead and/or dying neurons, in the region of the brain damaged due to the TBI.
• the number of dead and/or dying neurons in the region of the brain damaged due to the TBI is reduced by a method of treating a TBI in a subject in need thereof according to aspects of the present disclosure which includes delivering a therapeutically effective amount of NeuroDl to glial cells of the subject.
• Signs and/or symptoms of TBI in a subject include presence of reactive astrocytes in the region of the brain damaged due to the TBI.
• the number of reactive astrocytes in the region of the brain damaged due to the TBI is reduced by a method of treating a TBI in a subject in need thereof according to aspects of the present disclosure which includes delivering a therapeutically effective amount of NeuroDl to glial cells of the subject.
• the therapeutically effective amount of NeuroDl in the glial cells results in a greater number of functional neurons in the subject having a TBI, compared to an untreated subject having a TBI, whereby the TBI is treated.
• a therapeutically effective amount of NeuroDl in the glial cells results in a greater number of functional neurons in an area of the brain of the subject affected by TBI, compared to an untreated subject having a TBI, whereby the TBI is treated.
• the subject in need of treatment may be human or non-human mammalian, but can be non-mammalian as well.
• 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, such as but not limited to, mice and rats; as well as non-mammalian animals such as, but not limited to, birds, poultry, reptiles, amphibians. According to aspects of the present disclosure, the subject is human.
• the subject is a male. According to aspects of the present disclosure, the subject is a female. According to aspects of the present disclosure, the subject is gender neutral. According to aspects of the present disclosure, the subject is a premature newborn. According to aspects of the present disclosure, a premature newborn is born before 36 weeks gestation. According to aspects of the present disclosure, the subject is a term newborn. According to aspects of the present disclosure, a term newborn is below about 2 months old. According to aspects of the present disclosure, the subject is a neonate. According to aspects of the present disclosure, the subject is a neonate is below about 1 month old. According to aspects of the present disclosure, the subject is an infant. According to aspects of the present disclosure, an infant is between 2 months and 24 months old.
• an infant is between 2 months and 3 months, between 2 months and 4 months, between 2 months and 5 months, between 3 months and 4 months, between 3 months and 5 months, between 3 months and 6 months, between 4 months and 5 months, between 4 months and 6 months, between 4 months and 7 months, between 5 months and 6 months, between 5 months and 7 months, between 5 months and 8 months, between 6 months and 7 months, between 6 months and 8 months, between 6 months and 9 months, between 7 months and 9 months, between 7 months and 10 months, between 8 months and 9 months, between 8 months and 10 months, between 8 months and 11 months, between 9 months and 10 months, between 9 months and 11 months, between 9 months and 12 months, between 10 months and 11 months, between 10 months and 11 months, between 10 months and 12 months, between 10 months and 13 months, between 11 months and 12 months, between 11 months and 12 months, between 10 months and 11 months, between 10 months and 12 months, between 10 months and 13 months, between 11 months and 12 months, between 11 months and 12 months, between 11 months and 12 months, between 10 months and 13 months
• the subject is a toddler.
• a toddler is between 1 year and 4 years old.
• a toddler is between 1 year and 2 years, between 1 year and 3 years, between 1 year and 4 years, between 2 years and 3 years, between 2 years and 4 years, and between 3 years and 4 years old.
• the subject is a young child.
• a young child is between 2 years and 5 years old.
• a young child is between 2 years and 3 years, between 2 years and 4 years, between 2 years and 5 years, between 3 years and 4 years, between 3 years and 5 years, and between 4 years and 5 years old.
• the subject is a child.
• a child is between 6 years and 12 years old.
• a child is between 6 years and 7 years, between 6 years and 8 years, between 6 years and 9 years, between 7 years and 8 years, between 7 years and 9 years, between 7 years and 10 years, between 8 years and 9 years, between 8 years and
• an adolescent is between 13 years and 19 years old.
• an adolescent is between 13 years and 14 years, between 13 years and 15 years, between 13 years and 16 years, between 14 years and 15 years, between 14 years and 16 years, between 14 years and 17 years, between 15 years and 16 years, between 15 years and 17 years, between 15 years and 18 years, between 16 years and 17 years, between 16 years and 18 years, between 16 years and 19 years, between 17 years and 18 years, between 17 years and 19 years, and between 18 years and 19 years old.
• the subject is a pediatric subject. According to aspects of the present disclosure, a pediatric subject between 1 day and 18 years old.
• a pediatric subject is between 1 day and 1 year, between 1 day and 2 years, between 1 day and 3 years, between 1 year and 2 years, between 1 year and 3 years, between 1 year and 4 years, between 2 years and 3 years, between 2 years and 4 years, between 2 years and 5 years, between 3 years and 4 years, between 3 years and 5 years, between 3 years and 6 years, between 4 years and 5 years, between 4 years and 6 years, between 4 years and 7 years, between 5 years and 6 years, between 5 years and 7 years, between 5 years and 8 years, between 6 years and 7 years, between 6 years and 8 years, between 6 years and 9 years, between 7 years and 8 years, between 7 years and 9 years, between 7 years and 10 years, between 8 years and 9 years, between 8 years and 10 years, between 8 years and 11 years, between 9 years and 10 years, between 9 years and 11 years, between 9 years and 12 years, between 10 years and 11 years, between 10 years and 11 years, between 10 years and 12 years, between 10 years and 13 years, between 11 years and 12 years, between 11 years and
• the subject is a geriatric subject.
• a geriatric subject is between 65 years and 95 or more years old.
• a geriatric subject is between 65 years and 70 years, between 65 years and 75 years, between 65 years and 80 years, between 70 years and 75 years, between 70 years and 80 years, between 70 years and 85 years, between 75 years and 80 years, between 75 years and 85 years, between 75 years and 90 years, between 80 years and 85 years, between 80 years and 90 years, between 80 years and 95 years, between 85 years and 90 years, and between 85 years and 95 years old.
• a subject in need thereof is an adult.
• an adult subject is between 20 years and 95 or more years old. According to aspects of the present disclosure, an adult subject is between 20 years and 25 years, between 20 years and 30 years, between 20 years and 35 years, between 25 years and 30 years, between 25 years and 35 years, between 25 years and 40 years, between 30 years and 35 years, between 30 years and 40 years, between 30 years and 45 years, between 35 years and 40 years, between 35 years and 45 years, between 35 years and 50 years, between 40 years and 45 years, between 40 years and 50 years, between 40 years and 55 years, between 45 years and 50 years, between 45 years and 55 years, between 45 years and 60 years, between 50 years and 55 years, between 50 years and 60 years, between 50 years and 65 years, between 55 years and 60 years, between 55 years and 65 years, between 55 years and 70 years, between 60 years and 65 years, between 60 years and 70 years, between 60 years and 75 years, between 65 years and 70 years, between 65 years and 75 years, between 65 years and 80 years, between 70 years and 75 years, between 70 years and 75 years, between 70 years and
• a subject is between 1 year and 5 years, between 2 years and 10 years, between 3 years and 18 years, between 21 years and 50 years, between 21 years and 40 years, between 21 years and 30 years, between 50 years and 90 years, between 60 years and 90 years, between 70 years and 90 years, between 60 years and 80 years, or between 65 years and 75 years old.
• a subject is a young old subject (65 to 74 years old).
• a subject is a middle old subject (75 to 84 years old).
• a subject is an old subject (>85 years old).
• Methods of treatment of TBI in a subject include administration of a therapeutically effective amount of NeuroDl to the subject in the local region of the TBI, at or near the location the brain injury site, according to aspects of the present disclosure.
• Methods of treatment of TBI in a subject include administration of a therapeutically effective amount of NeuroDl to the subject in the local region of the TBI, in or near a glial scar caused by the TBI, according to aspects of the present disclosure.
• Methods of treatment of TBI in a subject include administration of a therapeutically effective amount of NeuroDl to the subject in the local region of the TBI, in or near a region of gliosis, particularly astrogliosis and/or microgliosis, according to aspects of the present disclosure.
• gliosis includes “astrogliosis” and “microgliosis” and refers to an increase in astrocytes and reactive astrocytes, i.e. astrogiosis, and an increase in microglia and hypertrophic microglia, i.e. microgliosis, due to brain damage.
• astrogiosis astrogiosis
• microglia and hypertrophic microglia i.e. microgliosis
• gliosis is believed to be a protective reaction of glial cells in response to brain damage, providing beneficial effects such as insulating the injury area, removing debris of dead cells, and protecting the remaining healthy cells.
• gliosis can impede neural regeneration and produce negative effects on the local microenvironment, leading to further neurodegeneration.
• beneficial outcomes include regeneration of functional neurons to replace, or at least partially replace, the neurons lost due to TBI, reduction in the number of reactive astrocytes by conversion of the reactive astrocytes to functional neurons thereby modulating the negative effects of gliosis, repair of the damaged neural network caused by the TBI, and rebalancing the microenvironment disrupted by the TBI.
• administration of a therapeutically effective amount of NeuroDl ameliorates the effects of TBI in a subject in need thereof.
• administration of a therapeutically effective amount of NeuroDl has enhanced effects when administered to reactive astrocytes compared to quiescent astrocytes.
• administration of a therapeutically effective amount of NeuroDl can be between 3 days to 60 days, between 5 days to 45 days, between 8 days to 30 days following the TBI in the subject.
• administration can be 2 days to 1 year or later following the TBI in the subject.
• administration of a therapeutically effective amount of NeuroDl can be between 3 days and 5 days, between 3 days and 10 days, between 3 days and 15 days, between 5 days and 10 days, between 5 days and 15 days, between 5 days and 20 days, between 10 days and 15 days, between 10 days and 20 days, between 10 days and 25 days, between 15 days and 20 days, between 15 days and 25 days, between 15 days and 30 days, between 20 days and 25 days, between 20 days and 30 days, between 20 days and 35 days, between 25 days and 30 days, between 25 days and 35 days, between 25 days and 40 days, between 30 days and 35 days, between 30 days and 40 days, between 30 days and 45 days, between 35 days and 40 days, between 35 days and 45 days, between 35 days and 50 days, between 40 days and 45 days, between 40 days and 50 days, between 40 days and 55 days, between 45 days and 50 days, between 45 days and 55 days, between 45 days and 60 days, between 50 days and 60 days, or between 55 days and 60 days.
• administration of a therapeutically effective amount of NeuroDl can be between 5 days and 10 days, between 5 days and 15 days, between 5 days and 20 days, 10 days and 15 days, between 10 days and 20 days, between 10 days and 25 days, between 15 days and 20 days, between 15 days and 25 days, between 15 days and 30 days, between 20 days and 25 days, between 20 days and 30 days, between 20 days and 35 days, between 25 days and 30 days, between 25 days and 35 days, between 25 days and 40 days, between 30 days and 35 days, between 30 days and 40 days, between 30 days and 45 days, between 35 days and 40 days, between 35 days and 45 days, or between 40 days and 45 days.
• administration of a therapeutically effective amount of NeuroDl can be between 8 days and 10 days, between 8 days and 15 days, between 8 days and 20 days, 10 days and 15 days, between 10 days and 20 days, between 10 days and 25 days, between 15 days and 20 days, between 15 days and 25 days, between 15 days and 30 days, between 20 days and 25 days, between 20 days and 30 days, or between 25 days and 30 days.
• providing the exogenous NeuroDl includes providing exogenous NeuroDl to the at least one reactive astrocyte at a first treatment time in the range of about 1 day to about 10 days after the TBI.
• exogenous NeuroDl is provided to the at least one reactive astrocyte between 1 day and 2 days, between 1 day and 3 days, between 1 day and 4 days, between 2 days and 3 days, between 2 days and 4 days, between 2 days and 5 days, between 3 days and 4 days, between 3 days and 5 days, between 3 days and 6 days, between 4 days and 5 days, between 4 days and 6 days, between 4 days and 7 days, between 5 days and 6 days, between 5 days and 7 days, between 5 days and 8 days, between 6 days and 7 days, between 6 days and 8 days, between 6 days and 9 days, between 7 days and 8 days, between 7 days and 9 days, between 7 days and 10 days, between 8 days and 9 days, between 8 days and 10 days, or between 9 days and 10 days.
• exogenous NeuroDl is provided to the at least one reactive astrocyte at a treatment time of 1 day after the TBI. According to aspects of the present disclosure, exogenous NeuroDl is provided to the at least one reactive astrocyte at a treatment time of 2 days after the TBI. According to aspects of the present disclosure, exogenous NeuroDl is provided to the at least one reactive astrocyte at a treatment time of 3 days after the TBI. According to aspects of the present disclosure, exogenous NeuroDl is provided to the at least one reactive astrocyte at a treatment time of 4 days after the TBI.. According to aspects of the present disclosure, exogenous NeuroDl is provided to the at least one reactive astrocyte at a treatment time of 5 days after the TBI..
• exogenous NeuroDl is provided to the at least one reactive astrocyte at a treatment time of 6 days after the TBI. According to aspects of the present disclosure, exogenous NeuroDl is provided to the at least one reactive astrocyte at a treatment time of 7 days after the TBI. According to aspects of the present disclosure, exogenous NeuroDl is provided to the at least one reactive astrocyte at a treatment time of 8 days after the TBT. According to aspects of the present disclosure, exogenous NeuroDl is provided to the at least one reactive astrocyte at a treatment time of 9 days after the TBI. According to aspects of the present disclosure, exogenous NeuroDl is provided to the at least one reactive astrocyte at a treatment time of 10 days after the TBI.
• the TBI causes a period of astrogliosis in the damaged region
• providing the exogenous NeuroDl includes providing exogenous NeuroDl to the at least one reactive astrocyte at a first treatment time during the period of astrogliosis or within 4 weeks after the period of astrogliosis.
• the exogenous NeuroDl is provided to the at least one reactive astrocyte at a second treatment time after the first treatment time and during the period of astrogliosis or within 4 weeks after the period of astrogliosis.
• the exogenous NeuroDl is provided to the at least one reactive astrocyte at a third treatment time after the second treatment time and during the period of astrogliosis or within 4 weeks after the period of astrogliosis. More than three treatments are optionally provided, such as a fourth treatment at a fourth treatment time after the third treatment, a fifth treatment at a fifth treatment time after the fourth treatment, and so on relating to sixth, seventh, eighth, ninth, and tenth, or more, treatments including administration of exogenous NeuroDl, during the period of astrogliosis or within 4 weeks after the period of astrogliosis.
• Combinations of therapies treating TBI in a subject are administered according to aspects of the present disclosure.
• an additional pharmaceutical agent or therapeutic treatment administered to a subject to treat TBI in an individual subject in need thereof include treatments such as, but not limited to, repairing a skull fracture, removing a blood clot, relieving pressure inside the skull, administration of one or more anti-inflammation agents, administration of one or more anti-anxiety agents, and administration of one or more anti-coagulant agents, administration of one or more anticonvulsants, administration of one or more antidepressants, administration of one or more muscle relaxants, physical therapy, speech therapy, and cognitive therapy.
• NeuroDl treatment is administered to a subject having a TBI as diagnosed and/or assessed by a medical examination.
• medical examination refers to any examination of a subject effective to diagnose or assess the subject for putative TBI, including neurological examination and physical examination.
• the medical examination includes an imaging technique and/or an electrophysiol ogical technique and NeuroDl treatment is administered to a subject having a TBI as diagnosed and/or assessed by an imaging technique and/or an electrophysiol ogical technique.
• Electrophysiology techniques such as electroencephalography (EEG) can be used to assess functional changes in neural firing caused by neuronal cell death or injury due to TBI.
• Imaging techniques such as magnetic resonance imaging (MRI), fMRI, Near Infrared Spectroscopy, position emission tomography (PET) scan, computerized axial tomography (CAT) scan, and ultrasound, can be used to assess structural and/or functional changes caused by neuronal cell death or injury due to TBI.
• presence of non-functional neurons due to TBI are detected by a functional assay, such as fMRI.
• fMRI refers to functional magnetic resonance imaging, an imaging procedure that detects and measure brain activity by detecting associated changes in blood flow.
• Methods of medical examination may be used singularly, or in any combination, to diagnose and/or assess a TBI in the subject.
• methods of medical examination may be used singularly, or in any combination, to assess efficacy of NeuroDl treatment of a TBI in the subject.
• NeuroDl treatment of a subject is monitored during or after treatment to monitor progress and/or final outcome of the treatment.
• Post-treatment assay for successful functional neuron integration and restoration of tissue microenvironment is diagnosed by restoration or near-restoration of normal electrophysiology, brain tissue structure, and neuronal function.
• Non-invasive methods to assay neuronal function include EEG.
• Neuronal function may be non- invasively assayed via Near Infrared Spectroscopy and fMRI.
• Non-invasive methods to assay brain tissue structure include MRI, CAT scan, PET scan, or ultrasound.
• Behavioral assays may be used to non-invasively assay for restoration of brain function following TBI.
• the behavioral assay should be matched to the loss of function caused by the TBI. For example, if the TBI caused paralysis, the patient's mobility and limb dexterity should be tested. If the TBI caused loss or slowing of speech, patient's ability to communicate via spoken word should be assayed. Restoration of normal behavior post-NeuroDl treatment indicates successful creation and integration of effective neuronal circuits.
• Assays to evaluate treatment with NeuroDl may be performed at any point, such as 1 day, 2 days, 3 days, one week, 2 weeks, 3 weeks, one month, or later, after NeuroDl treatment. Such assays may be performed prior to NeuroDl treatment in order to establish a baseline comparison if desired.
• NeuroDl is administered at the periphery of the injury site where a glial scar will develop if the subject is untreated or where a glial scar is already present.
• Glial scar location may be determined by assaying tissue structure or function.
• non-invasive methods to assay structural and/or functional changes caused by TBI including MRI, fMRI, CAT scan, or ultrasound.
• Functional assay may include EEG recording and/or fMRI.
• NeuroDl is administered as an expression vector containing a nucleic acid sequence encoding NeuroDl.
• an expression vector containing a nucleic acid sequence encoding NeuroDl is delivered by injection, into the brain of a subject.
• an expression vector containing a nucleic acid sequence encoding NeuroDl is delivered by stereotactic injection, into the brain of a subject.
• a viral vector including a nucleic acid encoding NeuroDl is delivered by injection into the central or peripheral nerve tissue of a subject.
• injection into the central or peripheral nerve tissue is selected from the group consisting of intracerebral injection, spinal cord injection, injection into the cerebrospinal fluid, and injection into the peripheral nerve ganglia.
• Alternative viral delivery methods include but not limited to intravenous injection, intranasal infusion, intramuscle injection, intrathecal injection, and intraperitoneal injection.
• a viral vector including a nucleic acid encoding NeuroDl is delivered by injection into the brain of a subject.
• a viral vector including a nucleic acid encoding NeuroDl is delivered by stereotactic injection into the brain of a subject.
• Method and compositions for treating a neurological condition in a subject in need thereof are provided according to aspects of the present disclosure which include providing a viral vector comprising a nucleic acid encoding NeuroDl; and delivering the viral vector to the brain of the subject, whereby the viral vector infects glial cells of the brain producing infected glial cells and whereby exogenous NeuroDl is expressed in the infected glial cells at a therapeutically effective level, wherein the expression of NeuroDl in the infected cells results in a greater number of functional neurons in the subject with a TBI compared to an untreated subject having a TBI, whereby the TBI is treated.
• the number of reactive glial cells is reduced, resulting in fewer neuroinhibitory factors released, less neuroinflammation, more blood vessels that are also evenly distributed, thereby making local environment more permissive to neuronal growth or axon penetration, hence alleviating at least one sign and/or symptom of TBI.
• Adeno-associated virus (AAV) vectors are particularly useful in methods according to aspects of the present disclosure and will infect both dividing and non- dividing cells, at an injection site.
• AAV are ubiquitous, noncytopathic, replication- incompetent members of ssDNA animal virus of parvoviridae family.
• an “AAV vector” refers to an AAV packaged with a DNA vector construct.
• an AAV vector is selected from the group consisting of AAV vector serotype 1, AAV vector serotype 2, AAV vector serotype 3, AAV vector serotype 4, AAV vector serotype 5, AAV vector serotype 6, AAV vector serotype 7, AAV vector serotype 8, AAV vector serotype 9, AAV vector serotype 10, AAV vector serotype 11, and AAV vector serotype 12.
• an AAV vector is selected from the group consisting AAV serotype 2, AAV serotype 5, and AAV serotype 9.
• an AAV vector is AAV serotype 1.
• an AAV vector is AAV serotype 2.
• an AAV vector is AAV serotype 3.
• an AAV vector is AAV serotype 4.
• an AAV vector is AAV serotype 5.
• an AAV vector is AAV serotype 6.
• an AAV vector is AAV serotype 7.
• an AAV vector is AAV serotype 8.
• an AAV vector is AAV serotype 9.
• an AAV vector is AAV serotype 10.
• an AAV vector is AAV serotype 11.
• an AAV vector is AAV serotype 12.
• a “FLEX” switch approach is used to express NeuroDl in infected cells according to aspects of the present disclosure.
• the terms “FLEX” and “flip-excision” are used interchangeably to indicate a method in which two pairs of heterotypic, antiparallel loxP-type recombination sites are disposed on either side of an inverted NeuroDl coding sequence which first undergo an inversion of the coding sequence followed by excision of two sites, leading to one of each orthogonal recombination site oppositely oriented and incapable of further recombination, achieving stable inversion, see for example Schnutgen et al ., Nature Biotechnology 21 :562-565, 2003; and Atasoy el al , J.
• NeuroDl is administered to a subject in need thereof by administration of 1) an adeno-associated virus expression vector including a DNA sequence encoding a site-specific recombinase under transcriptional control of an astrocyte-specific promoter such as GFAP or SI 00b or AldhlLl; and 2) an adeno-associated virus expression vector including a DNA sequence encoding NeuroDl under transcriptional control of a ubiquitous (constitutive) promoter or a neuron-specific promoter wherein the DNA sequence encoding NeuroDl is inverted and in the wrong orientation for expression of NeuroDl until the site-specific recombinase inverts the inverted DNA sequence encoding NeuroDl, thereby allowing expression of NeuroDl.
• an adeno-associated virus expression vector including a DNA sequence encoding a site-specific recombinase under transcriptional control of an astrocyte-specific promoter such as GFAP or SI 00b or AldhlLl
• Site-specific recombinases and their recognition sites include, for example, Cre recombinase along with recognition sites loxP and lox2272 sites, or FLP-FRT recombination, or their combinations.
• AAV particle refers to packaged capsid forms of the AAV virus that transmits its nucleic acid genome to cells.
• a concentration of 10 10 - 10 14 AAV particles/ml, 1-1000 m ⁇ of volume is injected at a controlled flow rate of 0.1-5.0 m ⁇ /minute.
• an AAV particle is injected at a volume between 1 ⁇ L and 100 ⁇ L, between 1 ⁇ L and 200 ⁇ L, between 1 ⁇ L and 300 ⁇ L, between 100 ⁇ L and 200 ⁇ L, between 100 ⁇ L and 300 ⁇ L, between 100 ⁇ L and 400 ⁇ L, between 200 ⁇ L and 300 ⁇ L, between 200 ⁇ L and 400 ⁇ L, between 200 ⁇ L and 500 ⁇ L, between 300 ⁇ L and 400 ⁇ L, between 300 ⁇ L and 500 ⁇ L, between 300 ⁇ L and 600 ⁇ L, between 400 ⁇ L and 500 ⁇ L, between 400 ⁇ L and 600 ⁇ L, between 400uL and 700 ⁇ L, between 500 ⁇ L and 600 ⁇ L, between 500 ⁇ L and 700 ⁇ L, between 500 ⁇ L and 800 ⁇ L, between 600 ⁇ L and 700 ⁇ L, between 600 ⁇ L and 800 ⁇ L, between 600 ⁇ L and 900 ⁇ L, between 700 ⁇ L and 100 ⁇ L, between
• the flow rate is between 0.1 ⁇ L/minute and 0.2 ⁇ L/minute, between 0.1 ⁇ L/minute and 0.3 ⁇ L/minute, between 0.1 ⁇ L/minute and 0.4 ⁇ L/minute, between
• an AAV vector including a nucleic acid encoding NeuroDl under transcriptional control of a ubiquitous (constitutive) promoter or a neuron-specific promoter wherein the DNA sequence encoding NeuroDl is inverted and in the wrong orientation for expression of NeuroDl and further includes sites for recombinase activity by a site specific recombinase, until the site-specific recombinase inverts the inverted DNA sequence encoding NeuroDl, thereby allowing expression of NeuroDl, is delivered by stereotactic injection into the brain of a subject along with an AAV encoding a site specific recombinase.
• the site of stereotactic injection is in or near a glial scar caused by disruption of normal blood flow in the CNS.
• a composition comprises a first recombinant expression vector comprising a glial cell specific promoter operably linked to a nucleic acid encoding a site specific recombinase and a second recombinant expression vector comprising a promoter operably linked to a nucleic acid sequence encoding NeuroDl, a nucleic acid sequence encoding a reporter gene, an enhancer, and a regulatory element.
• a composition comprises a first recombinant AAV expression vector comprising a glial cell specific promoter operably linked to a nucleic acid encoding a site specific recombinase and a second recombinant AAV expression vector comprising a promoter operably linked to a nucleic acid sequence encoding NeuroDl, a nucleic acid sequence encoding a reporter gene, an enhancer, and a regulatory element.
• the site-specific recombinase is Cre recombinase and the sites for recombinase activity are recognition sites loxP and lox2272 sites.
• the term “NeuroDl” encompasses human NeuroDl protein, identified here as SEQ ID NO: 2 and mouse NeuroDl protein, identified here as SEQ ID NO: 4.
• the term “NeuroDl” encompasses variants of NeuroDl protein, such as variants of SEQ ID NO: 2 and SEQ ID NO: 4, which may be included in methods and compositions of the present disclosure.
• variant refers to naturally occurring genetic variations and recombinantly prepared variations, each of which contain one or more changes in its amino acid sequence compared to a reference NeuroDl protein, such as SEQ ID NO: 2 or SEQ ID NO: 4, wherein the variant retains the functional properties of the reference protein. Such changes include those in which one or more amino acid residues have been modified by amino acid substitution, addition or deletion.
• variant encompasses orthologs of human NeuroDl, including for example mammalian and bird NeuroDl, such as, but not limited to NeuroDl orthologs from a non-human primate, cat, dog, sheep, goat, horse, cow, pig, bird, poultry animal and rodent such as but not limited to mouse and rat.
• mouse NeuroDl exemplified herein as amino acid sequence SEQ ID NO: 4 is an ortholog of human NeuroDl.
• 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, wherein the variant retains the functional properties of the reference protein.
• Mutations can be introduced using standard molecular biology techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.
• One of skill in the art will recognize that one or more amino acid mutations can be introduced without altering the functional properties of the NeuroDl protein.
• one or more amino acid substitutions, additions, or deletions can be made without altering the functional properties of the NeuroDl protein of SEQ ID NO: 2 or 4.
• Conservative amino acid substitutions can be made in a NeuroDl protein to produce a NeuroDl protein variant, wherein the variant retains the functional properties of the reference protein.
• Conservative amino acid substitutions are art recognized substitutions of one amino acid for another amino acid having similar characteristics.
• each amino acid may be described as having one or more of the following characteristics: electropositive, electronegative, aliphatic, aromatic, polar, hydrophobic and hydrophilic.
• a conservative substitution is a substitution of one amino acid having a specified structural or functional characteristic for another amino acid having the same characteristic.
• Acidic amino acids include aspartate, glutamate; basic amino acids include histidine, lysine, arginine; aliphatic amino acids include isoleucine, leucine and valine; aromatic amino acids include phenylalanine, glycine, tyrosine and tryptophan; polar amino acids include aspartate, glutamate, histidine, lysine, asparagine, glutamine, arginine, serine, threonine and tyrosine; and hydrophobic amino acids include alanine, cysteine, phenylalanine, glycine, isoleucine, leucine, methionine, proline, valine and tryptophan; and conservative substitutions include substitution among amino acids within each group. Amino acids may also be described in terms of relative size, alanine, cysteine, aspartate, glycine, asparagine, proline, threonine, serine, valine, all typically considered to be small.
• NeuroDl variants can include synthetic amino acid analogs, amino acid derivatives and/or non-standard amino acids, illustratively including, without limitation, alpha-aminobutyric acid, citrulline, canavanine, cyanoalanine, diaminobutyric acid, diaminopimelic acid, dihydroxy-phenylalanine, djenkolic acid, homoarginine, hydroxyproline, norleucine, norvaline, 3-phosphoserine, homoserine, 5- hydroxytryptophan, 1-methylhistidine, 3-methylhistidine, and ornithine.
• synthetic amino acid analogs amino acid derivatives and/or non-standard amino acids
• amino acid derivatives illustratively including, without limitation, alpha-aminobutyric acid, citrulline, canavanine, cyanoalanine, diaminobutyric acid, diaminopimelic acid, dihydroxy-phenylalanine, djenkolic acid, homoarginine,
• the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence).
• the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
• the determination of percent identity between two sequences can also be accomplished using a mathematical algorithm.
• a preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, PNAS 87:2264 2268, modified as in Karlin and Altschul, 1993, PNAS. 90:5873 5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403.
• Gapped BLAST are utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389 3402.
• PSI BLAST is used to perform an iterated search which detects distant relationships between molecules.
• the default parameters of the respective programs e.g., of XBLAST and NBLAST
• the default parameters of the respective programs are used (see, e.g, the NCBI website).
• Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11 17. Such an algorithm is incorporated in 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 is used.
• 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.
• NeuroDl protein encompasses fragments of the NeuroDl protein, such as fragments of SEQ ID NOs. 2 and 4 and variants thereof, operable in methods and compositions of the present disclosure.
• NeuroDl proteins and nucleic acids may be isolated from natural sources, such as the brain of an organism or cells of a cell line which expresses NeuroDl. Alternatively, NeuroDl protein or nucleic acid may be generated recombinantly, such as by expression using an expression construct, in vitro or in vivo. NeuroDl proteins and nucleic acids may also be synthesized by well-known methods. [00150] NeuroDl included in methods and compositions of the present disclosure is preferably produced using recombinant nucleic acid technology. Recombinant NeuroDl production includes introducing a recombinant expression vector encompassing a nucleic acid sequence, such as a DNA sequence or RNA sequence, encoding NeuroDl into a host cell in vitro or in vivo.
• a nucleic acid sequence encoding NeuroDl is introduced into a host cell to produce NeuroDl according to embodiments of the disclosure encodes SEQ ID NO: 2, SEQ ID NO: 4, or a variant thereof.
• the nucleic acid sequence identified herein as SEQ ID NO: 1 encodes SEQ ID NO: 2 and is included in an expression vector and expressed to produce NeuroDl.
• the nucleic acid sequence identified herein as SEQ ID NO: 3 encodes SEQ ID NO: 4 and is included in an expression vector and expressed to produce NeuroDl.
• nucleic acid sequences substantially identical to SEQ ID NOs: 1 and 3 encode NeuroDl and variants of NeuroDl, and that such alternate nucleic acids may be included in an expression vector and expressed to produce NeuroDl and variants of NeuroDl.
• a fragment of a nucleic acid encoding NeuroDl protein can be used to produce a fragment of a NeuroDl protein.
• expression vector refers to a recombinant vehicle for introducing a nucleic acid encoding NeuroDl into a host cell in vitro or in vivo where the nucleic acid is expressed to produce NeuroDl.
• an expression vector including SEQ ID NO: 1 or 3 or a substantially identical nucleic acid sequence encoding SEQ ID NO: 2 or SEQ ID NO: 4, or a variant thereof is expressed to produce NeuroDl in cells, in vitro or in vivo, containing the expression vector.
• the term “recombinant” is used to indicate a nucleic acid construct in which two or more nucleic acids are linked and which are not found linked in nature.
• Expression vectors include, but are not limited to plasmids, viruses, BACs and YACs. Particular viral expression vectors illustratively include those derived from adenovirus, adeno-associated virus, retrovirus, and lentivirus.
• An expression vector contains a nucleic acid that includes 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.
• operably linked refers to a nucleic acid in functional relationship with a second nucleic acid.
• operably linked encompasses functional connection of two or more nucleic acid molecules, such as a nucleic acid to be transcribed and a regulatory element.
• regulatory element refers to a nucleotide sequence which controls some aspect of the expression of an operably linked nucleic acid.
• Exemplary regulatory elements include an enhancer, such as, but not limited to: woodchuck hepatitis virus posttranscriptional regulatory element (WPRE); an internal ribosome entry site (IRES) or a 2A domain; an intron; an origin of replication; a polyadenylation signal (pA); a promoter; a transcription termination sequence; and an upstream regulatory domain, which contribute to the replication, transcription, post- transcriptional processing of an operably linked nucleic acid sequence.
• WPRE woodchuck hepatitis virus posttranscriptional regulatory element
• IVS internal ribosome entry site
• promoter refers to a DNA sequence operably linked to a nucleic acid sequence to be transcribed such as a nucleic acid sequence encoding NeuroDl.
• 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.
• a promoter is generally positioned upstream of the nucleic acid sequence transcribed to produce the desired molecule, and provides a site for specific binding by RNA polymerase and other transcription factors.
• 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.
• a portion of the 5 non-coding region can be isolated and used to drive expression of an operably linked nucleic acid.
• about 500-6000 bp of the 5 non-coding region of a gene is used to drive expression of the operably linked nucleic acid.
• a portion of the 5 non-coding region of a gene containing a minimal amount of the 5 non-coding region needed to drive expression of the operably linked nucleic acid is used.
• Assays to determine the ability of a designated portion of the 5’ non-coding region of a gene to drive expression of the operably linked nucleic acid are well-known in the art.
• promoters used to drive expression of NeuroDl are “ubiquitous” or “constitutive” promoters, that drive expression in many, most, or all cell types into which the expression vector is transferred, in vitro or in vivo.
• Non-limiting examples of ubiquitous promoters that can be used in expression of NeuroDl are cytomegalovirus promoter; simian virus 40 (SV40) early promoter; rous sarcoma virus promoter; adenovirus major late promoter; beta actin promoter; glyceraldehyde 3 -phosphate dehydrogenase; glucose-regulated protein 78 promoter; glucose-regulated protein 94 promoter; heat shock protein 70 promoter; beta-kinesin promoter; ROSA promoter; ubiquitin B promoter; eukaryotic initiation factor 4A1 promoter and elongation Factor I promoter; all of which are well- known in the art and which can be isolated from primary sources using routine methodology or obtained from commercial sources.
• Promoters can be derived entirely from a single gene or can be chimeric, having portions derived from more than one gene.
• Combinations of regulatory sequences may be included in an expression vector and used to drive expression of NeuroDl.
• a non-limiting example included in an expression vector to drive expression of NeuroDl is the CAG promoter which combines the cytomegalovirus CMV early enhancer element, chicken beta-actin promoter, and the splice acceptor of the rabbit beta-globin gene.
• promoters used to drive expression of NeuroDl according to methods described herein are those that drive expression preferentially in glial cells, particularly astrocytes and/or NG2 cells. Such promoters are termed “astrocyte-specific” and/or “NG2 cell-specific” promoters.
• Non-limiting examples of astrocyte-specific promoters are glial fibrillary acidic protein (GFAP) promoter and aldehyde dehydrogenase 1 family, member LI (AldhlLl) promoter.
• GFAP glial fibrillary acidic protein
• Human GFAP promoter is shown herein as SEQ ID NO: 6.
• Mouse AldhlLl promoter is shown herein as SEQ ID NO: 7.
• NG2 cell-specific promoter is the promoter of the chondroitin sulfate proteoglycan 4 gene, also known as neuron-glial antigen 2 (NG2).
• Human NG2 promoter is shown herein as SEQ ID NO: 8.
• Particular promoters used to drive expression of NeuroDl according to methods described herein are those that drive expression preferentially in reactive glial cells.
• Non-limiting examples of reactive glial cells include reactive astrocytes and reactive NG2 cells.
• a reactive glial cell is a reactive astrocyte.
• a reactive glial cell is a reactive NG2 cell.
• promoters used to drive expression of NeuroDl are termed “reactive astrocyte-specific” promoters.
• promoters used to drive expression of NeuroDl are termed “reactive NG2 cell-specific” promoters.
• a non-limiting example of a “reactive astrocyte-specific” promoter is the promoter of the lipocalin 2 (lcn2) gene.
• Mouse lcn2 promoter is shown herein as SEQ ID NO: 5.
• Homologues and variants of ubiquitous and cell type-specific promoters may be used in expressing NeuroDl.
• Promoter homologues and promoter variants can be included in an expression vector for expressing NeuroDl according to the present disclosure.
• the terms “promoter homologue” and “promoter variant” refer to a promoter which has substantially similar functional properties to confer the desired type of expression, such as cell type-specific expression of NeuroDl or ubiquitous expression of NeuroDl, on an operably linked nucleic acid encoding NeuroDl compared to those disclosed herein.
• a promoter homologue or variant has substantially similar functional properties to confer cell type-specific expression on an operably linked nucleic acid encoding NeuroDl compared to GFAP, S100b, Aldh1L1, NG2, lcn2 and CAG promoters.
• promoter variant refers to either an isolated naturally occurring or a recombinantly prepared variation of a reference promoter, such as, but not limited to, GFAP, S100b, Aldh1L1, NG2, lcn2 and pCAG promoters.
• promoters from other species are functional, e.g. the mouse AldhlLlpromoter is functional in human cells. Homologues and homologous promoters from other species can be identified using bioinformatics tools known in the art, see for example, Xuan et al ., 2005, Genome Biol 6:R72; Zhao et al ., 2005, Nucl Acid Res 33:D103-107; and Halees et al. 2003, Nucl. Acids. Res. 2003 31: 3554-3559.
• homologues and variants of cell type-specific promoters of NeuroDl and/or ubiquitous promoters have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater, nucleic acid sequence identity to the reference developmentally regulated and/or ubiquitous promoter and include a site for binding of RNA polymerase and, optionally, one or more binding sites for transcription factors.
• a nucleic acid sequence which 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 high stringency hybridization conditions.
• nucleic acid sequences encoding additional proteins can be included in an expression vector.
• additional proteins include non-NeuroDl proteins such as reporters, including, but not limited to, beta-galactosidase, green fluorescent protein and antibiotic resistance reporters.
• the recombinant expression vector encodes at least NeuroDl 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.
• the recombinant expression vector encodes at least NeuroDl 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.
• a reporter gene is included in a recombinant expression vector encoding NeuroDl.
• a reporter gene may be included to produce a peptide or protein that serves as a surrogate marker for expression of NeuroDl from the recombinant expression vector.
• reporter gene refers to gene that is easily detectable when expressed, for example by chemiluminescence, fluorescence, colorimetric reactions, antibody binding, inducible markers 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), red fluorescent protein (RFP), MmGFP (Zernicka-Goetz et al., Development, 124:1133-1137, 1997, dsRed, luciferase and beta-galactosidase (lacZ).
• mCherry is a monomeric red fluorescent protein derived from dsRed used as a reporter according to aspects of the present disclosure.
• SEQ ID NO: 9 is an example of a nucleic acid comprising a CAG promoter operably linked to a nucleic acid encoding NeuroDl, a nucleic acid sequence encoding enhanced green fluorescent protein (EGFP), an enhancer, the woodchuck hepatitis post-transcriptional regulatory element (WPRE) and a. IRES separating the nucleic acid encoding NeuroDl and the nucleic acid encoding EGFP.
• EGFP enhanced green fluorescent protein
• WPRE woodchuck hepatitis post-transcriptional regulatory element
• SEQ ID NO: 9 is inserted into an expression vector for expression of NeuroDl and the reporter gene EGFP.
• the IRES and nucleic acid encoding EGFP are removed from SEQ ID NO: 9 and the remaining nucleic acid sequence including CAG promoter and operably linked nucleic acid encoding NeuroDl is inserted into an expression vector for expression of NeuroDl.
• the WPRE or another enhancer is optionally included.
• transfection The process of introducing genetic material into a recipient host cell, such as for transient or stable expression of a desired protein encoded by the genetic material in the host cell is referred to as “transfection,” or “transduction.”
• 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, and heat shock mediated transfection.
• Transduction refers to virus-mediated introduction of genetic material into a recipient host cell.
• Virus-mediated transduction may be accomplished using a viral vector such as those derived from adenovirus, AAV and lentivirus.
• a host cell is transfected or transduced ex-vivo and then re- introduced into a host organism.
• cells or tissues may be removed from a subject, transfected or transduced with an expression vector encoding NeuroDl and then returned to the subject.
• [00182] Expression of exogenous NeuroDl in the host glial cell to convert the glial cell to a functional neuron is achieved by introduction of mRNA encoding NeuroDl, or a functional fragment thereof, to the host glial cell in vitro or in vivo according to aspects of the present disclosure.
• Expression of NeuroDl using a recombinant expression vector is accomplished by introduction of the expression vector into a eukaryotic or prokaryotic host cell expression system such as an insect cell, mammalian cell, yeast cell, bacterial cell or any other single or multicellular organism recognized in the art.
• Host cells are optionally primary cells or immortalized derivative cells. Immortalized cells are those which can be maintained in-vitro for at least 5 replication passages.
• Host cells containing the recombinant expression vector are maintained under conditions wherein NeuroDl is produced.
• Host cells may be cultured and maintained using known cell culture techniques such as described in Celis, Julio, ed., 1994, Cell Biology Laboratory Handbook, Academic Press, N.Y.
• Various culturing 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 one of skill in the art.
• a recombinant expression vector including a nucleic acid encoding NeuroDl is introduced into glial cells of a subject. Expression of exogenous NeuroDl in the glial cells “converts” the glial cells into functional neurons.
• neuroDl neuroDl
• a variant thereof or a functional fragment thereof
• a functional neuronal phenotype a functional neuronal phenotype
• NeuroDl converted neurons and “converted neurons” are used herein to designate a cell including exogenous NeuroDl protein or a functional fragment thereof which has consequent functional neuronal phenotype.
• the term “phenotype” refers to well-known detectable characteristics of the cells referred to herein.
• the functional neuronal phenotype can be, but is not limited to, one or more of: neuronal morphology, expression of one or more neuronal markers, electrophysiological characteristics of neurons, synapse formation and release of neurotransmitter.
• neuronal phenotype encompasses but is not limited to: characteristic morphological aspects of a neuron such as presence of dendrites, an axon and dendritic spines; characteristic neuronal protein expression and distribution, such as presence of synaptic proteins in synaptic puncta, presence of MAP2 in dendrites, presence of one or more of: neuronal nuclear protein (NeuN), GABA, glutamate decarboxylase (GAD) such as GAD67, Forkhead-box-Gl (FoxGl), , T-brain-1 (Tbrl), Cuxl, Ctip2, parvalbumin (PV), calretinin (CR), neuropeptide Y (NPY), and somatostatin (SST); and characteristic electrophysiological signs such as spontaneous and evoked synaptic events.
• neuronal nuclear protein Neuronal nuclear protein
• GABA glutamate decarboxylase
• GAD67 GAD67
• FoxGl Forkhead-box-G
• glial phenotype such as astrocyte phenotype and reactive astrocyte phenotypes encompasses but is not limited to: characteristic morphological aspects of astrocytes and reactive astrocytes such as a generally “star- shaped” morphology; and characteristic astrocyte and reactive astrocyte protein expression, such as presence of glial fibrillary acidic protein (GFAP).
• characteristic morphological aspects of astrocytes and reactive astrocytes such as a generally “star- shaped” morphology
• characteristic astrocyte and reactive astrocyte protein expression such as presence of glial fibrillary acidic protein (GFAP).
• GFAP glial fibrillary acidic protein
• a recombinant expression vector including a nucleic acid encoding NeuroDl, a variant thereof, or a functional fragment thereof is introduced into astrocytes of a subject. Expression of exogenous NeuroDl, a variant thereof, or a functional fragment thereof, in the astrocytes cells “converts” the astrocytes into functional neurons.
• a recombinant expression vector including a nucleic acid encoding NeuroDl, a variant thereof, or a functional fragment thereof, thereof is introduced into reactive astrocytes of a subject. Expression of exogenous NeuroDl, a variant thereof, or a functional fragment thereof, in the reactive astrocytes “converts” the reactive astrocytes into functional neurons.
• a recombinant expression vector including a nucleic acid encoding NeuroDl, a variant thereof, or a functional fragment thereof is introduced into NG2 cells of a subject. Expression of exogenous NeuroDl, a variant thereof, or a functional fragment thereof, in the NG2 cells “converts” the NG2 cells into functional neurons.
• An expression vector including a nucleic acid encoding NeuroDl, a variant thereof, or a functional fragment thereof, DNA encoding NeuroDl, a variant thereof, or a functional fragment thereof, mRNA encoding NeuroDl, a variant thereof, or a functional fragment thereof, and/or NeuroDl protein, a variant thereof, full-length or a functional fragment thereof, is optionally associated with a carrier for introduction into a host cell in vitro or in vivo.
• the carrier is a particulate carrier such as lipid particles including liposomes, micelles, unilamellar or mulitlamellar vesicles; polymer particles such as hydrogel particles, polyglycolic acid particles or polylactic acid particles; inorganic particles such as calcium phosphate particles such as described in for example U.S. Patent No. 5,648,097; and inorganic/organic particulate carriers such as described for example in U.S. Patent No. 6,630,486.
• a particulate carrier can be selected from among a lipid particle; a polymer particle; an inorganic particle; an organic particle; and a hybrid inorganic/organic particle.
• a mixture of particle types can also be included as a particulate pharmaceutically acceptable carrier.
• a particulate carrier is typically formulated such that particles have an average particle size in the range of about 1 nm - 10 microns.
• a particulate carrier is formulated such that particles have an average particle size in the range of about 1 nm - 100 nm.
• liposomes and methods relating to their preparation and use may 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. 2005, 19, 311-30.
• Detection of expression of exogenous NeuroDl following introduction of a recombinant expression vector including a nucleic acid encoding the exogenous NeuroDl or a functional fragment thereof is accomplished using any of various standard methodologies including, but not limited to, immunoassays to detect NeuroDl, nucleic acid assays to detect NeuroDl nucleic acids and detection of a reporter gene co- expressed with the exogenous NeuroDl.
• nucleic acid refers to RNA or DNA molecules having more than one nucleotide in any form including single-stranded, double-stranded, oligonucleotide or polynucleotide.
• nucleotide sequence refers to the ordering of nucleotides in an oligonucleotide or polynucleotide in a single-stranded form of nucleic acid.
• NeuroDl nucleic acid refers to an isolated NeuroDl nucleic acid molecule and encompasses isolated NeuroDl nucleic acids having a sequence that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater, identity to the DNA sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3, or the complement thereof, or a fragment thereof, or an isolated nucleic acid, such as RNA or DNA, molecule having a sequence that hybridizes under high stringency hybridization conditions to the nucleic acid set forth as SEQ ID NO: 1 or SEQ ID NO: 3, a complement thereof, or a fragment thereof.
• isolated with reference to a NeuroDl nucleic acid molecule indicates that the molecule is not in the genome of an organism from which it originated under control of the NeuroDl promoter in that location.
• the nucleic acid of SEQ ID NO: 3 is an example of an isolated DNA molecule having a sequence that hybridizes under high stringency hybridization conditions to the nucleic acid set forth in SEQ ID NO: 1.
• a fragment of a NeuroDl nucleic acid is any fragment of a NeuroDl nucleic acid that is operable in aspects of the present disclosure including a NeuroDl nucleic acid.
• a nucleic acid probe or primer able to hybridize to a target NeuroDl RNA or DNA molecule, such as mRNA or cDNA can be used for detecting and/or quantifying the RNA or DNA, such as mRNA or cDNA, encoding NeuroDl protein.
• a nucleic acid probe can be an oligonucleotide of at least 10, 15, 30, 50 or 100 nucleotides in length and sufficient to specifically hybridize under stringent conditions to NeuroDl RNA or DNA, such as mRNA or cDNA, or a complementary sequence thereof.
• a nucleic acid primer can be an oligonucleotide of at least 10, 15 or 20 nucleotides in length and sufficient to specifically hybridize under stringent conditions to the RNA or DNA, such as mRNA or cDNA, or complementary sequence thereof.
• nucleic acid includes a nucleotide sequence described as having a “percent complementarity” to a specified second nucleotide sequence.
• a nucleotide sequence may have 80%, 90%, or 100% complementarity to a specified second nucleotide sequence, indicating that 8 of 10, 9 of 10 or 10 of 10 nucleotides of a sequence are complementary to the specified second nucleotide sequence.
• the nucleotide sequence 3’-TCGA-5’ is 100% complementary to the nucleotide sequence 5’-AGCT-3'
• the nucleotide sequence 3’-TCGA- is 100% complementary to a region of the nucleotide sequence 5’-TTAGCTGG-3'
• hybridization and “hybridizes” refer to pairing and binding of complementary nucleic acids. Hybridization occurs to varying extents between two nucleic acids depending on factors such as the degree of complementarity of the nucleic acids, the melting temperature, Tm, of the nucleic acids and the stringency of hybridization conditions, as is well known in the art.
• stringency of hybridization conditions refers to conditions of temperature, ionic strength, and composition of a hybridization medium with respect to particular common additives such as formamide and Denhardt’s solution.
• Intermediate stringency conditions are exemplified by conditions under which nucleic acids having intermediate complementarity, about 50-84% complementarity, as well as those having a high degree of complementarity, hybridize.
• low stringency hybridization conditions are those in which nucleic acids having a low degree of complementarity hybridize.
• hybridization and “specifically hybridizes” 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.
• Stringency of hybridization and washing conditions depends on several factors, including the Tm of the probe and target and ionic strength of the hybridization and wash conditions, as is well-known to the skilled artisan.
• Hybridization and conditions to achieve a desired hybridization stringency are described, for example, in Sambrook et al ., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2001; and Ausubel, F. et al., (Eds.), Short Protocols in Molecular Biology, Wiley, 2002.
• An example of high stringency hybridization conditions is hybridization of nucleic acids over about 100 nucleotides in length in a solution containing 6X SSC, 5X Denhardt’s solution, 30% formamide, and 100 micrograms/ml denatured salmon sperm at 37°C overnight followed by washing in a solution of 0.1X SSC and 0.1% SDS at 60°C for 15 minutes.
• SSC is 0.15M NaCl/0.015M Na citrate.
• Denhardt’s solution is 0.02% bovine serum albumin/0.02% FICOLL/0.02% polyvinylpyrrolidone.
• SEQ ID NO: 1 and SEQ ID NO: 3 will hybridize to the complement of substantially identical targets and not to unrelated sequences.
• inventive compositions and methods are illustrated in the following examples. These examples are provided for illustrative purposes and are not considered limitations on the scope of inventive compositions and methods.
• Wild type (WT) C57BL/6J and FVB/N-Tg(GFAP::GFP) 14Mes/J transgenic mice were purchased from Jackson Laboratory. Mice were housed in a 12 hr light/dark cycle and supplied with sufficient food and water. Adult mice (25-35 g) of both genders, aged 4-6 months old, were used in this example.
• mice were anesthetized with ketamine/xylazine (120 mg/kg ketamine; 8 mg/kg xylazine) by intraperitoneal (IP) injection. After being fully anesthetized, each mouse was transferred onto a stereotaxic apparatus and the head fixed on shape-matched foam. The foam was made by soft plastic materials to absorb superfluous impact as well as to stabilize the animal’s head. An incision was made along the midline to fully expose the impact site on the skull, which is above mouse motor cortex with coordinates of 1.0 mm anterior to the Bregma and 1.5 mm lateral to the midline. The ear bars were removed to avoid injury to the ear canals before impact induction.
• IP intraperitoneal
• the electro-magnetic controlled device, Impactor One was purchased from Leica Biosystems® for TBI induction. Impact force of larger than 5.0 m/s was avoided to prevent skull fracture and death.
• An impactor tip of 2 mm diameter was used at velocity of 5 m/s, dwelling time of 200 ms, and impact depth of 1.0 mm, to cause focal closed head injury. The bottom plane of impactor tip was adjusted to be tangential to the impact site on the skull, in order to deliver the impact force evenly to the skull and reduce the risk of skull fracture. Animals with an obvious skull fracture are excluded from the experiments and euthanized immediately.
• mice 7 days after closed head injury, mice were randomly selected for injection administration of either a virus encoding NeuroDl or a control virus. Mice were anesthetized with ketamine/xylazine (120 mg/kg ketamine; 8 mg/kg xylazine) by intraperitoneal injection and placed in a stereotaxic apparatus. An incisor bar with a nose bridge holder and two ear bars were used to fix the head. After a midline incision was made, a small hole of ⁇ 1 mm was drilled in the skull at the center of impact site (coordinates: 1.0 mm anterior, 1.5 mm left lateral to Bregma).
• the selected virus 1.5 ⁇ L (AAV9)hGFAP::GFP or (AAV9)hGFAP::NeuroDl-GFP, or 3 ⁇ L retrovirus carrying NeuroDl- GFP or GFP control, was injected into the injured brain region using a motorized micro pump injector at a speed of 0.15 ⁇ L/min for 10 min with a 5 ⁇ L Hamilton brand glass syringe with a 33 Gauge needle. After injection, the needle was maintained in place for an additional 3 minutes before being fully withdrawn. Post- surgery, mice recovered on heating pad until free movement was observed. Mice were singly housed and carefully monitored daily for at least one week or until sacrifice. [00222] Neural projection tracing by virus or dye
• adeno-associated virus (AAV) with hSyn::Cre plus CAG: :FLEX-mCherry-P2A-mCherry was injected into thalamus (coordinates: 2.0 mm posterior, 1.1 mm left lateral to Bregma; 2.8 mm ventral to skull surface).
• cholera toxin B subunit fused with 647 fluorescent probe (CTB-647) was injected in the cortex contralateral to injury site (coordinates: 1.0 mm anterior, 1.5 mm right lateral to Bregma, 1.6 mm ventral to skull surface). Animals were sacrificed 7 days later and brain samples collected for analysis.
• the plasmid pAAV-GFAP-hChR2(H134R)-mCherry was obtained from Addgene (plasmid # 27055; RRID:Addgene_27055).
• pAAV-hGFAP::GFP and pAAV-hGFAP::NeuroDl-P2A-GFP vectors cDNAs coding GFP or NeuroDl were produced by PCR using the retroviral constructs as described in detail in Guo et al., Cell Stem Cell 14, 188-202, 2014.
• the GFP gene or NeuroDl fused with P2A-GFP gene was subcloned into the pAAV-GFAP-hChR2(H134R)-mCherry vector with hChR2(H134R)- mCherry cut out between Kpnl and BsrGI sites.
• the Cre gene was obtained by PCR from hGFAP-Cre (Addgene plasmid #40591) and inserted into AAV phSyn(S)-FlpO-bGHpA (Addgene plasmid # 51669) between Kpnl and Bmtl sites with FlpO replaced to generate pAAV-hSyn:Cre vector.
• the pAAV-FLEX-mCherry-P2A-mCherry vector was constructed as described in detail in Chen et al., Mol Ther., 2020 Jan 8;28(l):217-234. Plasmid constructs were sequenced for verification.
• Recombinant AAV9 was produced in 293AAV cells (Cell Biolabs, San Diego, CA, USA). Polyethylenimine (PEI, linear, MW 25,000) was used for transfection of triple plasmids: the pAAV expression vector, pAAV9-RC (Cell Biolabs, San Diego, CA, USA) and pHelper (Cell Biolabs, San Diego, CA, USA). 72 hours post-transfection, cells were scraped in their medium and centrifuged, frozen, and thawed four times by placing them alternately in dry ice or ethanol and a 37°C water bath.
• PEI Polyethylenimine
• AAV crude lysate was purified by centrifugation at 54,000 rpm for 1 hour in discontinuous iodixanol gradients with a Beckman SW55Ti rotor.
• the virus-containing layer was extracted, and viruses were concentrated by Millipore Amicon Ultra Centrifugal Filters.
• Virus titers were 2.2xlO u genome copies per milliliter (GC/mL) for hGFAP::GFP, 2.3 x10 11 GC/mL for hGFAP::NDl-GFP, 4.6x10 11 GC/mL for hSyn::Cre, and 1.6x10 12 GC/mL for CAG::FLEX-mCherry-P2A-mCherry, determined by QuickTiter AAV Quantitation Kit (Cell Biolabs, San Diego, CA, USA).
• the pCAG-NeuroDl-IRES-GFP and pCAG-GFP were constructed as previously described (Guo et al., Cell Stem Cell , 14:188-202 (2014)).
• gpg helper-free HEK cells were transfected with the target plasmid together with vesicular stomatitis virus G protein (VSV-G) vector to produce the retroviruses expressing NeuroDl or GFP.
• VSV-G vesicular stomatitis virus G protein
• the titer of retroviral particles was about 10 7 parti cles/mL, determined after transduction of HEK cells.
• Brain slices were washed 3 times with phosphate-buffered saline (PBS) followed by permeablization in 2% Triton X-100 in PBS for 10 minutes. Then brain sections were blocked in 5 % normal donkey serum and 0.3% Triton X-100 in PBS for 2 hours. The primary antibodies were added into blocking buffer and incubated with brain sections for overnight at 4°C. Primary antibodies were rinsed off with PBS 3 times followed by secondary antibody incubation for 2 hours at room temperature (RT). After being washed with PBS, brain sections were mounted onto a glass slide with an anti-fading mounting solution (Invitrogen). Images were acquired with confocal microscopes (Olympus FV1000 or Zeiss LSM800). To ensure antibody specificity, only secondary antibody was used for immunostaining as a side-by-side control, with no distinct signal detected.
• PBS phosphate-buffered saline
• mice 28 days after NeuroDl virus injection following closed head injury (CHI), the animals were placed in a running wheel. Thirty minutes after actively running, the mice were placed back into the home cage. One hour later they were sacrificed and perfused for c-Fos immunostaining.
• CHI closed head injury
• mice were anaesthetized with 2.5% avertin, and then perfused with NMDG-based cutting solution containing (in mM): 93 NMDG, 93 HC1, 2.5 KC1, 1.25 NaH2PO 4 , 30 NaHCCO 3 , 20 HEPES, 15 glucose, 12 N-Acetyl-L-cysteine, 5 sodium ascorbate, 2 thiourea, 3 sodium pyruvate, 7 MgSO 4 , and 0.5 CaCl 2 , at pH 7.3-7.4, at 300 mOsm, and bubbled with 95% O2 / 5% CO 2 .
• NMDG-based cutting solution containing (in mM): 93 NMDG, 93 HC1, 2.5 KC1, 1.25 NaH2PO 4 , 30 NaHCCO 3 , 20 HEPES, 15 glucose, 12 N-Acetyl-L-cysteine, 5 sodium ascorbate, 2 thiourea, 3 sodium pyruvate
• Coronal sections of 300 ⁇ m thickness were cut around AAV-injected cortical areas with a vibratome (VT1200S, Leica, Germany) at room temperature. Slices were collected and incubated at 33.0 ⁇ 1.0 °C in oxygenated NMDG cutting solution for 10-15 minutes. Then, slices were transferred to holding solutions with continuous 95% 02 / 5% CO2 bubbling and containing (in mM): 92 NaCl, 2.5 KC1, 1.25 NaH2PO 4 , 30 NaHCO 3 , 20 HEPES, 15 glucose, 12 N-Acetyl-L-cysteine, 5 sodium ascorbate, 2 thiourea, 3 sodium pyruvate, 2 MgSO 4 , and 2 CaCl 2 .
• a single slice was transferred to the recording chamber continuously perfused with standard aCSF (artificial cerebral spinal fluid) saturated by 95% O2 / 5% CO 2 at 33.0 ⁇ 1.0 °C.
• the standard aCSF contained (in mM): 124 NaCl, 2.5 KC1, 1.25 NaH2PO 4 , 26 NaHCO 3 , 10 glucose, 1.3 MgSO 4 , and 2.5 CaCl 2 .
• pipette solution contained (in mM): 120 Cs-methanesulfonate, lO KCl, 10 Na-phosphocreatine, 10 HEPES, 5 QX-314, 1 EGTA, 4 MgATP and 0.3 Na 2 GTP, pH 7.3 adjusted with KOH, 280-290 mOsm.
• 0.5% biocytin Sigma, Cat.B4261 was added to the pipette solution.
• the cell membrane potentials were held at -70 mV (the reversal potential of GABAA receptors) for sEPSC recording, and 0 mV (the reversal potential of ionotropic glutamate receptors) for sIPSC recording, respectively.
• Data were collected with a MultiClamp 700A amplifier and analyzed with pClamp 9.0 and Clampfit 10.6 software (Molecular Devices).
• the cortical areas around injury site from 750 ⁇ m to 2250 ⁇ m lateral to the midline were defined as total injury area for analysis.
• the superficial layer with width less than 600 ⁇ m and depth less than 450 ⁇ m from the impact center was defined as injury core.
• the middle layer with width 600-1000 ⁇ m and depth 450-900 ⁇ m from the impact center was defined as peri-injury area.
• mice brains were imaged by the Z-stack and tile function of Olympus FV-1000 with 40x oil lens after immunostaining.
• the range of Z-stack was set to be 5 layers with 1.5 mih step size around the center plane of the mounted slice.
• 3 squares of Z-stack images (resolution: 512x512, 0.621 pm/pixel) were selected inside the injury core or the peri-injury area for quantification.
• an electromagnetic controlled device Leica impactor one, shown diagrammatically in Fig. 1 A left, was used to induce a precisely controlled CHI, a type of TBI, to exposed skull above mouse motor cortex, see Fig. 1A right.
• Figures 2A, 2B, and 2C demonstrate neuronal death and degeneration at injury site after CHI.
• FIG. 2A 3 brain regions close to the impact center were considered as the injury core, located mostly inside the smallest semi-circular area defined by a dashed line. 5 regions next to the injury core were taken as peri-injury area, located mostly inside the larger semi-circular area defined by a dashed line. All the regions inside the dashed rectangular boxes defined by dashed lines were taken as region of interest (ROI) for analysis.
• ROI region of interest
• the brain sample at early time points after CHI such as 6 hours and 4 days after injury, were collected and assayed to detect a biomarker of cell apoptosis - Terminal deoxynucleotidyl transferase dUTP nick end labeling, TUNEL.
• TUNEL Terminal deoxynucleotidyl transferase dUTP nick end labeling
• the CHI with the primary injury by mechanical force or the following secondary injury damaged the cells and induced apoptosis.
• other brain areas like the regions in the same hemisphere far away from the impact site or the contralateral side, no TUNEL signal was detected.
• CHI myelin basic protein
• NF200 molecular weight neurofilament proteins
• the astrocytes around the injury core became greatly reactive compared to the non-injury side or the sham group, see Fig. 1C and Fig. IF.
• the cell proliferation marker, Ki67 staining the cell proliferation marker, Ki67, at different time points after CHI, it was found that the proliferation rate of astrocytes reached a peak at 4 days post-injury and go quiescent after 7 days post-injury, see Fig. 1G and Fig. 1H.
• the microglia population, marked by Ibal staining, see Fig. 1G appears to have a proliferation curve peaking earlier, at 1 day post-injury, see Fig. 1H.
• an AAV vector, recombinant serotype AAV9 was constructed to express NeuroDl in mouse cortical astrocytes under the direct control of a human GFAP promotor with enhanced green fluorescence protein (GFP) as indicator of expression, the construct designated hGFAP: :NeuroDl-P2A-GFP (also called ( AAV)GF AP : :ND 1 -GFP).
• Figures 3 A, 3B, 3C, 3D, 3E, 3F, 3G, and 3H show astrocyte-to-neuron conversion in situ by NeuroDl (NDl) after closed head injury in mouse neocortex.
• FIG. 3A and Fig. 3B an AAV expression vector expressing NeuroDl (AAV)GFAP::ND1-GFP or a control expression vector (AAV)GFAP::GFP) was injected into the injury site 7 days after CHI. Following administration of these vectors, cells were examined to determine which were infected by each virus and which express the encoded gene under control of the GFAP promotor.
• Figure 3C is a set of representative images showing the injured cortex 7 days after injection of AAV-GFAP::GFP virus (control group, left panel) or injection of AAV- GFAP::ND1-GFP virus (NDl group, right panel).
• FIG. 4A Figures 4A, 4B, 4C, and 4D show a transitional stage of astrocyte-to-neuron conversion, maturation of converted neurons, and conversion induced by retrovirus carrying NeuroDl.
• Fig. 3F- Day 7 i.e. 7 dpi
• GFP+ cells lose the astrocytic marker (GFAP) and gained the neuronal marker (NeuN), see Fig. 3F and Fig. 3H.
• Brain sections from animals in the NDl group were co-immunostained for the immature neuron marker, Tujl, and the mature neuron marker, MAP2, see Fig. 4B. Most converted neurons show higher Tuj 1 and lower MAP2 at early time points, but lower Tujl and higher MAP2 at later time points. This could reflect the converted neurons would undergo a maturation process, which may be similar to the developmental stage of the neural stem cells.
• astrocyte to neuron conversion was confirmed using a retrovirus vector, which would specifically infect dividing cells and could exclude the leakage issue.
• the plasmids were constructed to express NDl under control of a CAG promotor as previously described in Guo et al ., Cell Stem Cell , 14:188-202 (2014). Seven days after retrovirus injection, many NeuN+ and GFP+ cells with neuronal morphology were found in brains of mice to which the NDl- and GFP-expressing retrovirus was administered “NDl retrovirus” group compared to a control expressing GFP only, see Fig. 4C and Fig. 4D.
• the converted neurons can develop into different subtypes with cortical characteristics.
• glial cells are converted to neurons, it was investigated whether the converted neurons acquire the same molecular profiles as the endogenous neurons.
• the cells were immunostained to detect the forebrain marker, Forkhead-box-Gl (FoxGl), and forebrain neuronal marker, T-brain-1 (Tbrl).
• FoxGl is a transcription factor widely spread in all the regions originated from the telencephalon.
• Tbrl is involved in neuronal differentiation and migration in mice, especially in glutamatergic neurons.
• Figures 5A, 5B, 5C, and 5D show that the converted neurons could acquire cortical characteristics consistent with local microenvironment.
• the cells were immunostained to detect the cortical superficial layer marker Cuxl and deep layer marker Ctip2, see Fig. 5B, and Fig. 5C. After ND1 treatment, these two markers still had the same distribution as in uninjured cortex, see Fig. 5B, and Fig. 5C.
• 24.6% ⁇ 6.8% of the GFP+ and NeuN+ cells showed colocalization of Cuxl signal.
• the colocalization percentage was 11.0% ⁇ 1.5%, see Fig. 5D.
• the converted neurons were assayed to determine if they were excitatory or inhibitory neurons.
• Figures 6A, 6B, and 6C show that the converted neurons could differentiate into different subtypes.
• each single neuron plays its role by receiving, integrating and transmitting electrical signals. Therefore, the electrophysiological properties of converted neurons were investigated at 4 weeks after virus injection.
• Figures 7A, 7B, 7C, 7D, 7E, 7F, and 7G show that the ND1 converted neurons are functionally mature.
• sEPSC spontaneous excitatory postsynaptic currents
• sIPSC spontaneous inhibitory postsynaptic currents
• sEPSC amplitudes There was a significant difference of sEPSC amplitudes between the NDl group and control GFP virus group (p ⁇ 0.001), which was respectively 13.3 ⁇ 1.1 pA and 7.4 ⁇ 0.6 pA, see Fig. 7F.
• sIPSC neither the amplitude (NDl: 19.4 ⁇ 1.8 pA, control: 20.4 ⁇ 2.4 pA) nor the frequency (NDl: 1.2 ⁇ 0.3 Hz, control: 1.0 ⁇ 0.2 Hz) had significant difference between the two groups, see Fig. 7G.
• sEPSC showed an evident difference between converted neurons and control
• electrophysiological properties of converted neurons at more time points including at 1 week, 2 weeks, 4 weeks, and 8 weeks after virus injection (weeks post-injection, wpi)
• the frequency of sEPSC in converted neurons was higher at early time points (1, 2, and 4 wpi), then went down at 8 wpi, see Fig. 8A.
• the amplitude of sEPSC was low at 1 wpi, then went up high at 2 wpi and 4 wpi, and then went down at 8 wpi, see Fig. 8B.
• the frequency and amplitude of sEPSC at the later time point, 8 wpi were comparable to control, see Fig. 8 A and Fig. 8B.
• the frequency of sEPSC reflected the intensity of excitatory innervations which other neurons put on the converted one.
• the amplitude of sEPSC could be determined by the density of glutamate receptors on the postsynaptic membrane of converted neurons.
• FIGS. 9A, 9B, 9C, 9D, 9E, 9F, and 9H show that NDl -converted neurons integrate into local and remote neural networks.
• Figure 8D is a set of images illustrating colocalization of a synaptic marker (VGAT) with GFP and NeuN in the cell soma of converted neurons at 7 days after NeuroDl virus injection and CTB-647 injection on the contralateral side; CTB signal from contralateral side was also observed on the cell soma.
• Figure 8E is a set of images illustrating colocalization of a synaptic vesicle marker (SV2) with GFP and NeuN in the cell soma of converted neurons at 7 days after NeuroDl virus injection and CTB-647 injection on the contralateral side; CTB signal from contralateral side was also observed on the cell soma.
• SV2 synaptic vesicle marker
• Embodiment 1 A method of treating traumatic brain injury (TBI) comprising converting reactive astrocytes to functional neurons by providing exogenous neurogenic differentiation 1 (NeuroDl) to at least one reactive astrocyte in a damaged region of a subject’s brain.
• TBI traumatic brain injury
• NeuroDl exogenous neurogenic differentiation 1
• Embodiment 2 The method of embodiment 1, wherein the TBI is a closed head injury.
• Embodiment 3 The method of embodiments 1 or 2, wherein the damage region of the brain comprises non-functional neurons and reactive astrocytes due to the TBI.
• Embodiment 4 The method of embodiment 3, wherein the non-functional neurons are selected from the group consisting of dead and dying neurons.
• Embodiment 5 The method of embodiments 3 or 4, wherein the non- functional neurons are detected by a functional MRI (fMRI).
• fMRI functional MRI
• Embodiment 6 The method of any of embodiments 3 to 5, wherein the presence of non-functional neurons and reactive astrocytes in the damaged region are not primarily due to bleeding in the damaged region.
• Embodiment 7 The method of any of embodiments 3 to 6, wherein the presence of non-functional neurons and reactive astrocytes in the damaged region are not primarily due to ischemia in the damaged region.
• Embodiment 8 The method of any of embodiments 1 to 7, wherein providing the exogenous NeuroDl comprises administering a recombinant expression vector to the subject, wherein the recombinant expression vector comprises a nucleic acid sequence encoding NeuroDl.
• Embodiment 9 The method of any of embodiments 1 to 7, wherein providing the exogenous NeuroDl comprises administering a recombinant expression vector to the subject, wherein the recombinant expression vector is a viral expression vector comprising a nucleic acid sequence encoding NeuroDl.
• Embodiment 10 The method of any of embodiments 1 to 8, wherein providing the exogenous NeuroDl comprises administering a recombinant expression vector to the subject, wherein the recombinant expression vector is a recombinant adeno- associated virus expression vector, and wherein the recombinant adeno-associated virus vector comprises a nucleic acid sequence encoding NeuroDl.
• Embodiment 11 The method of any of embodiments 8 to 10, wherein the nucleic acid sequence encoding NeuroDl is operably linked to a promoter.
• Embodiment 12 The method of embodiment 11, wherein the promoter is a glial-cell specific promoter.
• Embodiment 13 The method of embodiment 12, wherein the glial-cell specific promoter is a glial fibrillary acidic protein (GFAP) promoter.
• GFAP glial fibrillary acidic protein
• Embodiment 14 The method of embodiment 13, wherein the GFAP promoter is a human GFAP (hGFP) promoter.
• hGFP human GFAP
• Embodiment 15 The method of any of embodiments 1 to 14, wherein no exogenous transcription factor other than NeuroDl is provided to the at least one reactive astrocyte.
• Embodiment 16 The method of any of embodiments 1 to 15, wherein the subject is human.
• Embodiment 17 The method of any of embodiments 1 to 16, wherein providing the exogenous NeuroDl comprises providing exogenous NeuroDl to the at least one reactive astrocyte at a first treatment time in the range of about two days to about ten days after the traumatic brain injury.
• Embodiment 18 The method of any of embodiments 1 to 17, wherein the traumatic brain injury causes a period of astrogliosis in the damaged region, and wherein providing the exogenous NeuroDl comprises providing exogenous NeuroDl to the at least one reactive astrocyte at a first treatment time during the period of astrogliosis or within 4 weeks after the period of astrogliosis.
• Embodiment 19 The method of embodiment 18, wherein providing the exogenous NeuroDl comprises providing exogenous NeuroDl to the at least one reactive astrocyte at a second treatment time after the first treatment time and during the period of astrogliosis or within 4 weeks after the period of astrogliosis.
• Embodiment 20 The method of embodiment 19, wherein providing the exogenous NeuroDl comprises providing exogenous NeuroDl to the at least one reactive astrocyte at a third treatment time after the second treatment time and during the period of astrogliosis or within 4 weeks after the period of astrogliosis.
• Embodiment 21 The method of any of embodiments 1 to 20, wherein the NeuroDl comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, a functional fragment of SEQ ID NO: 2, a functional fragment of SEQ ID NO: 4, an amino acid sequence having at least 85% identity to SEQ ID NO: 2, and an amino acid sequence having at least 85% identity to SEQ ID NO: 4.
• Embodiment 22 The method of embodiment 21, wherein the NeuroDl is encoded by a nucleic acid sequence comprising SEQ ID NO: 1, a nucleic acid sequence having at least 85% identity to SEQ ID NO: 1, a nucleic acid sequence comprising SEQ ID NO: 3, or a nucleic acid sequence having at least 85% identity to SEQ ID NO: 3.
• Embodiment 23 The method of any of embodiments 1 to 22, wherein providing the exogenous NeuroDl comprises injection into the damaged region of the brain.
• Embodiment 24 The method of any of embodiments 8 to 23, wherein the nucleic acid sequence encoding NeuroDl is present in a virus particle.
• Embodiment 25 The method of embodiment 24, wherein providing the exogenous NeuroDl comprises administering about 10 7 to about 10 14 virus particles to the damaged brain region of the subject.
• Embodiment 26 Use of a composition comprising neurogenic differentiation 1 (NeuroDl) in the manufacture of a medicament for converting reactive astrocytes to functional neurons in a damaged region of a subject’s brain, wherein the damaged region of the brain comprises non-functional neurons and reactive astrocytes, due to a traumatic brain injury (TBI).
• NeuroDl neurogenic differentiation 1
• Embodiment 27 The use of embodiment 26, wherein the non-functional neurons are selected from the group consisting of dead and dying neurons.
• Embodiment 28 The use of embodiments 26 or 27, wherein the traumatic brain injury is a closed head injury.
• Embodiment 29 The use of any of embodiments 26 to 28, wherein the NeuroDl is encoded by a nucleic acid sequence comprises a nucleic acid sequence having at least 85% identity to SEQ ID NO: 1.
• Embodiment 30 The use of any of embodiments 26 to 29, wherein the nucleic acid encoding NeuroDl comprises a nucleic acid sequence having at least 85% identity to SEQ ID NO: 3.
• Embodiment 31 The use of any of embodiments 26 to 30, wherein the NeuroDl comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, a functional fragment of SEQ ID NO: 2, a functional fragment of SEQ ID NO: 4, an amino acid sequence having at least 85% identity to SEQ ID NO: 2, and an amino acid sequence having at least 85% identity to SEQ ID NO: 4.
• compositions and methods described herein are presently representative of preferred embodiments, exemplary, and not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art. Such changes and other uses can be made without departing from the scope of the invention as set forth in the claims.

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