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Definitions

• the disclosure provides methods for treating a neurological disorder, such as a traumatic spinal cord injury or brain injury, or a disorder such as Parkinson’s disease, by administering a therapeutically effective amount of Gsxl protein (such as a Gsxl-cell penetrating peptide fusion protein) or nucleic acid molecule encoding Gsxl, thereby treating the neurological disorder.
• a neurological disorder such as a traumatic spinal cord injury or brain injury, or a disorder such as Parkinson’s disease
• SCI spinal cord injury
• NSPCs neural stem and progenitor cells
• NSPCs in the spinal cord largely differentiate into astrocytes and oligodendrocytes, with only a very small portion into neurons (Sabelstrom et al, Exp Neurol 260:44-49 (2014)).
• Efforts have been made to repair and regenerate the damaged spinal cord by stem cell therapy and forced expression of neurogenic transcription factors (Sox2, NeuroDl, and Olig2) to generate neurons in injured spinal cord (Chen et al, Brain Res Bull 135, 143-148 (2017)).
• these approaches provide limited or no functional improvement.
• injury-induced reactive astrocytes produce chondroitin sulfate proteoglycans (CSPGs), which prevent axonal growth and sprouting, and result in permanent functional deficits.
• CSPGs chondroitin sulfate proteoglycans
• Genomic Screened Homeo Box 1 (Gsxl or Gshl) is a neurogenic factor highly expressed in the central nervous system at the embryonic stage (Gong et al, Nature 425:917-925 (2003)).
• Gsxl and its homolog Gsx2 regulate proliferation and differentiation of neural stem/progenitor cells (NSPCs).
• NSCs neural stem/progenitor cells
• lentivirus-mediated gene expression system to transduce Gsxl into the adult mouse spinal cord with a lateral hemisection injury, results in Gsxl expression that promotes cell proliferation and activation of NSPCs at the injury site. Furthermore, lentivirus- mediated Gsxl expression at or near the injury site (Gsxl treatment) increases the number of glutamatergic and cholinergic neurons and decreases the number of GABAergic interneurons.
• Gsxl treatment attenuated glial scar formation and dramatically improved locomotor function in the injured mice.
• Genome-wide transcriptome analysis reveals that Gsxl treatment induces the Notch signaling pathway, which correlates with NSPC activation, neuronal differentiation, and provides molecular insight for Gsxl -mediated functional recovery.
• the neurological disorder can be a spinal cord injury, a brain injury, or both, such as one resulting from head and/or spinal cord trauma, such as one caused by a vehicle crash, fall, act of violence, or sports.
• the neurological disorder is a neurodegenerative disease, such as Parkinson’s disease, Alzheimer’s disease, stroke, ischemia, epilepsy, Huntington's disease, multiple sclerosis, or amyotrophic lateral sclerosis.
• Such methods can increase neurogenesis, decrease inflammation, decrease cell death, reduce astrogliosis, reduce glial scaring, increase locomotion of the subject, or combinations thereof.
• the method increases neurogenesis, reduces astrogliosis, reduces glial scaring, and increases locomotion of the subject.
• the methods include administering to the subject a therapeutically effective amount of a Gsxl protein (such as one that includes at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2 or 4) or administering to the subject a therapeutically effective amount of a nucleic acid molecule encoding Gsxl (such as one that includes at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1 or 3), thereby treating the neurological disorder.
• a Gsxl protein such as one that includes at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2 or 4
• a nucleic acid molecule encoding Gsxl such as one that includes at least 80%, at least 85%, at least 90%, at least 95%, at least
• the Gsxl protein administered is a Gsxl fusion protein, such as one that includes a Gsxl domain (such as one that includes at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2 or 4) and a cell penetrating peptide (CPP) domain (such as one that includes at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any of SEQ ID NOS: 61-79), wherein the Gsxl domain and the CPP domain can be linked indirectly ( e.g ., via a linker, such as SEQ ID NO: 80 or 81) or directly.
• a linker such as SEQ ID NO: 80 or 81
• the nucleic acid molecule encoding Gsxl encodes a Gsxl -CPP fusion protein, such as one that encodes a Gsxl domain (such as one that includes at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1 or 3, or encodes a protein comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2 or 4) operably linked to a CPP domain (such as one that encodes a CPP domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any of SEQ ID NOS: 61-79).
• the Gsxl protein, Gsxl-CPP fusion protein, or the nucleic acid molecule encoding one of these proteins is isolated
• Administration can include injection, such as injection into the CNS (e.g., spinal cord or brain).
• CNS e.g., spinal cord or brain.
• the nucleic acid molecule encoding Gsxl (such as a Gsxl-CPP fusion protein) includes or is part of a plasmid or viral vector, such as a lentiviral vector or adeno- associated viral (AAV) vector.
• the nucleic acid molecule encoding Gsxl (such as a Gsxl-CPP fusion protein) can be operably linked to a promoter, and enhancer, or both.
• Exemplary promoters include a constitutive promoter (e.g, CMV) or a central nervous system (CNS)-specific promoter (e.g, a synapasin 1 (Synl) promoter, glial fibrillary acidic protein (GFAP) promoter, nestin (NES) promoter, myelin-associated oligodendrocyte basic protein (MOBP) promoter, myelin basic protein (MBP) promoter, tyrosine hydroxylase (TH) promoter, or a forkhead box A2 (FOXA2) promoter).
• An exemplary enhancer is a neural-specific enhancer, such as NotchlCR2 (e.g, Tzatzalos et al, Dev Biol. 372(2):2l7-28, 2012) or Olig2CR5 (Hao et al., Dev Biol. 393(1): 183-93, 2014).
• One or more doses of the Gsxl protein, Gsxl-CPP fusion protein, or nucleic acid molecule encoding Gsxl can be administered.
• at least two separate administrations of the therapeutically effective amount of Gsxl protein, Gsxl-CPP fusion protein, or nucleic acid molecule encoding Gsxl are given to the subject, such as separated by at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 9 months, or at least one year.
• the Gsxl protein, Gsxl-CPP fusion protein, or nucleic acid molecule encoding Gsxl is administered within 1 hour, within 2 hours, within 3 hours, within 4 hours, within 5 hours, within 6 hours, within 12 hours, within 24 hours, within 48 hours, within 72 hours, within 96 hours, within 1 week, within 2 weeks, within 3 weeks, within 4 weeks, within 1 month, within 2 months, or within 3 months of the onset of the neurological disorder (e.g, within this amount of time following the traumatic event leading to the neurological disorder).
• Other neurological disorder therapeutic agents can also be administered.
• compositions which can be used with the disclosed methods.
• the composition includes an isolated Gsxl protein comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2 or 4 and a liposome, wherein the Gsxl protein is encapsulated in the liposome.
• the composition includes a fusion protein that includes (1) a Gsxl domain comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2 or 4 and (2) a cell penetrating peptide domain (such as one having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any of SEQ ID NOS: 61-79).
• a fusion protein can be encapsulated in a liposome.
• the composition includes a nucleic acid molecule encoding a Gsxl comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1 or 3 and a liposome, wherein such nucleic acid molecule is encapsulated in the liposome and includes or is part of a plasmid or viral vector, such as a lentiviral vector or adeno-associated viral (AAV) vector.
• a plasmid or viral vector such as a lentiviral vector or adeno-associated viral (AAV) vector.
• the composition includes a nucleic acid molecule encoding a Gsxl- CPP fusion protein, such as such as one that encodes a Gsxl domain (such as one that includes at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1 or 3, or encodes a protein comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2 or 4) operably linked to a CPP domain (such as one that encodes a CPP domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any of SEQ ID NOS: 61-79), and a liposome, wherein such nucleic acid molecule is encapsulated in the liposome and includes or is part of a plasmid or viral vector
• FIGS. 2A-2E Transduction of lenti-Gsxl-RFP is successful in delivering
• FIGS. 3A-3C RNA-Seq Analysis.
• A Number of biological replicates used for each group (SCI+Ctrl and SCI+Gsxl) at 3 different times points (3 DPI, 14 DPI, and 35 DPI) for RNA- Seq analysis.
• B Total number of differentially expressed genes (DEGs; p ⁇ 0.05) that are upregulated and downregulated at 3 DPI, 14 DPI, and 35 DPI.
• C Volcano plot at 3 DPI, 14 DPI, and 35 DPI indicating differentially expressed genes.
• FIG. 4 Functional enrichment of gene ontology (GO) terms for differentially expressed genes (DEGs) at 3 DPI.
• Enrichment terms for biological process represented as a scatter plot in a two dimensional semantic space using REVIGO. Circle size indicates the loglO(p-value) of the GO terms.
• FIGS. 5A-5I Gsxl expression increases NSPC activation after SCI.
• B Quantification of all Nestin+ cells and
• C Nestin+/RFP+ co-labeled cells.
• E List of differentially expressed genes that promote Notch signaling after lenti-Gsxl treatment compared to lenti-Ctrl treatment from RNA- Seq analysis.
• FIGS. 6A-6G Gsxl induces neurogenesis in the adult spinal cord after SCI.
• (D) Quantification of virally transduced cells co-labeled DCX, GFAP, or PDGFRa; n 6.
• Gene expression box plot of (E) DCX, (F) GFAP, and (G) PDGFRa at 35 DPI between SCI+Ctrl and SCI+Gsxl group. Each dot represents the gene expression as log2(count per million) for one biological replicate sample. Mean ⁇ SEM; * p ⁇ 0.05; Students’ t-test.
• FIGS. 7A-7D Gsxl induces neurogenesis at similar level in dorsal and ventral region of the spinal cord tissue.
• Confocal images of cross-sections (dorsal and ventral) of spinal cord tissues at 14 DPI show the expression of viral reporter RFP and early neuronal marker
• A Doublecortin DCX
• B astrocyte marker GFAP
• C oligodendrocyte progenitor marker PDGFRa. Arrows indicate cell marker+/RFP+ co-labeled cells.
• (D) Schematic and low beautiful view of the spinal cord. Scale bar 100 pm.
• FIG. 8A Functional enrichment of gene ontology (GO) terms for differentially expressed genes (DEGs) at 14 DPI.
• Enrichment terms for biological process represented as a scatter plot in a two dimensional semantic space using REVIGO. Circle size indicates the loglO(p-value) of the GO terms.
• FIGS. 8B-8C Gsxl treatment does not change the number of oligodendrocytes after SCI. Hemisection SCI was performed on 8-12 weeks old mice around T10. Immediately after lentivirus injection encoding Ctrl or Gsxl gene along with RFP reporter.
• FIGS. 9A-9F Gsxl induces glutamatergic and cholinergic interneurons and decreases GABAergic intemeurons.
• FIGS. 10A-10I Attenuated astrogliosis and glial scar formation.
• DEGs Differentially expressed genes between SCI+Ctrl and SCI+Gsxl that are associated with reactive astrocytes (RA) (e.g, Mmmpl3, Mmp2, Nes, Axin2, Plaur, and Ctnnbl), scar forming astrocytes (SA) (e.g., Slit2 and Sox9) and both with RA and SA (e.g., Gfap and Vim) at 14 DPI and 35 DPI.
• RA reactive astrocytes
• SA scar forming astrocytes
• SA e.g., Slit2 and Sox9
• Gfap and Vim e.g., Gfap and Vim
• FIGS. 11A-11G Improved locomotor functional recovery after SCI. Lateral hemisection SCI was performed on 8-12 weeks old mice around T9-T10 level immediately followed by the injection of lentivirus encoding Gsxl along with RFP reporter (lenti-Gsxl-RFP). Lentivirus encoding only the reporter RFP was used as a control (lenti-Ctrl-RFP). Locomotor function was assessed by BMS score at least twice weekly up to 56 DPI. (A) Representative images of hindlimb walking status at 56 DPI and (B) the BMS scores of left hindlimb (n > 6).
• FIGS. 12A-12C Gsxl treatment promotes signaling for axon growth and 5-HT neuronal activity after hemisection SCI.
• A IPA heat map of differentially expressed genes involved in CREB signaling in neurons at 3 DPI, 14 DPI, and 35 DPI between SCI+Ctrl and SCI+Gsxl; n>3.
• FIG. 13 Functional enrichment of gene ontology (GO) terms for differentially expressed genes (DEGs) at 35 DPI.
• Enrichment terms for biological process represented as a scatter plot in a two dimensional semantic space using REVIGO. Circle size indicates the loglO(p-value) of the GO terms.
• FIG. 14 Effect of GSX1 on various pathways, leading to increased locomotion in a mammal with SCI.
• nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.
• sequence listing submitted herewith, generated on July 22, 2019, 32 kb, is herein incorporated by reference in its entirety.
• SEQ ID NOS: 1 - 2 are exemplary human Gsxl coding and protein sequences, respectively.
• SEQ ID NOS: 3 - 4 are exemplary mouse Gsxl coding and protein sequences, respectively.
• SEQ ID NOS: 5 - 60 are primers used to detect expression of genes shown in Table 2 using RT-qPCR analysis.
• SEQ ID NOS: 61 - 79 are exemplary cell -penetrating peptides.
• SEQ ID NOS: 80 -81 are exemplary linker peptides.
• a Gsxl nucleic acid molecule or protein such as a Gsxl-CPP fusion protein
• routes of administration include, but are not limited to, injection (such as injection into the CNS, for example injection into the spine or brain, for example at or near the site of injury, for example rostral and/or caudal to the injury site).
• administration is an intrathecal injection (e.g ., of Gsxl or Gsxl-CPP nucleic acid molecule or protein) to treat SCI in lumbar/sacral region, a cisterna magna injection (e.g., of Gsxl or Gsxl-CPP nucleic acid molecule or protein) to treat SCI in cervical/thoracic region, or intraparenchymal or introcerebroventricular injection (e.g, of Gsxl nucleic acid molecule or protein) to treat traumatic brain injury.
• intrathecal injection e.g ., of Gsxl or Gsxl-CPP nucleic acid molecule or protein
• a cisterna magna injection e.g., of Gsxl or Gsxl-CPP nucleic acid molecule or protein
• intraparenchymal or introcerebroventricular injection e.g, of Gsxl nucleic acid molecule or protein
• Chimeric or fusion protein A protein that includes a first peptide (e.g., Gsxl) and a second peptide (e.g., a cell penetrating peptide, such as one or more of those provided in SEQ ID NOS: 61-79), where the first and second proteins are different.
• a chimeric polypeptide also encompasses polypeptides that include two or more non-conti guous portions derived from the same polypeptide.
• a chimeric protein is a Gsxl -cell penetrating peptide fusion protein (Gsxl-CPP), wherein the Gsxl portion can have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2 or 4 and wherein the cell penetrating peptide (CPP) is at the N- or C-terminus of Gsxl .
• the two or more different peptides can be joined directly or indirectly, for example using a linker (such as 1-30 amino acids).
• Complementarity The ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick base pairing or other non-traditional types.
• a percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary).
• Perfectly complementary means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.
• substantially complementary refers to a degree of complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions.
• Contacting can occur in vitro or ex vivo, for example, by adding a reagent to a sample (such as one containing neural cells), or in vivo by administering to a subject.
• Effective amount The amount of an agent (such as a Gsxl protein, Gsxl-CPP fusion protein, or nucleic acid molecule encoding such) that is sufficient to effect beneficial or desired results.
• an agent such as a Gsxl protein, Gsxl-CPP fusion protein, or nucleic acid molecule encoding such
• a therapeutically effective amount may vary depending upon one or more of: the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can be determined by one of ordinary skill in the art.
• the beneficial therapeutic effect can include amelioration of a disease, symptom, disorder, or pathological condition; reducing or preventing the onset of a disease, symptom, disorder or condition; and generally counteracting a disease, symptom, disorder or pathological condition.
• an“effective amount” is an amount sufficient to (1) decrease inflammation, for example at or near the injury site, such as decrease the number of infiltrated macrophages (such as a decrease of at least 5%, at least 10%, at least 15%, at least 20%, at least 50%, at least 75%, or at least 90% for example relative to no administration of the Gsxl protein, Gsxl-CPP fusion protein, or nucleic acid molecule encoding such), (2) increase the number of neural stem/progenitor cells (NSPCs) (e.g., as determined by measuring expression of nestin and/or Sox2), for example at or near the injury site, (for example an increase of at least 5%, at least 10%, at least 15%, at least 20%, at least 50%, at least 75%, at least 90%, or at least 100%, for example relative to no administration of the Gsxl protein, Gsxl-CPP fusion protein, or nucleic acid molecule encoding such), (3) increase differentiation of NSPCs towards a neuronal
• ASIA American Spinal Injury Association
• ASIA American Spinal Injury Association
• a decrease cell death for example at or near the injury site, such as decreasing the number of cleaved caspase3 positive cells (such as a decrease of at least 5%, at least 10%, at least 15%, at least 20%, at least 50%, at least 75%, or at least 90% for example relative to no administration of the Gsxl protein , Gsxl-CPP fusion protein, or nucleic acid molecule encoding such).
• Expression of a gene can be regulated anywhere in the pathway from DNA to RNA to protein. Regulation can include controls on transcription, translation, RNA transport and processing, degradation of intermediary molecules such as mRNA, or through activation, inactivation, compartmentalization or degradation of specific protein molecules after they are produced.
• nucleic acid molecule or protein can be altered relative to a normal (wild type) nucleic acid molecule or protein (such as in a normal non-recombinant cell).
• Alterations in gene expression include but are not limited to: (1) overexpression (e.g, upregulation); (2) underexpression (e.g., downregulation); or (3) suppression of expression. Alternations in the expression of a nucleic acid molecule can be associated with, and in fact cause, a change in expression of the corresponding protein.
• Genomic Screened Homeo Box (GSX1) ( e.g ., OMIM 616542): Also known as Gshl. This gene encodes a protein involved in pituitary development. The mouse protein is 261 amino acids, and the human protein is 264 amino acids, and the two proteins share about 96% sequence homology. The human GSX1 gene maps to chromosome l3ql2.2.
• Gsxl sequences are publically available, for example from the GenBank® sequence database (e.g., Accession Nos. NP_663632. l, NP_032204.l, XP_006068096.2, and
• NP_001178592.1 provide exemplary Gsxl protein sequences, while Accession Nos.
• NM_l45657.2, NM_008l78.2, XM_006068034.2, and NM_00l 191663.1 provide exemplary Gsxl nucleic acid sequences).
• Gsxl variants such as those having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% sequence identity to these GenBank® sequences (such as to SEQ ID NO: 1, 2, 3 or 4).
• Such Gsxl sequences can be used to generate therapeutic recombinant nucleic acid molecules and proteins, for example to treat a neurological disorder, such as a SCI, using the methods provided herein.
• a Gsxl protein is part of a fusion protein, such as a Gsxl-CPP fusion protein.
• Increase or Decrease A statistically significant positive or negative change, respectively, in quantity from a control value.
• An increase is a positive change, such as an increase at least 50%, at least 100%, at least 200%, at least 300%, at least 400% or at least 500% as compared to the control value.
• a decrease is a negative change, such as a decrease of at least 20%, at least 25%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% decrease as compared to a control value. In some examples the decrease is less than 100%, such as a decrease of no more than 90%, no more than 95% or no more than 99%.
• An“isolated” biological component such as a protein or nucleic acid, or cell
• An“isolated” biological component has been substantially separated, produced apart from, or purified away from other biological components in the cell or tissue of an organism in which the component occurs, such as other cells, chromosomal and extrachromosomal DNA and RNA, and proteins.
• Nucleic acids and proteins that have been“isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins (such as Gsxl proteins and nucleic acid molecules) prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids and proteins.
• Isolated proteins, nucleic acids, or cells in some examples are at least 50% pure, such as at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 100% pure.
• Linker A moiety or group of moieties that joins or connects two or more discrete separate peptide or proteins, such as monomer domains, for example to generate a chimeric protein. In one example a linker is a substantially linear moiety.
• Exemplary linkers that can be used to generate the chimeric proteins provided herein include but are not limited to: peptides, nucleic acid molecules, peptide nucleic acids, and optionally substituted alkylene moieties that have one or more oxygen atoms incorporated in the carbon backbone.
• a linker can be a portion of a native sequence, a variant thereof, or a synthetic sequence.
• Linkers can include naturally occurring amino acids, non-naturally occurring amino acids, or a combination of both.
• a linker is composed of at least 5, at least 10, at least 15 or at least 20 amino acids, such as 5 to 10, 5 to 20, or 5 to 50 amino acids.
• the linker is a polyalanine.
• the linker is a flexible linker, such as one that includes Gly and Ser residues ( e.g ., GSGSGS (SEQ ID NO: 80) or GGSGGGGSGG, SEQ ID NO: 81).
• Non-naturally occurring or engineered Terms used herein as interchangeably and indicate the involvement of the hand of man.
• the terms, when referring to nucleic acid molecules or polypeptides indicate that the nucleic acid molecule or the polypeptide is at least substantially free from at least one other component with which they are naturally associated in nature and as found in nature.
• the terms can indicate that the nucleic acid molecules or polypeptides is one having a sequence not found in nature.
• a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
• a promoter is operably linked to a coding sequence (such as a Gsxl coding sequence) if the promoter affects the transcription or expression of the coding sequence.
• operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.
• compositions and formulations suitable for pharmaceutical delivery of recombinant nucleic acid molecule or protein such as Gsxl or Gsxl-CPP fusion protein.
• parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
• pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
• Polypeptide, peptide and protein refer to polymers of amino acids of any length.
• the polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
• the terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation,
• amino acid includes natural and/or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and
• Promoter An array of nucleic acid control sequences which direct transcription of a nucleic acid, such as a Gsxl or Gsxl-CPP fusion protein coding sequence.
• a promoter includes necessary nucleic acid sequences near the start site of transcription.
• a promoter also optionally includes distal enhancer or repressor elements.
• A“constitutive promoter” is a promoter that is continuously active and is not subject to regulation by external signals or molecules. In contrast, the activity of an“inducible promoter” is regulated by an external signal or molecule (for example, a transcription factor).
• the promoter used is native to the nucleic acid molecule it is expressing (endogenous promoter), for example, is endogenous to Gsxl.
• the promoter used is not native to the nucleic acid molecule it is expressing (exogenous promoter).
• a “tissue-specific promoter” is a promoter that direct expression of a nucleic acid molecule in particular cells or tissues, such as the central nervous system.
• Exemplary promoters that can be used to drive expression of Gsxl include: CMV promoter, SV40 promoter, beta actin promoter, or inducible Tetracycline (Tet) inducible lentiviral system (Tet on or off system).
• Recombinant or host cell A cell that has been genetically altered, or is capable of being genetically altered by introduction of an exogenous polynucleotide, such as a recombinant plasmid or vector.
• a host cell is a cell in which a recombinant nucleic acid molecule can be propagated and/or its DNA expressed. Such cells can be a neural cell.
• the term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term“host cell” is used.
• Regulatory element Includes promoters, enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g ., transcription termination signals, such as
• Regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells (e.g ., tissue-specific regulatory sequences).
• tissue-specific regulatory sequences may direct expression primarily in a desired tissue of interest, such as neural tissues or cells.
• Regulatory elements may also direct expression in a temporal -dependent manner, such as in a cell- cycle dependent or developmental stage-dependent manner, which may or may not also be tissue or cell-type specific.
• a Gsxl or Gsxl-CPP coding sequence is operably linked to a promoter, such as a constitutive promoter, such as a pol III promoter, pol II promoter, or pol I promoter.
• a promoter such as a constitutive promoter, such as a pol III promoter, pol II promoter, or pol I promoter.
• pol III promoters include, but are not limited to, U6 and Hl promoters.
• pol II promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer), the SV40 promoter, the dihydrofolate reductase promoter, the b-actin promoter, the phosphoglycerol kinase (PGK) promoter, CAG promoter, EIBC promoter, ROSA promoter, and the EFla promoter.
• a Gsxl coding sequence is operably linked to a tissue-specific promoter, such as a CNS-specific promoter.
• enhancer elements such as WPRE; CMV enhancers; the R-U5' segment in LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(l):466-472,
• a Gsxl coding sequence is operably linked to an enhancer, such as a neural-specific enhancer (e.g., NotchlCR2 or Olig2CR5).
• a neural-specific enhancer e.g., NotchlCR2 or Olig2CR5
• a Gsxl or Gsxl-CPP coding sequence is operably linked to both a promoter and an enhancer, such as a constitutive promoter (e.g, CMV) and a neural -specific enhancer (e.g., Notch 1CR2 or Olig2CR5).
• a constitutive promoter e.g, CMV
• a neural -specific enhancer e.g., Notch 1CR2 or Olig2CR5
• Sequence identity/similarity The similarity between amino acid (or nucleotide) sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are.
• BLAST Basic Local Alignment Search Tool
• NCBI National Center for Biotechnology Information
• blastp blastn
• blastx blastx
• tblastn tblastx
• Variants of protein and nucleic acid sequences are typically characterized by possession of at least about 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity counted over the full length alignment with the amino acid sequence using the NCBI Blast 2.0, gapped blastp set to default parameters.
• the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters,
• Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 95%, at least 98%, or at least 99% sequence identity.
• homologs and variants When less than the entire sequence is being compared for sequence identity, homologs and variants will typically possess at least 80% sequence identity over short windows of 10-20 amino acids, and may possess sequence identities of at least 85% or at least 90% or at least 95% depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are available at the NCBI website on the internet.
• sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologs could be obtained that fall outside of the ranges provided.
• Subject A mammal, such as a human or veterinary subject. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets.
• the subject is a non-human mammalian subject, such as a monkey or other non-human primate, mouse, rat, rabbit, pig, goat, sheep, dog, cat, boar, bull, horse, or cow.
• the subject is a laboratory animal/organism, such as a mouse, rabbit, or rat.
• the subject has a neurological disorder, such as a neurodegenerative disease or has suffered a traumatic brain injury or traumatic SCI that can be treated using the methods provided herein.
• Therapeutic agent refers to one or more molecules or compounds that confer some beneficial effect upon administration to a subject.
• the beneficial therapeutic effect can include enablement of diagnostic determinations; amelioration of a disease, symptom, disorder, or pathological condition; reducing the onset of a disease, symptom, disorder or condition; and generally counteracting a disease, symptom, disorder or pathological condition, such as a neurological disorder.
• Transduced, Transformed, Transfected A virus or vector“transduces” a cell when it transfers nucleic acid molecules into a cell.
• a cell is“transformed” or“transfected” by a nucleic acid transduced into the cell when the nucleic acid becomes stably replicated by the cell, either by incorporation of the nucleic acid into the cellular genome, or by episomal replication.
• nucleic acid molecule can be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, particle gun acceleration and other methods in the art.
• the method is a chemical method (e.g ., calcium-phosphate transfection or polyethyleneimine (PEI) transfection), physical method (e.g., electroporation, microinjection, particle bombardment), fusion (e.g., liposomes), receptor-mediated endocytosis (e.g., DNA-protein complexes, viral envelope/capsid-DNA complexes) and biological infection by viruses such as recombinant viruses (Wolff, J. A., ed, Gene Therapeutics, Birkhauser, Boston,
• Transgene An exogenous gene, for example supplied by a vector (such as a viral vector).
• a transgene includes a Gsxl or Gsxl-CPP coding sequence, for example operably linked to a promoter sequence.
• Transgenic A cell or animal (e.g, human or mouse) carrying a transgene.
• Treating, Treatment, and Therapy Any success or indicia of success in the attenuation or amelioration of a pathology or condition, including any objective or subjective parameter such as abatement or diminishing of symptoms.
• the treatment may be assessed by objective or subjective parameters; including the results of a physical examination, and other clinical tests, and the like.
• treatment using the disclosed methods (1) decreases inflammation, for example at or near the injury site, such as decrease the number of infiltrated macrophages (such as a decrease of at least 5%, at least 10%, at least 15%, at least 20%, at least 50%, at least 75%, or at least 90% for example relative to no administration of the Gsxl protein, Gsxl-CPP fusion protein, or nucleic acid molecule encoding such), (2) increases the number of neural stem/progenitor cells (NSPCs) (e.g., as determined by measuring expression of nestin and/or Sox2), for example at or near the injury site, (for example an increase of at least 5%, at least 10%, at least 15%, at least 20%, at least 50%, at least 75%, at least 90%, or at least 100%, for example relative to no administration of the Gsxl protein, Gsxl-CPP fusion protein, or nucleic acid molecule encoding such), (3) increases differentiation of NSPCs towards a neuronal linage,
• Gsxl protein administration of the Gsxl protein, Gsxl-CPP fusion protein, or nucleic acid molecule encoding such).
• Upregulated When used in reference to the expression of a molecule, such as a gene or a protein (e.g., Gsxl), refers to any process which results in an increase in production of a gene product.
• a gene product can be RNA (such as mRNA, rRNA, tRNA, and structural RNA) or protein. Therefore, upregulation includes processes that increase transcription of a gene or translation of mRNA and thus increase the presence of proteins or nucleic acids. The disclosed methods, can be used to upregulate Gsxl.
• Examples of processes that increase transcription include those that increase transcription initiation rate, those that increase transcription elongation rate, those that increase processivity of transcription and those that decrease transcriptional repression.
• Gene upregulation can include increasing expression above an existing level.
• Examples of processes that increase translation include those that increase translational initiation, those that increase translational elongation and those that increase mRNA stability. Upregulation includes any detectable increase in the production of a gene product.
• detectable Gsxl protein or nucleic acid expression in a cell increases by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 100%, at least 200%, at least 400%, or at least 500% as compared to a control (such an amount of protein or nucleic acid expression detected in a corresponding normal or non-recombinant cell).
• a control is a relative amount of expression in a normal cell (e.g ., a non-recombinant CNS cell, such as a neural cell).
• the desired activity is expression of a Gsxl nucleic acid molecule to treat a neurological disorder.
• Vector A nucleic acid molecule into which a foreign nucleic acid molecule can be introduced without disrupting the ability of the vector to replicate and/or integrate in a host cell.
• Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double- stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g., circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art.
• a vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication.
• a vector can also include one or more selectable marker genes (such as antibiotic resistance or a fluorescent protein), and other genetic elements.
• An integrating vector is capable of integrating itself into a host nucleic acid.
• An expression vector is a vector that contains the regulatory sequences to allow transcription and translation of inserted gene or genes.
• vector refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques.
• viral vector refers to a viral vector, wherein virally-derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g., retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses).
• Viral vectors also include polynucleotides carried by a virus for transfection into a host cell.
• the vector is a lentivirus (such as 3rd generation integration-deficient lentiviral vectors) or adeno- associated viral (AAV) vector.
• lentivirus such as 3rd generation integration-deficient lentiviral vectors
• AAV adeno- associated viral
• vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
• Other vectors e.g, non-episomal mammalian vectors
• vectors are capable of directing the expression of genes to which they are operatively-linked, such as a Gsxl or Gsxl-CPP coding sequence. Such vectors are referred to herein as "expression vectors.” Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
• Recombinant expression vectors can include a nucleic acid provided herein (such as a Gsxl or Gsxl-CPP coding sequence) in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors can include one or more regulatory elements, which may be selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed.
• a nucleic acid provided herein such as a Gsxl or Gsxl-CPP coding sequence
• operably linked is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows for expression of the nucleotide sequence (e.g ., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
• the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression desired, the size of the transgenic cargo, etc.
• a vector can be introduced into host cells to thereby produce transcripts, proteins, or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., Gsxl or Gsxl-CPP).
• Gsxl treatment at or near the injury site promotes the activation of NSPCs and the generation of specific subtypes of interneurons (e.g, glutamatergic and cholinergic neurons). Gsxl expression also inhibits reactive astrogliosis and glial scar formation, and leads to a dramatic locomotor functional recovery in mice with lateral hemisection SCI.
• RNA-Seq and RT-qPCR analysis demonstrates that Gsxl treatment alters the expression of genes associated with cell proliferation, NSPC activation, neurogenesis, astrogliosis, and scar formation, which correlates with functional recovery after SCI.
• Sox2 and NeuroDl are general neurogenic transcription factors, but not specific transcription factors for spinal neuronal genesis; 2) Sox2-induced neurons resemble GABAergic interneurons. The additional inhibitory intemeurons might have caused a further imbalance of the excitation/inhibition ratio; and 3) functional recovery may require the generation of various specific cell types, e.g ., glutamatergic and cholinergic intemeurons.
• Gsxl treatment inhibits the generation of GABAergic intemeurons.
• Gsxl treatment-induced reduction of GABAergic intemeurons may contribute to the restoration of the excitation/inhibition ratio.
• Gsxl regulates Notch signaling via its interaction with a Notch 1 enhancer.
• an increase in Gsxl and Notch 1 leads to a higher level of glutamate
• Notch signaling is a canonical pathway required for NSPC proliferation and self-renewal, as well as for prevention of untimely neuronal differentiation of NSPCs.
• the RNA- Seq and RT-qPCR data herein show that Gsxl transiently upregulates Notch and Nanog signaling pathways during an acute stage of SCI (FIGS. 5E-5G).
• Gsxl treatment increased the number of glutamatergic and cholinergic neurons and decreased the GABAergic intemeurons at the injury site (FIGS. 9A-9F). These upregulated signaling pathways (FIGS. 5A-5I) support the activation and expansion of endogenous NSPCs.
• Gsxl treatment significantly decreases in the expression of genes associated with reactive astrocytes (RA) and scar forming astrocytes (SA). Gsxl-induced NSPC differentiation into neuronal lineage may be at the expense of the astrocyte lineage. Reduction in astrogliosis leads to attenuation of scar formation (FIGS. 10A-10I).
• Gsxl-induced neurons For Gsxl-induced neurons to be functional, they need to establish proper connections.
• the methods provided herein upregulate axon guidance signaling, Netrin signaling, CREB signaling pathway, and synaptogenesis (FIGS. 6A-6G, 12A, 12B).
• Gsxl in the injured spinal cord to (i) reduce glial scar (such as a reduction of at least 20%, at least 25%, or at least 30%, for example relative to no administration of a Gsxl protein, Gsxl-CPP fusion protein, or nucleic acid molecule encoding such), (ii) induce neurogenesis (such as an increase in neurogenesis of at least 20%, at least 40%, or at least 50%, for example relative to no administration of a Gsxl protein, Gsxl-CPP fusion protein, or nucleic acid molecule encoding such), and/or (iii) improve locomotion after SCI (such as an increase in locomotion of at least 50%, at least 100%, at least 200%, or at least 300%, for example relative to no administration of a Gsxl protein, Gsxl-CPP fusion protein, or nucleic acid molecule encoding such).
• reduce glial scar such as a reduction of at least 20%, at least 25%, or at least 30%, for example relative
• Gsxl is a therapeutic agent for the treatment of SCI and other central nervous related injuries (FIG. 14).
• the methods can include administering to the subject a therapeutically effective amount of Gsxl protein, Gsxl-CPP fusion protein, or a nucleic acid molecule encoding Gsxl or Gsxl-CPP, thereby treating the neurological disorder.
• the administration is via injection, such as injection into the CNS ( e.g ., spinal cord or brain).
• the Gsxl protein, Gsxl-CPP fusion protein, or a nucleic acid molecule encoding Gsxl or Gsxl-CPP can be administered near or at the site of the brain or spinal cord injury, such as rostral and/or caudal to the injury site.
• a Gsxl protein is administered that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2 or 4.
• a Gsxl-CPP fusion protein comprising a Gsxl protein and a cell penetrating peptide is administered, wherein the Gsxl portion of the fusion protein has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2 or 4.
• the CPP domain of the Gsxl-CPP fusion protein has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOS: 61-79.
• a Gsxl portion of a Gsxl-CPP fusion protein has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2 or 4, and the CPP domain of the Gsxl-CPP fusion protein has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOS: 61-79.
• the CPP domain of the Gsxl-CPP fusion protein is C-terminal to the Gsxl domain.
• the CPP domain of the Gsxl-CPP fusion protein is N-terminal to the Gsxl domain. In some examples, the CPP domain of the Gsxl-CPP fusion protein is directly linked to the Gsxl domain. In some examples, the CPP domain of the Gsxl-CPP fusion protein is indirectly linked to the Gsxl domain, for example via a peptide linker, such as a linker of 1 to 30 amino acids, such as a linker having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 80 or 81.
• a peptide linker such as a linker of 1 to 30 amino acids, such as a linker having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 80 or 81.
• a Gsxl encoding nucleic acid molecule is administered, wherein the nucleic acid molecule encoding Gsxl encodes a protein comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2 or 4.
• the nucleic acid molecule encoding Gsxl includes at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1 or 3.
• a Gsxl-CPP encoding nucleic acid molecule is administered, wherein the nucleic acid molecule encoding Gsxl-CPP includes a portion that encodes a Gsxl domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2 or 4.
• the nucleic acid molecule encoding Gsxl-CPP includes a portion that encodes a CPP domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOS: 61 to 79.
• the nucleic acid molecule encoding the Gsxl domain of Gsxl-CP includes at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1 or 3.
• Gsxl and Gsxl-CPP coding sequences can include other elements.
• the nucleic acid molecule encoding Gsxl or Gsxl-CPP is part of a plasmid or viral vector, such as a lentiviral vector or adeno-associated viral vector.
• the nucleic acid molecule encoding Gsxl or Gsxl-CPP is operably linked to a promoter, such as a constitutive promoter (e.g ., CMV, beta actin, or a native Gsxl promoter), or a tissue-specific promoter, such as a central nervous system (CNS)-specific promoter (e.g., a synapasin 1 (Synl) promoter, glial fibrillary acidic protein (GFAP) promoter, nestin (NES) promoter, myelin-associated oligodendrocyte basic protein (MOBP) promoter, myelin basic protein (MBP) promoter, tyrosine hydroxylase (TH) promoter, or a forkhead box A2 (FOXA2) promoter).
• a promoter such as a constitutive promoter (e.g ., CMV, beta actin, or a native Gsxl promoter), or a tissue-specific promoter, such as a central
• the nucleic acid molecule encoding a Gsxl-CPP fusion protein is operably linked to a promoter, such as a constitutive promoter (e.g., CMV, beta actin, or a native Gsxl promoter), wherein the Gsxl portion of the Gsxl-CPP fusion protein has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2 or 4.
• a constitutive promoter e.g., CMV, beta actin, or a native Gsxl promoter
• Exemplary neurological disorders that can be treated with the disclosed methods include spinal cord injuries, brain injuries, or both.
• the spinal cord injury, brain injury, or both is caused by trauma from an external force, such as a blow or jolt to the head or a penetrating head injury, such as a vehicle crash (e.g, car, motorcycle, ATV, or bike), fall, act of violence (e.g., gun-shot wound or stab wound), or sports (e.g, a collision or fall resulting during football, soccer, baseball, hockey, diving, skiing, rugby, lacrosse, horseback riding, or basketball).
• a spinal cord injury usually begins with a sudden, traumatic blow to the spine that fractures or dislocates vertebrae.
• the spinal cord injury can be at the cervical, thoracic, lumbar, sacral, or coccyx region of the spine, such as a C4, C6, T6, T9, T10, or Ll injury.
• the subject treated with the disclosed methods has quadriplegia or paraplegia.
• the neurological disorder is a traumatic brain injury (TBI), which occurs due to a sudden acceleration or deceleration with the cranium or a combination of movement and sudden impact. Damage occurs both at the time of injury, as well as minutes to days later, for example, due to changes in blood flow and pressure within the cranium. TBI is classified from mild (including concussion) to severe.
• the neurological disorder that can be treated with the disclosed methods is a neurodegenerative disorder, such as Parkinson’s disease, Alzheimer’s disease, stroke, ischemia, epilepsy, Huntington's disease, multiple sclerosis, or amyotrophic lateral sclerosis.
• Such neurodegenerative disorders are an abnormality in the nervous system of a mammalian subject, in which neuronal integrity is threatened, for example when neuronal cells display decreased survival or when the neurons can no longer propagate a signal.
• the therapeutically effective amount of Gsxl protein, Gsxl-CPP fusion protein, or nucleic acid molecule encoding such is present in a pharmaceutical composition, such as one that includes a pharmaceutically acceptable carrier, such as saline or water.
• the method includes least two separate administrations of the therapeutically effective amount of Gsxl protein, Gsxl-CPP fusion protein, or nucleic acid molecule encoding such, such as at least 3, at least 4, at least 5, at least 10, or at least 20 separate administrations.
• the at least two separate administrations are separated by at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 9 months, or at least one year.
• administering occurs within 1 hour, within 2 hours, within 3 hours, within 4 hours, within 5 hours, within 6 hours, within 12 hours, within 24 hours, within 48 hours, within 72 hours, within 96 hours, within 1 week, within 2 weeks, within 3 weeks, within 4 weeks, within 1 month, within 2 months, or within 3 months of the onset of the neurological disorder.
• the disclosed methods can further include administering to the subject a therapeutically effective amount of another neurological disorder therapeutic agent.
• the method includes selecting a subject with a neurological disorder, such as a traumatic spinal cord or brain injury, or a neurodegenerative disease. These subjects can be selected for treatment with a Gsxl protein, Gsxl-CPP fusion protein, or nucleic acid molecule encoding such.
• treating a neurological disorder using the disclosed methods includes one or more of (1) decreasing inflammation, for example at or near the injury site, such as decreasing the number of infiltrated macrophages (such as a decrease of at least 5%, at least 10%, at least 15%, at least 20%, at least 50%, at least 75%, or at least 90% for example relative to no administration of the Gsxl protein, Gsxl-CPP protein, or nucleic acid molecule encoding such), (2) increasing the number of neural stem/progenitor cells (NSPCs) (e.g., as determined by measuring expression of nestin, Ki67, and/or Sox2), for example at or near the injury site, (for example an increase of at least 5%, at least 10%, at least 15%, at least 20%, at least 50%, at least 75%, at least 90%, at least 100%, at least 200%, at least 300%, or at least 500%, for example relative to no administration of the Gsxl protein, Gsxl-CPP protein, or nucleic acid molecule
• such responses are achieved within about 3 days, within about 1 week, within about 2 weeks, within about 4 weeks, within about 8 weeks, within about 12 weeks, with in about 4 months, within about 6 months, or within about 52 weeks following treatment.
• the disclosed methods include measuring inflammation, cell proliferation, astrogliosis, glial scaring, neurogenesis, NSPC activation, and/or cell death, for example at or near an injury site, before and/or after treating a subject.
• the disclosed methods include measuring locomotion of the subject before and after treating a subject.
• the disclosed methods include measuring locomotion before and/or after treating a subject.
• functional outcome after spinal cord injury in humans can be determined or measured using the Modified Barthel Index (MB I), Functional Independence Measure (FIM), Quadriplegia Index of Function (QIF), and/or the Spinal Cord Independence Measure (SCIM). Examples of such methods are described in Furlan et al, Journal of
• the control is a value obtained prior to treatment.
• the control is a historical control or standard reference value or range of values (such as a previously tested control sample, such as a group of subjects with or without a neurological disorder).
• control is a reference value, such as a standard value obtained from a population of normal individuals, or individual known to have a neurological disorder (such as a SCI or TBI). Similar to a control population, the value the value obtained from the treated subject can be compared to the mean reference value or to a range of reference values (such as the high and low values in the reference group or the 95% confidence interval).
• the control is the subject (or group of subjects) treated with placebo compared to the same subject (or group of subjects) treated with the Gsxl protein, Gsxl-CPP protein, or nucleic acid molecule encoding such in a cross-over study.
• the control is the subject (or group of subjects) prior to treatment with the Gsxl protein, Gsxl-CPP protein, or nucleic acid molecule encoding such.
• Exemplary full-length Gsxl proteins are shown in SEQ ID NOS: 2 (human) and 4 (mouse).
• a Gsxl protein includes or consists of the protein sequence of SEQ ID NO: 2 or 4.
• a Gsxl protein includes or consists of the protein sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2 or 4.
• Exemplary Gsxl coding sequences are shown in SEQ ID NOS: 1 (human) and 3 (mouse).
• a Gsxl nucleic acid sequence includes or consists of the sequence of SEQ ID NO: 1 or 3, which in some examples is part of a plasmid or vector, and in some examples operably linked to a promoter (such as a constitutive or CNS-specific promoter).
• the disclosed methods utilize a Gsxl protein (such as a mammalian Gsxl protein), that is, a Gsxl protein is administered to the subject.
• a Gsxl-CPP fusion protein such as a fusion protein composed of a mammalian Gsxl protein and a cell penetrating peptide
• Gsxl proteins which can be used to generate the Gsxl domain of a Gsxl-CPP protein are shown in SEQ ID NOS: 2 (human) and 4 (mouse). Native or variant Gsxl proteins can be used.
• variant Gsxl peptides are produced by manipulating a Gsxl nucleotide sequence.
• a variant Gsxl sequence is used, such as one including amino acid substitutions, additions, deletions, or combinations thereof, as long as the protein retains the ability to increase neurogenesis, reduce astrogliosis and glial scar formation, and increase locomotion following spinal cord injury. Methods of measuring neurogenesis, astrogliosis and glial scar formation, and locomotion are described herein.
• Regions of Gsxl that are more likely to tolerate substitution can be determined by aligning sequences (e.g ., SEQ ID NOS: 2 and 4), wherein amino acids conserved between species are less likely to tolerate substitutions, while amino acids that vary at a particular positon are more likely to tolerate substitutions.
• Variant Gsxl proteins such as variants of SEQ ID NOS: 2 and 4, can contain one or more mutations, such as a single insertion, a single deletion, a single substitution.
• a variant Gsxl protein includes 1-20 insertions, 1-20 deletions, 1-20 substitutions, or any combination thereof.
• the variant Gsxl protein e.g., SEQ ID NO: 2 or 4
• SEQ ID NO: 2 or 4 has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid changes.
• SEQ ID NO: 2 or 4 has 1, 2, 3, 4, 5, 6, 7,
• amino acid changes such as 1-8 insertions, 1-15 deletions, 1-10 substitutions, or any combination thereof (e.g., 1-15, 1-4, or 1-5 amino acid deletions together with 1-10, 1-5 or 1-7 amino acid substitutions).
• One type of modification includes the substitution of amino acids for amino acid residues having a similar biochemical property, that is, a conservative substitution (such as 1-4, 1-8, 1-10, or 1-20 conservative substitutions). Typically, conservative substitutions have little to no impact on the activity of a resulting peptide.
• a conservative substitution is an amino acid substitution in SEQ ID NO: 2 or 4 that does not substantially affect the ability of the Gsxl peptide to increase neurogenesis, reduce astrogliosis and glial scar formation, and increase locomotion following spinal cord injury, in a mammal.
• An alanine scan can be used to identify which amino acid residues in a Gsxl protein (or Gsxl-CPP protein), such as SEQ ID NO: 2 or 4, can tolerate an amino acid substitution.
• these activities of Gsxl are not altered by more than 25%, for example not more than 20%, for example not more than 10%, when an alanine, or other conservative amino acid, is substituted for 1-4, 1-8, 1-10, or 1-20 native amino acids.
• amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative substitutions include: Ser for Ala; Lys for Arg; Gln or His for Asn; Glu for Asp; Ser for Cys; Asn for Gln; Asp for Glu; Pro for Gly; Asn or Gln for His; Leu or Val for He; Ile or Val for Leu; Arg or Gln for Lys; Leu or He for Met; Met, Leu or Tyr for Phe; Thr for Ser; Ser for Thr; Tyr for Trp; Trp or Phe for Tyr; and Ile or Leu for Val.
• substitutions that are less conservative, e.g., selecting residues that differ more significantly in their effect on maintaining: (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation; (b) the charge or hydrophobicity of the polypeptide at the target site; or (c) the bulk of the side chain.
• substitutions that in general are expected to produce the greatest changes in polypeptide function are those in which: (a) a hydrophilic residue, e.g., serine or threonine, is substituted for (or by) a hydrophobic residue, e.g., leucine, isoleucine, phenylalanine, valine or alanine; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysine, arginine, or histidine, is substituted for (or by) an electropositive side chain, e.g., lysine, arginine, or histidine, is substituted for (or by) an electropositive side chain, e.g., lysine, arginine, or histidine, is substituted for (or by) an electropositive side chain, e.g., lysine, arginine, or histidine
• electronegative residue e.g., glutamic acid or aspartic acid
• a residue having a bulky side chain e.g., phenylalanine
• one not having a side chain e.g., glycine.
• the effects of these amino acid substitutions can be assessed by analyzing the function of the Gsxl protein, such as SEQ ID NO: 2 or 4, or a or Gsxl-CPP protein, by analyzing the ability of the variant Gsxl protein to increase neurogenesis, reduce astrogliosis and glial scar formation, and increase locomotion following spinal cord injury, in a mammal.
• a Gsxl protein (or Gsxl-CPP protein) used in the disclosed methods is PEGylated at one or more positions.
• a Gsxl protein (or Gsxl-CPP protein) used in the disclosed methods includes an immunoglobin FC domain.
• the conserved FC fragment of an antibody can be incorporated either n-terminal or c-terminal of the Gsxl protein, and can enhance stability of the protein and therefore serum half-life.
• the FC domain can also be used as a means to purify the proteins on protein A or Protein G sepharose beads.
• Gsxl proteins can be tagged with cell penetrating peptide (CPP) to promote cellular uptake.
• CPP cell penetrating peptide
• the Gsxl protein used is a fusion or chimeric protein, that includes (1) a Gsxl protein, and (2) a cell penetrating peptide (referred to herein as a Gsxl-CPP fusion protein).
• the cell penetrating peptide domain can be at the N- or C-terminus of the Gsxl protein domain.
• Cell penetrating peptides are usually short peptides (40 amino acids or less) that are highly cationic and usually rich in arginine and lysine that can facilitate cellular intake/uptake of proteins.
• Exemplary cell penetrating peptides that can be used include hydrophilic peptides (e.g, TAT
• Gsxl proteins and Gsxl-CPP proteins
• Isolation and purification of recombinantly expressed Gsxl proteins (and Gsxl-CPP proteins) can be carried out by conventional means, such as preparative chromatography and immunological separations. Once expressed, Gsxl proteins (and Gsxl-CPP proteins) can be purified according to standard procedures, including ammonium sulfate precipitation, affinity columns, column chromatography, and the like (see, generally, R. Scopes, Protein Purification , Springer-Verlag, N.Y., 1982). Substantially pure compositions of at least about 90 to 95% homogeneity are disclosed herein, and 98 to 99% or more homogeneity can be used for
• Gsxl proteins(and Gsxl-CPP proteins) can also be constructed in whole or in part using standard peptide synthesis.
• Gsxl proteins (and Gsxl-CPP proteins) are synthesized by condensation of the amino and carboxyl termini of shorter fragments. Peptide bonds can be formed by activation of a carboxyl terminal end (such as by the use of the coupling reagent N, N'-dicylohexylcarbodimide).
• Gsxl coding sequences are shown in SEQ ID NOS: 1 (human) and 3 (mouse).
• a Gsxl encoding nucleic acid molecule includes or consists of the sequence of SEQ ID NO: 1 or 3.
• a Gsxl nucleic acid molecule encodes the protein of SEQ ID NO: 2 or 4, or a variant thereof (such as those described above).
• a Gsxl encoding nucleic acid sequence includes or consists of the sequence of SEQ ID NO: 1 or 3, which in some examples is part of a plasmid or vector, and in some examples operably linked to a promoter (such as a constitutive or CNS-specific promoter).
• the nucleic acid molecule encodes a Gsxl-CPP fusion protein, and can include a portion that encodes a Gsxl portion (e.g ., encodes the protein of SEQ ID NO: 2 or 4, or includes or consists of the sequence of SEQ ID NO: 1 or 3), and includes a portion that encodes a CPP (e.g., encodes any one of SEQ ID NOS: 61-79).
• a Gsxl portion e.g ., encodes the protein of SEQ ID NO: 2 or 4, or includes or consists of the sequence of SEQ ID NO: 1 or 3
• CPP e.g., encodes any one of SEQ ID NOS: 61-79.
• a Gsxl-CPP fusion protein is encoded by two or more separate nucleic acid molecules, such as separate vectors, such as a first nucleic acid molecule that encodes a Gsxl domain (e.g., encodes the protein of SEQ ID NO: 2 or 4, or includes or consists of the sequence of SEQ ID NO: 1 or 3), and a second nucleic acid molecule that encodes a CPP domain (e.g ., encodes any one of SEQ ID NOS: 61-79).
• a first nucleic acid molecule that encodes a Gsxl domain e.g., encodes the protein of SEQ ID NO: 2 or 4, or includes or consists of the sequence of SEQ ID NO: 1 or 3
• a second nucleic acid molecule that encodes a CPP domain e.g ., encodes any one of SEQ ID NOS: 61-79.
• the disclosed methods utilize a Gsxl (or Gsxl-CPP) nucleic acid sequence (such as a cDNA, genomic, or RNA sequences), that is, a Gsxl (or Gsxl-CPP) nucleic acid molecule is administered to the subject, and the Gsxl (or Gsxl-CPP) protein encoded expressed in the cell where the nucleic acid molecule is introduced.
• the Gsxl (or Gsxl-CPP) nucleic acid molecule can encode a native or variant Gsxl protein as described above.
• nucleic acid sequences coding for any Gsxl protein e.g., SEQ ID NO: 2 or 4
• Gsxl-CPP can be generated.
• such a sequence is optimized for expression in a host cell, such as a host cell used to express the Gsxl (or Gsxl-CPP) protein.
• Such nucleic acids can be used directly (e.g., administered to a subject), or used to produce a Gsxl (or Gsxl-CPP) protein which is administered to a subject.
• the nucleic acid molecule encoding a Gsxl protein comprises or consists of the sequence of SEQ ID NO: 1 or 3.
• cells, plasmids and viral vectors including such nucleic acids which can also include a promoter operably linked to the Gsxl (or Gsxl-CPP) coding sequence.
• a nucleic acid sequence the encodes for a Gsxl protein has at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1 or 3.
• the Gsxl portion of a Gsxl-CPP fusion protein is encoded by a nucleic acid the having at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1 or 3.
• sequences can readily be produced, using the amino acid sequences provided herein and that are publicly available, and the genetic code.
• one of skill can readily construct a variety of clones containing functionally equivalent nucleic acids, such as nucleic acids which differ in sequence but which encode the same Gsxl protein sequence.
• Nucleic acid molecules include DNA, cDNA, mRNA, and RNA sequences which encode a Gsxl protein. Silent mutations in the coding sequence result from the degeneracy (i.e.,
• leucine can be encoded by CTT, CTC, CTA, CTG, TTA, or TTG; serine can be encoded by TCT, TCC, TCA, TCG, AGT, or AGC; asparagine can be encoded by AAT or AAC; aspartic acid can be encoded by GAT or GAC; cysteine can be encoded by TGT or TGC; alanine can be encoded by GCT, GCC, GCA, or GCG; glutamine can be encoded by CAA or CAG; tyrosine can be encoded by TAT or TAC; and isoleucine can be encoded by ATT, ATC, or ATA. Tables showing the standard genetic code can be found in various sources (see, for example, Stryer, 1988, Biochemistry, 3 rd Edition, W.H. 5 Freeman and Co., NY).
• Codon preferences and codon usage tables for a particular species can be used to engineer isolated nucleic acid molecules encoding a Gsxl protein (such as one encoding a protein having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 2 or 4) that take advantage of the codon usage preferences of that particular species.
• a Gsxl protein such as one encoding a protein having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 2 or 4
• the Gsxl proteins used in the disclosed methods can be designed to have codons that are preferentially used by a particular organism of interest (such as a human or mouse).
• a nucleic acid encoding a Gsxl protein (such as a nucleic acid molecule having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1 or 3, or encoding a protein having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 2 or 4), or a Gsxl-CPP fusion protein, can be cloned or amplified by in vitro methods, such as the polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), the self-sustained sequence replication system (3 SR) and the Ob replicase amplification system (QB).
• PCR polymerase chain reaction
• LCR ligase chain reaction
• TAS transcription-based amplification system
• SR self-sustained sequence replication system
• nucleic acids encoding a Gsxl protein can be prepared by cloning techniques (such as those found in Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring, Harbor, N.Y., 1989, and Ausubel et al., (1987) in "Current Protocols in Molecular Biology,” John Wiley and Sons, New York, N.Y.).
• Nucleic acid sequences encoding a Gsxl protein (such as a nucleic acid molecule having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1 or 3, or encoding a protein having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 2 or 4), or a Gsxl-CPP fusion protein, can be prepared by any suitable method including, for example, cloning of appropriate sequences or by direct chemical synthesis by methods such as the phosphotriester method of Narang et al., Meth. Enzymol. 68:90-99, 1979; the phosphodiester method of Brown et al., Meth. Enzymol. 68: 109-151, 1979; the diethylphosphoramidite method of Beaucage et al.,
• a Gsxl protein (such as on having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 2 or 4) is prepared by inserting a cDNA which encodes a Gsxl protein (such as a nucleic acid molecule having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1 or 3) into a vector. The insertion can be made so that the Gsxl protein is read in frame so that the Gsxl protein is produced. Similar methods can be used to encode a Gsxl-CPP fusion protein.
• the Gsxl nucleic acid coding sequence (such as a nucleic acid molecule having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1 or 3, or encoding a protein having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 2 or 4) can be inserted into an expression vector including, but not limited to a plasmid, virus or other vehicle that can be manipulated to allow insertion or incorporation of sequences and can be expressed in either prokaryotes or eukaryotes.
• Hosts can include microbial, yeast, insect, plant and mammalian organisms.
• the vector can encode a selectable marker, such as a thymidine kinase gene, antibiotic resistance gene, or fluorescent protein. Similar methods can be used for a nucleic acid encoding a Gsxl-CPP fusion protein.
• Nucleic acid sequences encoding a Gsxl protein (such as a nucleic acid molecule having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1 or 3, or encoding a protein having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 2 or 4) can be operatively linked to expression control sequences.
• An expression control sequence operatively linked to a Gsxl protein coding sequence is ligated such that expression of the Gsxl coding sequence is achieved under conditions compatible with the expression control sequences.
• Exemplary expression control sequences include, but are not limited to promoters, enhancers, transcription terminators, a start codon (z.e., ATG) in front of a Gsxl protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons. Similar methods can be used for a nucleic acid encoding a Gsxl-CPP fusion protein. In one embodiment, vectors are used for expression in yeast such as S. cerevisiae, P.
• promoters for use in yeast expression systems include the constitutive promoters, plasma membrane H + -ATPase ( PMA1 ), glyceraldehyde-3- phosphate dehydrogenase ( GPD ), phosphoglycerate kinase- 1 ( PGK1 ), alcohol dehydrogenase- 1 ( ADH1 ), and pleiotropic drug-resistant pump (PDR5).
• inducible promoters are of use, such as GAL1-10 (induced by galactose), PH05 (induced by low extracellular inorganic phosphate), and tandem heat shock HSE elements (induced by temperature elevation to 37°C).
• Promoters that direct variable expression in response to a titratable inducer include the methionine- responsive MET3 and MET25 promoters and copper-dependent CUP1 promoters. Any of these promoters may be cloned into multicopy (2m) or single copy ⁇ CENT) plasmids to give an additional level of control in expression level.
• the plasmids can include nutritional markers (such as URA3 , ADE3 , HIS P and others) for selection in yeast and antibiotic resistance ⁇ AMP) for propagation in bacteria. Plasmids for expression on K. lactis are known, such as pKLACl.
• plasmids after amplification in bacteria, can be introduced into the corresponding yeast auxotrophs by methods similar to bacterial transformation.
• the nucleic acid molecules encoding a Gsxl protein (such as one having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1 or 3, or encoding a protein having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 2 or 4), or a Gsxl-CPP encoding nucleic acid molecule, can also be designed to express in insect cells.
• a Gsxl protein (such as one having at least 90%, at least 95%, t least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 2 or 4), or a Gsxl-CPP, can be expressed in a yeast strain.
• a yeast strain For example, seven pleiotropic drug-resistant transporters, YOR1, SNQ2, PDR5, YCF1, PDR10, PDR11, and PDR15, together with their activating transcription factors, PDR1 and PDR3, have been simultaneously deleted in yeast host cells, rendering the resultant strain sensitive to drugs.
• Yeast strains with altered lipid composition of the plasma membrane such as the erg6 mutant defective in ergosterol biosynthesis, can also be utilized.
• Proteins that are highly sensitive to proteolysis can be expressed in a yeast cell lacking the master vacuolar endopeptidase Pep4, which controls the activation of other vacuolar hydrolases.
• Heterologous expression in strains carrying temperature-sensitive (ts) alleles of genes can be employed if the corresponding null mutant is inviable.
• Viral vectors can also be prepared that encode a Gsxl protein (such as those that include a nucleic acid molecule having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1 or 3, or a nucleic acid molecule that encodes a protein having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 2 or 4), or encode a Gsxl-CPP.
• a Gsxl protein such as those that include a nucleic acid molecule having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1 or 3
• a nucleic acid molecule that encodes a protein having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%
• Exemplary viral vectors include polyoma, SV40, adenovirus, vaccinia virus, adeno-associated virus (AAV), herpes viruses including HSV and EBV, Sindbis viruses, alphaviruses and retroviruses of avian, murine, and human origin.
• Baculovirus Autographa californica multinuclear polyhedrosis virus;
• AcMNPV vectors can also be used.
• suitable vectors include retrovirus vectors, orthopox vectors, avipox vectors, fowlpox vectors, capripox vectors, suipox vectors, adenoviral vectors, herpes virus vectors, alpha virus vectors, baculovirus vectors, Sindbis virus vectors, vaccinia virus vectors and poliovirus vectors.
• Specific exemplary vectors are poxvirus vectors such as vaccinia virus, fowlpox virus and a highly attenuated vaccinia virus (MV A), adenovirus, baculovirus and the like.
• MV A highly attenuated vaccinia virus
• Pox viruses of use include orthopox, suipox, avipox, and capripox virus.
• Orthopox include vaccinia, ectromelia, and raccoon pox.
• One example of an orthopox of use is vaccinia.
• Avipox includes fowlpox, canary pox and pigeon pox.
• Capripox include goatpox and sheeppox.
• the suipox is swinepox.
• Other viral vectors that can be used include other DNA viruses such as herpes virus and adenoviruses, and RNA viruses such as retroviruses and polio.
• Viral vectors that encode a Gsxl protein can include at least one expression control element operationally linked to the nucleic acid sequence encoding the Gsxl protein.
• the expression control elements are inserted in the vector to control and regulate the expression of the nucleic acid sequence.
• expression control elements of use in these vectors includes, but is not limited to, lac system, operator and promoter regions of phage lambda, yeast promoters and promoters derived from polyoma, adenovirus, retrovirus or SV40. Additional operational elements include, but are not limited to, leader sequence, termination codons, polyadenylation signals and any other sequences necessary for the appropriate transcription and subsequent translation of the nucleic acid sequence encoding the Gsxl protein in the host system.
• the expression vector can contain additional elements necessary for the transfer and subsequent replication of the expression vector containing the nucleic acid sequence in the host system. Examples of such elements include, but are not limited to, origins of replication and selectable markers.
• Such vectors can be constructed using conventional methods (Ausubel et al., (1987) in “Current Protocols in Molecular Biology,” John Wiley and Sons, New York, N.Y.) and are commercially available. Similar vectors can be used with a nucleic acid encoding a Gsxl-CPP fusion protein.
• the viral vector that encodes a Gsxl protein (such as those that include a nucleic acid molecule having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1 or 3, or a nucleic acid molecule that encodes a protein having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 2 or 4) is a lentiviral or AAV vector, and includes a promoter operably linked to the Gsxl coding sequence.
• the promoter is a constitutive promoter, such as CMV, beta actin, or T7, or a tissue specific promoter, such as a s CNS-specific promoter (e.g., synapasin 1 (Synl) promoter, glial fibrillary acidic protein (GFAP) promoter, nestin (NES) promoter, myelin-associated oligodendrocyte basic protein (MOBP) promoter, myelin basic protein (MBP) promoter, tyrosine hydroxylase (TH) promoter, or a forkhead box A2 (FOXA2) promoter).
• a s CNS-specific promoter e.g., synapasin 1 (Synl) promoter, glial fibrillary acidic protein (GFAP) promoter, nestin (NES) promoter, myelin-associated oligodendrocyte basic protein (MOBP) promoter, myelin basic protein (MBP) promoter, tyrosine hydroxylase (
• Methods for preparing recombinant virus containing a heterologous DNA sequence encoding the Gsxl protein are known.
• Such techniques involve, for example, homologous recombination between the viral sequences flanking the Gsxl coding sequence in a donor plasmid and homologous sequences present in the parental virus.
• the vector can be constructed for example by using a unique restriction endonuclease site that is naturally present or artificially inserted in the parental viral vector to insert the heterologous DNA. Similar methods can be used for a nucleic acid encoding a Gsxl-CPP fusion protein.
• transfection methods include calcium phosphate coprecipitates, mechanical procedures such as microinjection, electroporation, insertion of a plasmid encased in liposomes, or viral vectors.
• Eukaryotic cells can also be co-transformed with polynucleotide sequences encoding a Gsxl protein (such as those that include a nucleic acid molecule having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1 or 3, or a nucleic acid molecule that encodes a protein having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 2 or 4), and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene.
• a selectable phenotype such as the herpes simplex thymidine kinase gene.
• Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein (see for example, Eukaryotic Viral Vectors , Cold Spring Harbor Laboratory, Gluzman ed., 1982).
• Expression systems such as plasmids and vectors can be used to produce Gsxl proteins in cells including higher eukaryotic cells such as the COS, CHO, HeLa and myeloma cell lines. Similar cells can be used with a nucleic acid encoding a Gsxl-CPP fusion protein.
• a nucleic acid molecule encoding a Gsxl protein or a Gsxl-CPP fusion protein can be used to transform cells and make transformed cells.
• cells expressing a Gsxl protein or a Gsxl- CPP fusion protein (such as those that include a nucleic acid molecule having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1 or 3, or a nucleic acid molecule that encodes a protein (or Gsxl portion thereof) having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 2 or 4), are disclosed.
• the cell is a mammalian cell present in a mammal, for example to treat a neurological disorder.
• the cell is in culture ( e.g ., ex vivo or in vitro ), for example used to express a therapeutic Gsxl protein or a Gsxl-CPP fusion protein, such as a eukaryotic or prokaryotic cell (e.g., bacteria, archea, plant, fungal, yeast, insect, and mammalian cells, such as Lactobacillus, Lactococcus, Bacillus (such as B. subtilis), Escherichia (such as E. coli), Clostridium, Saccharomyces or Pichia (such as S. cerevisiae or P. pastoris), Kluyveromyces lactis, Salmonella typhimurium, SF9 cells, C129 cells, 293 cells,
• a eukaryotic or prokaryotic cell e.g., bacteria, archea, plant, fungal, yeast
• Neurospora and immortalized mammalian neurological cell lines.
• Cells expressing a Gsxl protein or a Gsxl-CPP fusion protein are transformed or recombinant cells.
• Such cells can include at least one exogenous nucleic acid molecule that encodes a Gsxl protein or a Gsxl-CPP fusion protein (such as those that include a nucleic acid molecule having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1 or 3, or a nucleic acid molecules that encodes a protein (or Gsxl portion thereof) having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 2 or 4).
• progeny may not be identical to the parental cell since there may be mutations that occur during replication.
• the one exogenous nucleic acid molecule that encodes a Gsxl protein is stably transferred, meaning that the foreign DNA is continuously maintained in the transformed cell.
• Transformation of a host cell with recombinant nucleic acid may be carried out by conventional techniques.
• the host is prokaryotic, such as E. coli
• competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated with CaCh, MgCh or RbCl. Transformation can also be performed after forming a protoplast of the host cell if desired, using polyethylene glycol transformation, or by electroporation. Examples of commonly used mammalian host cell lines are HEK293T cells,
• VERO and HeLa cells VERO and HeLa cells, CHO cells, and WI38, BHK, and COS cell lines.
• compositions that include a Gsxl protein or a Gsxl-CPP fusion protein, or nucleic acid molecule encoding such, are provided.
• a composition includes an isolated Gsxl protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2 or 4, for example encapsulated in a liposome.
• a composition includes an isolated Gsxl -cell penetrating peptide (CPP) fusion protein comprising a Gxxl portion comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2 or 4, and a CPP portion, wherein the Gsxl portion and the CPP portion are optionally joined by a linker.
• the CPP portion has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOS: 61-79.
• the Gsxl-CPP fusion protein in a composition is encapsulated in a liposome.
• a composition includes an isolated nucleic acid molecule encoding a Gsxl protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1 or 3 or encoding a Gsxl protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2 or 4.
• a composition includes an isolated nucleic acid molecule encoding a Gsxl-CPP fusion protein, wherein a Gsxl portion of the Gsxl-CPP fusion protein is encoded by a nucleic acid molecule having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1 or 3 or encodes a Gsxl protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2 or 4, and wherein optionally a nucleic acid molecule encoding a CPP portion of the Gsxl- CPP fusion protein encodes a protein comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOS: 61-79.
• the nucleic acid molecule
• the pharmaceutical composition consists essentially of a Gsxl protein (such as a protein having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 2 or 4), a Gsxl fusion protein composed of (1) a Gsxl protein comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2 or 4 and (2) a cell penetrating peptide (such as a protein comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOS: 61 -79), or a nucleic acid encoding a Gsxl protein (such as a nucleic acid molecule having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or
• therapeutically effective agents are not included in the compositions.
• compositions can be formulated with an appropriate pharmaceutically acceptable carrier (such as water or saline), depending upon the particular mode of administration chosen.
• an appropriate pharmaceutically acceptable carrier such as water or saline
• compositions can be administered to a subject with a neurological disorder using the disclosed methods.
• the pharmaceutical composition is suitable for injection, such as injection into the CNS, for example at or near the site of injury (e.g ., rostral and/or caudal to the injury site).
• intraparenchymal, introcerebroventricular, or intrathecal (cisternal and lumbar) injections are used to target brain and/or spinal cord.
• the pharmaceutical composition can include a therapeutically effective amount of another agent.
• examples of such agents include, without limitation, those listed in section“F” below, or combinations thereof.
• parenteral formulations usually include injectable fluids that are
• non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
• pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, pH buffering agents, or the like, for example sodium acetate or sorbitan monolaurate.
• Excipients that can be included are, for instance, other proteins, such as human serum albumin or plasma preparations.
• the Gsxl protein, Gsxl-CPP fusion protein, or nucleic acid molecule encoding such is included in a controlled release formulation, for example, a microencapsulated formulation.
• a controlled release formulation for example, a microencapsulated formulation.
• biodegradable and biocompatible polymers, methods can be used, and methods of encapsulating a variety of synthetic compounds, proteins and nucleic acids can be used (see, for example, U.S. Patent Publication Nos. 2007/0148074; 2007/0092575; and
• the Gsxl protein, Gsxl-CPP fusion protein, or nucleic acid molecule encoding such is included in a nanodispersion system.
• a nanodispersion system includes a biologically active agent and a dispersing agent (such as a polymer, copolymer, or low molecular weight surfactant).
• Exemplary polymers or copolymers that can be used include polyvinylpyrrolidone (PVP), poly(D,L-lactic acid) (PLA), poly(D,L-lactic-co-glycolic acid (PLGA), poly(ethylene glycol).
• Exemplary low molecular weight surfactants include sodium dodecyl sulfate, hexadecyl pyridinium chloride, polysorbates, sorbitans, poly(oxyethylene) alkyl ethers, poly(oxyethylene) alkyl esters, and combinations thereof.
• the nanodispersion system includes PVP and ODP or a variant thereof (such as 80/20 w/w).
• the nanodispersion is prepared using the solvent evaporation method, see for example, Kanaze et al, Drug Dev. Indus. Pharm. 36:292-301, 2010; Kanaze et al., J. Appl. Polymer Sci. 102:460-471, 2006.
• a viral vector such as a lentiviral or AAV vector.
• the nucleotide sequence encoding a Gsxl protein (such as a protein having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 2 or 4, or such as a nucleic acid molecule having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1 or 3, or a nucleic acid molecule that encodes a protein having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 2 or 4) can be placed under the control of a promoter to increase expression of the Gsxl protein.
• a promoter to increase expression of the Gsxl protein.
• release delivery systems can be used. Examples include polymer based systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Patent No. 5,075, 109.
• Delivery systems also include non-polymer systems, such as lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and tri-glycerides; hydrogel release systems; silastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like.
• lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and tri-glycerides
• hydrogel release systems such as silastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like.
• Specific examples include, but are not limited to: (a) erosional systems in which the Gsxl protein, Gsxl-CPP fusion protein, or nucleic acid molecule encoding such, is contained in a form within a matrix such as those described in U.S. Patent Nos.
• a long-term sustained release implant can be suitable for treatment of chronic conditions, such as neurological disorders.
• Long-term release as used herein, means that the implant is constructed and arranged to deliver therapeutic levels of the active ingredient for at least 30 days, or at least 60 days.
• Long-term sustained release implants include some of the release systems described above. These systems have been described for use with nucleic acids (see U.S. Patent No. 6,218,371). For use in vivo , nucleic acids and peptides are relatively resistant to degradation (such as via endo- and exo-nucleases). Thus, modifications of a Gsxl protein, such as the inclusion of a C-terminal amide, can be used.
• the dosage form of the pharmaceutical composition can be determined by the mode of administration chosen.
• topical, inhalation, oral and suppository formulations can be employed.
• Topical preparations can include eye drops, ointments, sprays, patches and the like.
• Inhalation preparations can be liquid (e.g ., solutions or suspensions) and include mists, sprays and the like.
• Oral formulations can be liquid (e.g., syrups, solutions or suspensions), or solid (e.g, powders, pills, tablets, or capsules).
• Suppository preparations can also be solid, gel, or in a suspension form.
• conventional non-toxic solid carriers can include pharmaceutical grades of mannitol, lactose, cellulose, starch, or magnesium stearate.
• the therapeutically effective amount of the Gsxl protein, Gsxl-CPP fusion protein, or nucleic acid molecule encoding is the amount necessary to (1) decrease inflammation, for example at or near the injury site, such as decrease the number of infiltrated macrophages (such as a decrease of at least 5%, at least 10%, at least 15%, at least 20%, at least 50%, at least 75%, or at least 90% for example relative to no administration of the Gsxl protein, Gsxl-CPP fusion protein, or nucleic acid molecule encoding such), (2) increase the number of neural stem/progenitor cells (NSPCs) (e.g., as determined by measuring expression of nestin, and/or doublecortin), for example at or near the injury site, (for example an increase of at least 5%, at least 10%, at least 15%, at least 20%, at least 50%, at least 75%, at least 90%, or at least 100%, for example relative to no administration of the Gsxl protein, Gsxl
• compositions that include the Gsxl protein, Gsxl-CPP fusion protein, or nucleic acid molecule encoding such can be formulated in unit dosage form, suitable for individual administration of precise dosages.
• a unit dosage contains from about 1 mg to about 1 g of a Gsxl or Gsxl-CPP protein, such as about 10 mg to about 100 mg, about 50 mg to about 500 mg, about 100 mg to about 900 mg, about 250 mg to about 750 mg, or about 400 mg to about 600 mg.
• a therapeutically effective amount of a Gsxl or Gsxl-CPP protein is about 0.01 mg/kg to about 50 mg/kg, for example, about 0.5 mg/kg to about 25 mg/kg or about 1 mg/kg to about 10 mg/kg. In other examples, a therapeutically effective amount of a Gsxl or Gsxl-CPP fusion protein is about 1 mg/kg to about 5 mg/kg, for example about 2 mg/kg. In a particular example, a therapeutically effective amount of a Gsxl or Gsxl-CPP fusion protein is about 1 mg/kg to about 10 mg/kg, such as about 2 mg/kg.
• Gsxl protein or a Gsxl-CPP fusion protein of about 100 pg/kg to 10 mg/kg body weight or more (such as about 0.1-10 mg/kg, about 1-20 mg/kg, about 5-50 mg/kg, or about 10-100 mg/kg).
• the effective dosage is within narrower ranges of, for example, 5-40 mg/kg, 10-35 mg/kg or 20-25 mg/kg.
• the dosage is about 1-100 mg, such as about 1-10 mg, about 5-25 mg, about 10-50 mg, about 25-60 mg, or about 50-100 mg (for example, about 1 mg, 5, mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 70 mg, 80 mg, 90 mg, or 100 mg).
• the dose is about 20-60 mg, and in one non-limiting example, about 25 mg.
• compositions that include a Gsxl or Gsxl-CPP coding sequence can be formulated in unit dosage form, suitable for individual administration of precise dosages.
• the quantity of recombinant viral vector, carrying the nucleic acid coding sequence of Gsxl protein or Gsxl-CPP fusion protein to be administered is based on the titer of virus particles.
• a unit dosage e.g., 0.5-1.5 m ⁇
• a unit dosage contains about 10 5 to about 10 10 plaque forming units (pfu)/ml per mammal.
• the recipient subject is administered a dose of about 10 5 to about 10 10 pfu/ml per mammal of recombinant virus in the composition.
• the recipient subject is administered a dose of at least 10 5 pfu/ml per mammal, at least 10 6 pfu/ml per mammal at least 10 7 pfu/ml per mammal at least 10 8 pfu/ml per mammal, at least 10 9 pfu/ml per mammal, or at least 10 10 pfu/ml per mammal
• methods for administering the composition into mammals include, but are not limited to, injection of the composition into the affected tissue (such as into the brain or spinal cord) or intravenous, subcutaneous, intradermal or intramuscular administration of the virus.
• compositions of this disclosure that include a Gsxl or Gsxl-CPP protein can be administered to humans or other animals by any means, including orally, intravenously,
• the composition is administered via injection.
• site-specific administration of the composition can be used, for example by administering the Gsxl protein, a Gsxl-CPP fusion protein, or coding sequence to CNS tissue (for example the brain or spinal cord, for example at or near the area of injury, such as rostral and/or caudal to the injury site).
• administration is an intrathecal injection (e.g., of Gsxl protein, Gsxl-CPP fusion protein, or nucleic acid molecule encoding such) to treat SCI in lumbar/sacral region, a cistema magna injection (e.g, of Gsxl protein, Gsxl-CPP fusion protein, or nucleic acid molecule encoding such) to treat SCI in cervical/thoracic region, or intraparenchymal or introcerebroventricular injection ( e.g ., of Gsxl protein, Gsxl-CPP fusion protein, or nucleic acid molecule encoding such) to treat traumatic brain injury.
• intrathecal injection e.g., of Gsxl protein, Gsxl-CPP fusion protein, or nucleic acid molecule encoding such
• a cistema magna injection e.g, of Gsxl protein, Gsxl-CPP fusion protein, or nucle
• Treatment can involve a single administration, or multiple administrations (such as at least two separate administrations), such as doses over a period of a few days to months, or even years.
• a therapeutically effective amount of Gsxl protein, Gsxl-CPP fusion protein, or nucleic acid molecule encoding such can be administered in a single dose, or in several doses, for example daily, weekly, monthly, or yearly, during a course of treatment.
• treatment involves administration once monthly, once yearly, or every-other- month.
• the at least two separate administrations can be separated by at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 9 months, or at least one year.
• the first dose (and in some examples only dose) administrated occurs within 1 hour, within 2 hours, within 3 hours, within 4 hours, within 5 hours, within 6 hours, within 12 hours, within 24 hours, within 48 hours, within 72 hours, within 96 hours, within 1 week, within 2 weeks, within 3 weeks, within 4 weeks, within 1 month, within 2 months, or within 3 months of the onset of the neurological disorder, such as within 1 to 24 hours, 2 to 24 hours, 4 to 24 hours, or 1 to 96 hours of the onset of the neurological disorder.
• the Gsxl protein, Gsxl-CPP fusion protein, or nucleic acid molecule encoding such is administered in combination (such as sequentially, simultaneously, or contemporaneously) with one or more other agents, such as those useful in the treatment of a neurological disorder.
• administration in combination” or“co-administration” refers to both concurrent and sequential administration of the active agents.
• a Gsxl protein, a Gsxl-CPP fusion protein, or sequence encoding such is administered to a subject with a traumatic spinal cord or brain injury in combination with effective doses of one or more of stem cells, steroids (e.g., methylprednisolone), and iv fluids.
• the subject also receives surgery, hypothermia treatment, or both.
• Administration of a Gsxl protein or Gsxl coding sequence may also be in combination with lifestyle
• a Gsxl protein or Gsxl coding sequence is administered to a subject with a neurological disorder of the brain, such as Parkinson’s disease, Alzheimer’s disease, stroke (ischemic or hemorrhagic), ischemia, epilepsy, Huntington's disease, multiple sclerosis, amyotrophic lateral sclerosis, in combination with effective doses of one or more other therapeutic agents.
• a neurological disorder of the brain such as Parkinson’s disease, Alzheimer’s disease, stroke (ischemic or hemorrhagic), ischemia, epilepsy, Huntington's disease, multiple sclerosis, amyotrophic lateral sclerosis, in combination with effective doses of one or more other therapeutic agents.
• the method can further include administering a therapeutically effective amount of one or more of stem cells, deep brain stimulation, surgery (e.g ., pallidotomy or thalamotomy) benztropine mesylate (Cogentin), entacapone (Comtan), dopar, dopamine agonist (e.g., apomorphine (Apokyn), pramipexole (Mirapex), ropinirole HC1 (Requip), and rotigotine (Neupro)), larodopa, levodopa and carbidopa (Sinemet), rasagiline (Azilect), safmamide (Xadago), tasmar and trihexphenidyl (Artane).
• surgery e.g ., pallidotomy or thalamotomy
• benztropine mesylate Cosmeticin
• Comtan entacapone
• dopar dopar
• dopamine agonist e.g.
• the method can further include administering a therapeutically effective amount of one or more of stem cells, a cholinesterase inhibitor (e.g., Razadyne® (galantamine), Exelon® (rivastigmine), or Aricept® (donepezil)), an N-methyl D- aspartate (NMDA) antagonist (e.g., memantine), Celexa® (citalopram), Remeron® (mirtazapine), Zoloft® (sertraline), Wellbutrin® (bupropion), Cymbalta® (duloxetine), and Tofranil®
• a cholinesterase inhibitor e.g., Razadyne® (galantamine), Exelon® (rivastigmine), or Aricept® (donepezil)
• NMDA N-methyl D- aspartate
• memantine e.g., memantine
• Celexa® citalopram
• Remeron® mirtazapine
• the method can further include administering a therapeutically effective amount of one or more of a tissue plasminogen activator (e.g., Alteplase IV r-tPA) for an ischemic stroke, or surgery (e.g, install a coil or clip to stop blood loss) for a hemorrhagic stroke.
• a tissue plasminogen activator e.g., Alteplase IV r-tPA
• surgery e.g, install a coil or clip to stop blood loss
• the method can further include administering a therapeutically effective amount of one or more of a vasodilator, anticoagulant, (e.g., heparin, aspirin), nitrate, ACE inhibitor, ranolazine, and surgery.
• a vasodilator e.g., cardiac ischemia or mesenteric artery ischemia
• anticoagulant e.g., heparin, aspirin
• nitrate e.g., aspirin
• ACE inhibitor e.g., ACE inhibitor
• the method can further include administering a therapeutically effective amount of one or more of an anti-seizure or anti-epileptic medication (e.g., carbamazepine, valproate, lamotrigine, dilantin or phenytek, ohenobarbital, tegretol or Carbatrol, mysoline, zarontin, depakene, depakote, depakote ER, valium and similar tranquilizers such as Tranxene and Klonopin, felbatol, gabitril, keppra, lamictal, lyrica, neurontin, topamax, trileptal, and, zonegran), surgery, vagus nerve stimulation, deep brain stimulation, and a ketogenic diet.
• an anti-seizure or anti-epileptic medication e.g., carbamazepine, valproate, lamotrigine, dilantin or phenytek, ohenobarbital
• the method can further include administering a therapeutically effective amount of one or more of a monoamine depletor (e.g, tetrabenazine or amantadine), SSRI antidepressant (e.g, fluoxetine citalopram, paroxetine, and sertraline) or other anti-depressant (e.g, amitriptyline, mirtazapine, duloxetine, and venlafaxine), antipsychotic drug (e.g, quetiapine, risperidone or olanzapine), mood-stabilizing drug (e.g, valproate or carbamazepine), and a high protein diet.
• a monoamine depletor e.g, tetrabenazine or amantadine
• SSRI antidepressant e.g, fluoxetine citalopram, paroxetine, and sertraline
• other anti-depressant e.g, amitriptyline,
• the method can further include administering a therapeutically effective amount of one or more of stem cells, a corticosteroid (e.g, methylprednisolone or prednisone), an interferon beta blocker (e.g, copaxone, teriflunomide, or mitoxantrone).
• a corticosteroid e.g, methylprednisolone or prednisone
• an interferon beta blocker e.g, copaxone, teriflunomide, or mitoxantrone.
• the method can further include administering a therapeutically effective amount of one or more of a glutamate antagonist (e.g ., riluzole) and a neuroprotective agent (e.g, edaravone).
• a glutamate antagonist e.g ., riluzole
• a neuroprotective agent e.g, edaravone
• the pharmaceutical composition that includes a Gsxl protein, a Gsxl-CPP fusion protein, or sequence encoding such further includes one or more of these therapeutic agents.
• Administration of a Gsxl protein, a Gsxl-CPP fusion protein, or sequence encoding such may also be in combination with increased physical activity, speech or language therapy, occupational therapy, physical therapy, or combinations thereof.
• compositions which can be used with the disclosed methods.
• the composition includes an isolated Gsxl protein comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2 or 4 and a liposome, wherein the Gsxl protein is encapsulated in the liposome.
• the composition includes a Gsxl fusion protein composed of (1) a Gsxl protein comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2 or 4 and (2) a cell penetrating peptide (or a nucleic acid molecule encoding such a Gsxl-CPP fusion protein).
• Exemplary cell penetrating peptides that can be used include hydrophilic peptides (e.g, TAT [YGRKKRRQRRR; SEQ ID NO: 61], SynBl [RGGRLSYSRRRFSTSTGR; SEQ ID NO: 62], SynB3 [RRLSYSRRRF ; SEQ ID NO: 63], PTD-4 [PIRRRKKLRRLK ; SEQ ID NO: 64], PTD-5 [RRQRRT SKLMKR; SEQ ID NO: 65], FHV Coat- (35-49) [RRRRNRTRRNRRRVR; SEQ ID NO: 66], BMV Gag-(7-25)
• hydrophilic peptides e.g, TAT [YGRKKRRQRRR; SEQ ID NO: 61], SynBl [RGGRLSYSRRRFSTSTGR; SEQ ID NO: 62], SynB3 [RRLSYSRRRF ; SEQ ID NO: 63], PTD-4 [PIRRRKKLRRLK ;
• periodic sequences e.g, pVec, polyarginines RxN (4 ⁇ N ⁇ 17) chimera, polylysines KxN (4 ⁇ N ⁇ 17) chimera, (RAca)6R, (RAbu)6R, (RG)6R, (RM)6R, (RT)6R, (RS)6R, R10, (RA)6R, R7, and pep-l [ac- KETWWETWWTEW SQPKKKRKV -cya; SEQ ID NO: 79]), and CrlO (a cyclic pol-arginine CPP).
• Such a Gsxl protein or Gsxl-CPP fusion protein can be encapsulated in a liposome.
• a composition includes an isolated nucleic acid molecule encoding a Gsxl protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1 or 3 or encoding a Gsxl protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2 or 4.
• a composition includes an isolated nucleic acid molecule encoding a Gsxl-CPP fusion protein, wherein a Gsxl portion of the Gsxl-CPP fusion protein is encoded by a nucleic acid molecule having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1 or 3 or encodes a Gsxl protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2 or 4, and wherein optionally a nucleic acid molecule encoding a CPP portion of the Gsxl-CPP fusion protein encodes a protein comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOS: 61-79.
• the nucleic acid molecule
• compositions can further include other materials, such as a pharmaceutically acceptable carrier, such as water or saline, and/or other therapeutic agents, as described above.
• a pharmaceutically acceptable carrier such as water or saline
• other therapeutic agents such as described above.
• a host cell such as neural stem/progenitor cells (NSPCs), fibroblast cells, or embryonic stem cells (ESCs)
• NSPCs neural stem/progenitor cells
• fibroblast cells e.g., fibroblast cells
• ESCs embryonic stem cells
• a nucleic acid molecule provided herein such as a viral vector or plasmid encoding a Gsxl protein or Gsxl-CPP fusion protein.
• the Gsxl nucleic acid molecule can encode a Gsxl protein comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2 or 4.
• the nucleic acid molecule encoding Gsxl has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1 or 3, and in some examples is part of a plasmid or vector (such as a lentiviral or AAV vector), and can be operably linked to a promoter, enhancer element, or both.
• the cell includes an isolated nucleic acid molecule encoding a Gsxl-CPP fusion protein, wherein a Gsxl portion of the Gsxl-CPP fusion protein can be encoded by a nucleic acid molecule having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1 or 3 or encodes a Gsxl protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2 or 4.
• the portion of the nucleic acid molecule encoding a CPP portion of the Gsxl-CPP fusion protein can encodes a protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOS: 61-79.
• a nucleic acid molecule encoding a Gsxl-CPP fusion protein is part of a plasmid or vector (such as a lentiviral or AAV vector), and can be operably linked to a promoter, enhancer element, or both.
• the recombinant host cells are cultured ex vivo.
• fibroblast cells are used, they can be cultured in MEF medium [Dulbecco’s modified Eagle’s medium (DMEM, Hyclone), 10% fetal bovine serum (FBS) (Gibco),
• DMEM modified Eagle’s medium
• FBS fetal bovine serum
• NSPCs and ESCs can be cultured in neuron differentiation medium: DMEM/F12 (Life Technologies) with 1 x Pen/Strep, 25 pg/ml insulin,
• the resulting transformed (e.g ., recombinant) host cells which are thus programmed to a neuronal phenotype, can be introduced (e.g., transplanted) into a subject with a neurological disorder, such as a SCI or TBI, or Parkinson’s disease, Alzheimer’s disease, stroke, ischemia, epilepsy, Huntington's disease, multiple sclerosis, or amyotrophic lateral sclerosis.
• a neurological disorder such as a SCI or TBI, or Parkinson’s disease, Alzheimer’s disease, stroke, ischemia, epilepsy, Huntington's disease, multiple sclerosis, or amyotrophic lateral sclerosis.
• a neurological disorder such as a SCI or TBI, or Parkinson’s disease, Alzheimer’s disease, stroke, ischemia, epilepsy, Huntington's disease, multiple sclerosis, or amyotrophic lateral sclerosis.
• programmed cells can be used to reach such neurological disorders in a subject, such as a
• This example provides the materials and methods used to obtain the results shown in Examples 2-7.
• Lentiviruses encoding Gsxl and a reporter RFP (lenti-Gsxl-RFP) and control lentiviruses (encoding only the reporter RFP, lenti-Ctrl-RFP) (ABM® LV465366 and LV084) were generated by transfecting HEK293T cells with a mixture of target vector (lenti-Gsxl-RFP or lenti-Ctrl-RFP), envelope plasmid (pMD2.G/VSVG, Addgene 12259), and 3rd generation packaging plasmids (pMDLg/pRRE, Addgene 12251 and pRSV-Rev, Addgene 12253).
• HEK293T cells were cultured in high glucose Dulbecco’s Modified Eagle Media (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% nonessential amino acid (MEM NEAA lOOx Life Technology 11140050), and 1% Glutamax 1 100X (Life Technology 35050061). Transfection of the HEK293T (Human Embryonic Kidney) cells was performed when the culture reaches ⁇ 50-60% confluency. Virus containing supernatant was collected at day 2 and day 4 after transfection. Viruses were concentrated by precipitating the virus supernatant by polyethylene glycol 6000 (PEG6000) method (Kutner et al, NatProtoc 4, 495-505 (2009)). Viral titer was determined by infecting HEK293T cells (Kutner et ah, NatProtoc 4, 495-505 (2009)).
• PEG6000 polyethylene glycol 6000
• mice Young adult (8-12 weeks) mice were used, under compliance of Institutional Animal Care and Lise Committee (IACUC). All animals were housed in an animal care facility with l2-hour light/dark cycle. Mouse under each experimental condition were assigned randomly with equal number of male and female mice when possible.
• IACUC Institutional Animal Care and Lise Committee
• mice were first anesthetized with 5% isoflurane inhalation for 3-4 minutes and then maintained at 2.5% isoflurane for the remainder of the surgery. Next, the skin was disinfected with betadine scrub and 70% ethanol wipes.
• Laminectomy was performed around T9-T10 t expose the spinal cord.
• local anesthesia (0.125% Marcain) was applied and dorsal blood vessels were burned using the cauterizer.
• a lateral cut was performed to the left side of the spinal cord and the cut ends at the midline of the spinal cord for hemisection SCI.
• ⁇ l-2 pL of virus (lxlO 8 TU/ml) was injected about 1 mm rostral and caudal to lesion epicenter.
• Virus was injected at about 1 pL/min and the needle was left in place for 2-3 minutes to allow diffusion and prevent leakage or backflow.
• skin and muscle were cut to expose the spinal cord. Muscles were sutured and skin was stapled back together.
• 1 mg/kg Meloxicam, a pain killer, and 50 mg/kg Cefazoline, an antibiotic were administered subcutaneously.
• mice Animals were divided into the following three groups (3-6 mice/group): 1) sham mice (exposed the spine without injury, Sham); 2) SCI mice with injection of lenti-control-RFP
• BMS scale ranges from 0
• the BMS score assessment was given by three independent observers who are blinded to the type of treatment after a 2-3 minute observation per animal. The BMS assessment is performed before the injury and then twice a week for up to 8 weeks after injury.
• cryopreserved by embedding in Tissue-Tek® optimum cutting temperature (O.C.T.) and stored at - 80°C until needed. Sagittal or cross-sections (12 pm thickness) of spinal cord tissues were generated using a cryostat (ThermoScientific).
• Immunostaining was performed following a previously established protocol with minor modifications (Li et al ., Sci Rep 6, 38665 (2016)). Briefly, sections were treated with cold methanol for 10 mins at room temperature for fixation and antigen retrieval. All antibodies were diluted in blocking solution containing 0.05% Triton X-100, 2% donkey serum, 3% bovine serum albumin (BSA), and PBS (IX), pH 7.4. Sections were incubated with primary antibodies (Table 1) overnight at 4°C and washed three times for 10 min with PBS. Secondary antibodies (Table 1) were then incubated for 1 hour at room temperature and washed three times for 10 min with PBS.
• RNA 6000 Nano chip on the 2100 Bioanalyzer automated electrophoresis system (Agilent Technologies).
• RNA-sequencing were performed by Admera Health (South Plainfield, NJ). Total RNA was used for library preparation of each sample, which was
• the libraries were prepared using an Illumina MiSeq paired-end kit and sequenced as paired-end, 2x150 bp on the Illumina MiSeq. The sequencing run was performed according to the manufacturer’s instructions and generated a total of 40 million reads per sample.
• cDNA Complementary DNA
• qPCR was performed with Power SYBRTM Green PCR Master Mix and gene specific primers (Table 2) using StepOnePlus Real-Time PCR system (Applied Biosystem).
• GAPDH is used as a reference housekeeping gene.
• the Levak method is used to calculate the fold change, by normalizing it to the Sham.
• lentivirus lxlO 8 TU/ml
• lenti-Gsxl-RFP reporter red fluorescent protein
• mice Animals were randomly assigned to the following three groups (3-6 mice/group): 1) sham mice (exposed the spine without injury, Sham); 2) SCI mice with injection of lenti -Ctrl -RFP (SCI+Ctrl); and 3) SCI mice with lenti-Gsxl-RFP injection (SCI+Gsxl). It was first confirmed that the lentivirus-mediated Gsxl expression in the spinal cord tissue at 3 days-post injury (DPI) and 7 DPI by immunohistochemistry and RT-qPCR at 3 DPI (FIGS. 2A- 2E). Compared to the control, Gsxl treatment significantly increased the percentage of virally transduced RFP+ cells with Gsxl expression.
• DPI days-post injury
• RT-qPCR RT-qPCR
• Gsxl treatment increases cell proliferation in injured spinal cord
• SCI increases cell proliferation at the lesion site.
• DPI day-post injury
• the expression of the cell proliferation marker Ki67 was examined by immunohistochemistry followed by confocal imaging analysis (FIG. 1B).
• the RFP+ and Ki67+ cells were found to be located around the injection sites.
• Ki67+ cells A significant increase in the percentage of Ki67+ cells was observed in both injury groups that received viral injection compared to the Sham mice, with the highest increase in the SCI+Gsxl group (FIG. 1C).
• a significantly higher percentage of Ki67+/RFP+ co-labeled cells among RFP+ cells were found in the mice with lenti-Gsxl-RFP injections compared to mice that received lenti -Ctrl -RFP injections (FIG. 1D).
• the increase in Ki67 mRNA expression was validated by RT-qPCR analysis. Gsxl treatment induces a significantly higher level of Ki67 mRNA level ( ⁇ 4-fold; FIG.
• RNA-seq analysis identified 475, 1447, and 3946 differentially expressed genes (DEGs) at 3, 14 and 35 DPI, respectively (FIGS. 3B-3C).
• the top 40 DEGs (Table 3) were shown in heatmap at 3 DPI (FIG. 3D), 14 DPI (FIG. 3E), and 35 DPI (FIG. 3F).
• Gsxl treatment promotes NSPC activation after SCI
• NSPCs exist in quiescent states under normal conditions, but become activated after injury.
• Gsxl treatment the expression of NSPC markers (Nestin and Notchl) in the injured spinal cord at 3 DPI via immunohistochemistry and confocal imaging analysis was examined.
• the RFP+ and Nestin+ cells were found around the injection sites (FIG. 5 A).
• a significant increase in the percentage of Nestin+ cells was observed among DAPI+ cells at the lesion site in the injury groups that received viral injection (SCI+Ctrl and SCI+Gsxl) compared to the Sham mice, with the highest increase in the SCI+Gsxl group (FIG. 5B).
• a significantly higher percentage of Nestin+/RFP+ co-labeled cells among RFP+ cells were found in SCI+Gsxl group compared to SCI+Ctrl group (FIG. 5C).
• RT-qPCR analysis confirmed that Gsxl treatment significantly increased Nestin mRNA expression (FIG. 5D).
• IPA analysis showed a significant upregulation of the genes involved in the Notch signaling pathway (e.g ., Hes7 and Rbpj) and a downregulation of the transcription repressor gene Hesl (FIG. 5E).
• Immunohistochemistry analysis using anti-Notchl antibody on sagittal sections of the spinal cord tissues at 3 DPI showed a significant increase in the number of
• Notchl+/RFP+ cells in Gsxl treatment compared to the control (FIGS. 5F and 5G).
• the RT-qPCR analysis confirmed a significant increase in the mRNA expression of Notchl and Jagl after SCI (SCI+Ctrl and SCI+Gsxl) compared to the Sham group and a further increase in Notchl mRNA level in lenti-Gsxl treatment compared to the control (FIG. 5H).
• Nrarp a negative regulator of the Notch signaling pathway that physically interacts with Notch intracellular domain (NICD) and blocks Notch transcription
• NBD Notch intracellular domain
• FIG. 5H Gsxl treatment decreased expression of the Dell and Hesl gene (FIG. 5H).
• Dell promotes stem cell differentiation to glial lineage, while Hesl is a transcriptional repressor. The expression of Hesl results in premature neuronal differentiation.
• Nanog signaling pathway e.g., Akt2, Map2k2, Pik3cd, Pik3cg, and Rap2b
• Akt2, Map2k2, Pik3cd, Pik3cg, and Rap2b an increase in genes associated with activation of Nanog signaling pathway.
• Nanog is an essential pathway in embryonic stem cells (ESCs) and the Nanog gene is commonly expressed in NSPCs.
• the expression of genes in Notch/Nanog signaling pathways was not detected by 35 DPI.
• oligodendrocytes To determine whether Gsxl treatment alters cell fate determination in NSPC lineage development, sagittal section (FIGS. 6A-6D) and cross-section (FIGS. 7A-7D) of spinal cord tissues at 14 DPI were examined with an early neuronal marker doublecortin (DCX) (FIG.
• DCX early neuronal marker doublecortin
• DCX is mostly expressed in neuroblasts and immature neurons and is associated with adult neurogenesis, but not reactive gliosis.
• Gsxl treatment significantly increased the percentage of DCX+/RFP+ co-labeled cells and decreased the percentage of GFAP+/RFP+ and PDGFRa+/RFP+ co-labeled cells among RFP+ cells (FIG. 6D).
• Gsxl treatment increases the number of specific subtypes of interneurons
• FIG. 9A Sagittal sections of spinal cord tissues at 56 DPI were stained with a mature neuronal marker NeuN (FIG. 9A), a cholinergic neuronal marker ChAT (FIG. 9B), a glutamatergic interneuron marker vGlut2 (FIG. 9C), and a GABAergic interneuron marker GABA (FIG. 9D).
• Gsxl treatment reduces glial scar formation
• SCI causes activation of microglia and astrocytes, which leads to reactive astrogliosis and glial scar formation.
• Glial scar is mostly composed of reactive astrocytes (RA), non-neuronal cells (e.g ., pericytes and meningeal cells), and proteoglycan-rich extracellular matrix (ECM).
• RA reactive astrocytes
• non-neuronal cells e.g ., pericytes and meningeal cells
• ECM proteoglycan-rich extracellular matrix
• Activated astrocytes secrete chondroitin sulfate proteoglycan (CSPG), which constitutes the major component of the glial scar.
• CSPG chondroitin sulfate proteoglycan
• RNA-Seq analysis revealed that the expression of genes associated with RA (e.g., Mmpl3, Mmp2, Nes, Axin2, Slit2, Plaur, and Ctnnbl), scar-forming astrocytes (SA) (e.g, Slit2 and Sox9), and both RA+SA (e.g., Gfap and Vim) 51 were downregulated at 14 DPI and 35 DPI (FIGS. 10C-10E).
• RA e.g., Mmpl3, Mmp2, Nes, Axin2, Slit2, Plaur, and Ctnnbl
• SA scar-forming astrocytes
• RA+SA e.g., Gfap and Vim
• GFAP GFAP
• CSPG CSPG protein expression levels were determined by immunohistochemistry analysis using anti-GFAP and anti-CS56 antibodies, respectively.
• a baseline level of GFAP (FIG. 10F) but no detectable level of CSPG expression (FIG. 10H) were observed in the Sham group.
• injury induced a higher protein level of GFAP (FIG. 10G) and CS56 (FIG. 101) in the two SCI groups (i.e., SCI+Ctrl and SCI+Gsxl).
• Gsxl treatment greatly reduced GFAP+ and CS56+ immunostained area around the lesion site in the SCI+Gsxl group (FIGS. 10G, 101).
• Gsxl treatment improves locomotor function after SCI
• mice with a lateral hemisection SCI at the T10 level exhibited paralysis in the left hindlimb after injury (FIG. 11 A).
• locomotor behavior was assessed using an established open-field locomotion test and a BMS score scale (Basso et al., J Neurotrauma 23:635-659 (2006)) starting from the day before the injury (-1 DPI) to 56 DPI (8 weeks after SCI).
• a BMS score was assigned double- blindly by three observers. BMS score ranges from 0 (complete paralysis and no ankle movement) to 9 (normal walking).
• the Sham animals displayed a normal locomotor behavior with BMS score remained at ⁇ 9 from -1 to 56 DPI (FIG. 11B).
• stemcell.rutgers.edu/Treatment.mov stemcell.rutgers.edu/Control.mov;
• mice in the SCI+Ctrl group (n>6) had a significantly improved locomotor function with BMS score gradually increased from ⁇ 0 to ⁇ 5 by 30 DPI (FIG. 11B).
• Gsxl -treated animals showed near normal locomotion (with BMS score reaching ⁇ 6-7) compared to the Sham mice (FIG. 11B).
• RT-qPCR analysis was used to assess expression of a selected set of genes involved in axon growth.
• RNA-SEQ, IP A, and gene ontology analysis on DEGs was performed.
• Gsxl treatment led to the activation of Netrin signaling (FIG. 11D; FIG. 12B), and axonal guidance pathways (FIG. 11E), and CREB signaling in neurons (FIG. 12A).
• CREB is a transcription factor responsible for axon growth and regeneration 55.
• RNA-Seq and IPA analysis further identified an increase in the expression of genes that promote synaptogenesis (FIG. 11F) and a decrease in the expression of genes known to inhibit synaptogenesis (FIG. 11G) in Gsxl treatment at 35 DPI.

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