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  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
  • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
  • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
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  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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  • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
  • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
  • A61L27/383—Nerve cells, e.g. dendritic cells, Schwann cells
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  • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
  • A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
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  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
  • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
  • A61L27/3886—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells comprising two or more cell types
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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0618—Cells of the nervous system
• • A—HUMAN NECESSITIES
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  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
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  • C12N2740/00—Reverse transcribing RNA viruses
  • C12N2740/00011—Details
  • C12N2740/10011—Retroviridae
  • C12N2740/16011—Human Immunodeficiency Virus, HIV
  • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
  • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

• SCI Spinal cord injury
• SCI at the cervical (neck) level frequently results in life-threatening respiratory deficits, which can be attributed in large part to the direct compromise of the phrenic motor circuitry that controls the diaphragm - the primary respiratory muscle.
• Other devastating effects of SCI include paraplegia and quadriplegia. With more than 250,000 incidents of SCI occurring world-wide each year, there is an urgent need to develop treatments capable of improving survival, function, and quality of life in affected individuals.
• NPCs neural progenitor cells
• Angiogenesis is thought to be an essential component of tissue repair.
• surprisingly little effort has been made to enhance angiogenesis / vasculogenesis in the context of SCI.
• vascular growth factors e.g. VEGF and FGF
• FGF vascular growth factors
• ECs naive endothelial cells
• the present invention derives, in part, from several surprising discoveries that are described in more detail in the Examples section of this patent specification.
• E40RFU ECs engineered endothelial cells that express an adenovirus E40RF1 sequence
• the present invention provides various new and improved compositions and methods for use in the repair of spinal cord injury.
• the present invention provides methods of treating spinal cord injury (SCI) in a subject in need thereof, the methods comprising:
• compositions comprising (a) E40RF1+ endothelial cells (ECs), and (b) neural cells. Such compositions may be useful in treating SCI in subjects in need thereof.
• the“treatment” achieved using the methods and compositions of the invention comprises neural repair.
• the“treatment” achieved using the methods and compositions of the invention comprises growth and/or extension of neurons and/or axons through a spinal cord lesion.
• the“treatment” achieved using the methods and compositions of the invention comprises growth and/or extension of motor neurons and/or axons through a spinal cord lesion.
• the“treatment” achieved using the methods and compositions of the invention comprises growth and/or extension of sensory neurons and/or axons through a spinal cord lesion.
• the“treatment” achieved using the methods and compositions of the invention comprises growth and/or extension of serotonergic neurons and/or axons through a spinal cord lesion. In some embodiments the“treatment” achieved using the methods and compositions of the invention comprises growth and/or extension of phrenic neurons and/or axons through a spinal cord lesion. In some embodiments the“treatment” achieved using the methods and compositions of the invention comprises growth and/or extension of neurons and/or axons through a spinal cord lesion wherein the neurons and/or axons become synaptically integrated into the subject’s central nervous system. In some embodiments the “treatment” achieved using the methods and compositions of the invention comprises an increase in transmittal of electrical signals across a spinal cord lesion.
• the“treatment” achieved using the methods and compositions of the invention comprises an improvement in a motor function that was compromised or lost as the result of the spinal cord lesion. In some embodiments the“treatment” achieved using the methods and compositions of the invention comprises an improvement in a sensory function that was compromised or lost as the result of the spinal cord lesion. In some embodiments the“treatment” achieved using the methods and compositions of the invention comprises an improvement in diaphragm function and/or breathing. [0008] In each of such methods and compositions, various different types of ECs can be used. For example, in some embodiments the ECs are vascular ECs.
• the ECs are primary ECs, while in other embodiments the ECs are cultured EC cells from an EC cell line.
• the ECs are mammalian ECs.
• the ECs are primate ECs.
• the ECs are human ECs.
• the ECs are other mammalian ECs, such as rabbit, rat, mouse, guinea pig, goat, pig, sheep, cow, horse, cat or dog ECs.
• the ECs are umbilical vein ECs (ETVECs).
• the ECs are human umbilical vein ECs (EtUVECs).
• the ECs are central-nervous system ECs. In some embodiments the ECs are brain ECs. In some embodiments the ECs are spinal cord ECs. In some embodiments the ECs are olfactory bulb ECs. In some embodiments the ECs are peripheral-nervous system ECs. In some embodiments the ECs are allogeneic with respect to the subject into which they are to be transplanted / administered. In some embodiments the ECs are autologous with respect to the subject into which they are to be transplanted / administered. In some embodiments the ECs have the same MHC/HLA type as the subject into which they are to be transplanted / administered.
• the ECs are mitotically inactive. In some embodiments the ECs are differentiated ECs. In some embodiments the ECs are adult ECs. In some embodiments the ECs are differentiated from induced pluripotent stem cells (iPSCs). In some embodiments the ECs are differentiated from iPSCs induced from cells including, but not limited to, skin, fibroblasts, hepatocytes, lymphoblasts, astrocytes, peripheral blood mononuclear cells. In some embodiments the ECs are produced by trans-differentiation from a differentiated non-endothelial cell type. In some embodiments the ECs have been previously cultured in a 3D matrix. In some embodiments the ECs have not been previously cultured in a 3D matrix.
• the neural cells are primary neural cells.
• the neural cells are cultured from a neural cell line or from a primary source.
• the neural cells are mammalian neural cells.
• the neural cells are primate neural cells.
• the neural cells are human neural cells.
• the neural cells are other mammalian cells, such as rabbit, rat, mouse, guinea pig, goat, pig, sheep, cow, horse, cat or dog neural cells.
• the neural cells are neuronal cells.
• the neural cells are glial cells.
• the neural cells are neural stem cells (NSCs). In some embodiments the neural cells are neural progenitor cells (NPCs). In some embodiments the neural cells are neural progenitor cells (NPCs) derived from the spinal cord. In some embodiments the neural cells are neural progenitor cells (NPCs) derived from the olfactory bulb. In some embodiments the neural cells are neural progenitor cells (NPCs) derived from the spinal cord or the olfactory bulb. In some embodiments the neural cells are neural progenitor cells (NPCs) derived from the developing spinal cord. In some embodiments the neural cells are neural progenitor cells (NPCs) derived from the developing olfactory bulb.
• NSCs neural progenitor cells
• the neural cells are neural progenitor cells (NPCs) derived from the developing spinal cord or the developing olfactory bulb.
• NPCs neural progenitor cells
• the neural cells are lineage-restricted neuronal progenitor cells or glial progenitor cells.
• the neural cells are allogeneic with respect to the subject into which they are to be transplanted / administered.
• the neural cells are autologous with respect to the subject into which they are to be transplanted / administered.
• the neural cells have the same MHC/HLA type as the subject into which they are to be transplanted / administered.
• the neural cells are mitotically inactive.
• the neural cells are differentiated neural cells.
• the neural cells are adult neural cells.
• the neural cells are differentiated from induced pluripotent stem cells (iPSCs).
• iPSCs induced pluripotent stem cells
• the neural cells are differentiated from iPSCs induced from cells including, but not limited to, skin, fibroblasts, hepatocytes, lymphoblasts, astrocytes, peripheral blood mononuclear cells.
• the neural cells are produced by trans-differentiation from a differentiated non-neural cell type.
• the neural cells have been previously cultured in a 3D matrix. In some embodiments the neural cells have not been previously cultured in a 3D matrix.
• the subjects that can be treated with the methods and compositions of the present invention include any subjects having a spinal cord injury (SCI).
• the subject is a mammal.
• the subject is a primate.
• the subject is a human.
• the subject is a rabbit, rat, mouse, guinea pig, goat, pig, sheep, cow, horse, cat or dog.
• each of the methods and compositions of the invention involves endothelial cells that contain an adenovirus E40RF1 polypeptide - i.e.“E40RF1+ ECs.”
• the E40RF1+ ECs contain a nucleic acid molecule that encodes an adenovirus E40RF1 polypeptide.
• nucleic acid molecule is present in a vector.
• the vector is a retroviral vector.
• the retroviral vector is a lentiviral vector.
• the retroviral vector is a Maloney murine leukemia virus (MMLV) vector.
• the nucleic acid that encodes the adenovirus E40RF1 polypeptide is integrated into the genomic DNA of the ECs.
• the E40RF1+ ECs and/or neural cells can be administered using any suitable means known in the art for local delivery of cells or agents to the site of a spinal cord injury.
• the E40RF1+ ECs and/or neural cells are administered by local injection.
• the E40RF1+ ECs and/or neural cells are administered by local infusion.
• the E40RF1+ ECs and/or neural cells are administered by local surgical implantation methods.
• the E40RF1+ ECs and/or neural cells are administered in a biocompatible matrix material (e.g. a biocompatible and/or biodegradable matrix, such as a solid 3D implant or a liquid matrix).
• the E40RF1+ ECs and/or neural cells are not administered in a biocompatible matrix material.
• the E40RF1+ ECs and/or neural cells are not administered in a solid 3D biocompatible matrix.
• the E40RF1+ ECs and/or neural cells can be administered in any suitable carrier composition known in the art.
• the cells may be administered in a composition comprising a physiological saline.
• the cells may be administered in a biocompatible matrix material - such as one that remains liquid during the administration process, or one that is a solid 3D implant during the administration process.
• the E40RF1+ ECs and/or neural cells can be administered together or separately.
• the E40RF1+ ECs and/or neural cells can also be administered concurrently or at different times.
• the E40RF1+ ECs and/or neural cells will be administered to the subject only once, while in other embodiments the E40RF1+ ECs and/or neural cells may be administered to the subject multiple times.
• the ratio of the E40RF1+ ECs to neural cells that is administered can be varied. In some embodiments a 1 : 1 ratio of E40RF1+ ECs to neural cells is used. However, E40RF1+ EC to neural cell ratios of about 1 : 10, or about 1 :9, or about 1 :8, or about 1 :7, or about 1 :6, or about 1 :5, or about 1 :4, or about 1 :3, or about 1 :2, or about 2: 1, or about 3: 1, or about 4: 1, or about 5: 1, or about 6: 1, or about 7: 1, or about 8: 1, or about 9: 1, or about 10: 1, can also be used.
• the numbers of E40RF1+ ECs and neural cells that are administered can also be varied.
• the number of E40RF1+ ECs administered should be an“effective amount” as defined herein.
• the number of neural cells administered should be an“effective amount” as defined herein.
• the total number of cells that is administered is in the range of about 500,000 cells to about 10,000,000 (10 million) cells. In some embodiments, such as those where cells are administered to small animals such as rodents, the total number of cells that is administered is in the range of about 500,000 cells to about 2,000,000 (2 million) cells.
• the total number of cells that is administered is in the range of about 5,000,000 (5 million) cells to about 10,000,000 (10 million) cells.
• the progress of the treatment can be monitored at various times (for example, beginning with immediate assessment, continuing on a daily basis for the first week and, twice a week thereafter for an indefinite amount of time or up to completion of pre-set experimental time-point) using various different methods.
• Example of such methods include, but are not limited to, methods that enable anatomical repair to be visualized (e.g. using medical imaging techniques, or histological assessment when appropriate), and methods that enable functional improvements to be observed (e.g. by measuring one or more sensory or motor functions affected by the SCI).
• the timing of the administration of the E40RF1+ ECs and/or the neural cells to the subject can be any suitable time after the creation of the injury. In the case of human subjects, a physician will typically make a determination about the timing of the administration.
• the E40RF1+ ECs and/or the neural cells are administered to the subject within the acute phase after the creation of the SCI injury. In some embodiments, the E40RF1+ ECs and/or the neural cells are administered to the subject within the subacute phase after the creation of the SCI injury. In some embodiments, the E40RF1+ ECs and/or the neural cells are
• the E40RF1+ ECs and/or the neural cells are administered to the subject within the chronic phase after the creation of the SCI injury. In some embodiments, the E40RF1+ ECs and/or the neural cells are administered to the subject within about 1 week of the creation of the SCI injury. In some embodiments, the E40RF1+ ECs and/or the neural cells are administered to the subject within about 2 week2 of the creation of the SCI injury. In some embodiments, the E40RF1+ ECs and/or the neural cells are administered to the subject within about 3 weeks of the creation of the SCI injury. In some embodiments, the E40RF1+ ECs and/or the neural cells are administered to the subject within about 4 weeks of the creation of the SCI injury.
• FIG. 1A-E Schematic diagrams of methods and timelines employed in the experiments described in the Examples section of this patent specification.
• Fig. 1A Schematic diagram of NPC isolation (from developing spinal cord), culture, freezing, storage and thawing prior to transplantation.
• Fig. IB Schematic diagram of EC isolation (from spinal cord), selection and culture prior to transplantation.
• Fig. 1C Schematic diagram of combined NPC and EC transplantation at the site of a spinal cord injury by injection. The diagram also illustrates anterograde and retrograde tracing methods.
• Fig. ID Schematic diagram of typical experimental timeline.
• Fig. IE Additional schematic diagram of the SCI injury model used in the experiments described in Examples 1-3.
• the left-hand panel shows the cervical spinal cord and the anatomy of the phrenic motor circuit following a lateralized cervical (C) 3/4 contusion injury.
• Inspiratory neurons in the ventral respiratory column (VRC) (i) innervate phrenic motoneurons (ii) and spinal interneurons (SpINs; iii).
• Contusive injury (iv) disrupts both white and grey matter, denervating the motor pool caudal to injury (v).
• the right-hand panel shows schematically the injection of endothelial cells (ECs) and neural progenitor cells (NPC) into the contusion cavity (vi), for example lweek post-injury.
• ECs endothelial cells
• NPC neural progenitor cells
• FIG. 2 A-C Results of phenotypic analysis of transplanted NPCs and ECs showing differentiation into glial fibrillary acidic protein (GFAP) positive glia 6 weeks after transplantation, as described in more detail in the Examples section of this patent
• GFAP glial fibrillary acidic protein
• FIG. 2A Transplanting GFP-expressing NPCs and ECs results in high expression of GFAP positive glia (Fig. 2B) 6 weeks after transplantation.
• Fig. 2C shows a scatter plot used for calculating the Manders colocalization coefficient, where Quadrant 1 (Ql) represents pixels that have high GFAP intensities and low GFP intensities; Q2 represents pixels with high intensity levels in both GFAP and GFP channels and Q4 represents high GFP and low GFAP intensities. Q3 represents pixels that have low intensity levels in both channels.
• FIG. 3 A-D Results of transplantation of NPCs and ECs showing enhanced serotonergic (5HT-positive) growth through the lesion cavity, as described in more detail in the Examples section of this patent specification.
• Transplantation of NPCs with endothelial cells (ECs) results in enhanced serotonergic growth through the lesion cavity.
• Transplanted GFP labeled NPCs and ECs survive 6 weeks after transplantation (Fig. 3A), yield GFAP positive glia (Fig. 3B) and result in increased vascularization throughout the lesion cavity as depicted by Rat Endothelial Cell Antigen (RECA) staining (Fig. 3C).
• the combinatorial transplant results in host serotonergic (5HT) growth through the lesion cavity (Fig. 3D).
• Fig. 3A-D the white arrows show growing axons. Scale bars are as indicated.
• Fig. 4 Results of transplantation of NPCs and ECs showing recovery of diaphragm function 6 weeks after transplantation, as described in more detail in the Examples section of this patent specification. Diaphragm function was assessed 6 weeks after transplantation using terminal diaphragm electromyography (dEMGs) during baseline (normal breathing) and under a respiratory challenge (hypoxia, 10% 02). The percent change (i.e. the animal’s ability to respond to the respiratory challenge) is represented with each dot being an average of a 40 second recording from each animal. The bar graphs represent the average of each indicated group.
• dEMGs terminal diaphragm electromyography
• “and/or” is to be taken as specific disclosure of each of the two specified features or components with or without the other.
• the term“and/or” as used in a phrase such as“A and/or B” is intended to include A and B, A or B, A (alone), and B (alone).
• the term“and/or” as used in a phrase such as“A, B, and/or C” is intended to include A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone); and C (alone).
• the term“allogeneic” means deriving from, originating in, or being members of the same species, where the members are genetically related or genetically unrelated but genetically similar.
• An“allogeneic transplant” refers to transfer of cells from a donor subject to a recipient subject, where the recipient subject is the same species as the donor subject. In some allogeneic transplantation methods, the donor subject and the recipient subject have the same MHC/HLA type - i.e. the donor subject and the recipient subject are MHC-matched or HLA-matched.
• cells are: (a) obtained from a first/donor subject, (b) optionally maintained and/or cultured and/or expanded and/or modified ex vivo , and (c) subsequently transplanted into a second/recipient subject of the same species as the first/donor subject.
• endothelial cells are obtained from a first/donor subject, genetically modified ex vivo to render them E40RF1+, and then transplanted into a second/recipient subject of the same species as the first/donor subject.
• autologous means deriving from or originating in the same subject.
• An“autologous transplant” refers to administration of a subject’s own cells to the subject - i.e. in the case of autologous transplantation, the“donor” and the“recipient” of the transplanted cells are the same individual. In some autologous transplantation
• cells are: (a) obtained from a subject, (b) optionally maintained and/or cultured and/or expanded and/or modified ex vivo , and (c) subsequently transplanted back into the same subject.
• endothelial cells are obtained from a subject, genetically modified ex vivo to render them E40RF1+, and then transplanted back into the same subject.
• EC refers to an endothelial cell
• E40RF1 refers to open reading frame 1 of the early 4 region of an adenovirus genome.
• the term“effective amount” refers to an amount of a specified agent or cell population (e.g. an E40RF1 polypeptide, a nucleic acid molecule encoding an E40RF1 polypeptide, or a population of E40RF1+ engineered endothelial cells or a population of neural cells), as described herein, that is sufficient to achieve the stated purpose described herein.
• a specified agent or cell population e.g. an E40RF1 polypeptide, a nucleic acid molecule encoding an E40RF1 polypeptide, or a population of E40RF1+ engineered endothelial cells or a population of neural cells
• an effective amount of a nucleic acid molecule (e.g. in a vector) to be introduced/delivered to the endothelial cells may be one that results in detectable expression of E40RF1 protein in the endothelial cells.
• an effective amount of such cells or cell combinations may be one that results in a detectable degree of, or a detectable improvement of, one or more indicators of SCI repair, including, but not limited to, growth of axons through or around a SCI lesion and recovery of sensory or motor function in one or more sensory or motor systems.
• An appropriate“effective amount” in any individual case may be determined empirically, for example using standard techniques known in the art, such as dose or cell number escalation studies, and may be determined considering such factors as the planned use, the planned mode of delivery/administration, desired frequency of
• an“effective amount” may be determined using assays such as those described in the Examples section of this patent disclosure to assess effects on SCI repair.
• assays include, but are not limited to, those based on studying anatomical indicators of SCI repair and those based on studying functional indicators of SCI repair.
• engineered when used in relation to cells herein refers to cells that have been engineered by man to result in the specified phenotype (e.g. E40RFl + ), or to express a specified nucleic acid molecule or polypeptide.
• engineered cells is not intended to encompass naturally occurring cells, but is, instead, intended to encompass, for example, cells that comprise a recombinant nucleic acid molecule, or cells that have otherwise been altered artificially (e.g. by genetic modification), for example so that they express a polypeptide that they would not otherwise express, or so that they express a polypeptide at substantially higher levels than that observed in non-engineered endothelial cells.
• isolated refers to a product, compound,
• composition, or cell population (including a population of one cell type or of multiple specified cell types) that is separated from at least one other product, compound, composition or cell population with which it is associated in its naturally occurring state, whether in nature or as made synthetically.
• the abbreviation“NPC” refers to a neural progenitor cell.
• the abbreviation“NSC” refers to a neural stem cell.
• recombinant refers to nucleic acid molecules that are generated by man (including by a machine) using methods of molecular biology and genetic engineering (such as molecular cloning), and that comprise nucleotide sequences that would not otherwise exist in nature.
• recombinant nucleic acid molecules are to be distinguished from nucleic acid molecules that exist in nature - for example in the genome of an organism.
• a nucleic acid molecule that comprises a complementary DNA or“cDNA” copy of an mRNA sequence, without any intervening intronic sequences such as would be found in the corresponding genomic DNA sequence, would thus be considered a recombinant nucleic acid molecule.
• a recombinant E40RF1 nucleic acid molecule might comprise an E40RF1 coding sequence operatively linked to a promoter and/or other genetic elements with which that coding sequence is not ordinarily associated in a naturally- occurring adenovirus genome.
• subject refers to, except where indicated, mammals such as humans and non-human primates, as well as rabbits, rats, mice, goats, pigs, and other mammalian species to be treated using the compositions or methods described herein.
• the phrase“substantially pure” as used herein in relation to a cell population refers to a population of cells of a specified type (e.g. as determined by expression of one or more specified cell markers, morphological characteristics, or functional characteristics), or of specified types (plural) in embodiments where two or more different cell types are used together, that is at least about 50%, preferably at least about 75-80%, more preferably at least about 85-90%, and most preferably at least about 95% of the cells making up the total cell population.
• a“substantially pure cell population” refers to a population of cells that contain fewer than about 50%, preferably fewer than about 20-25%, more preferably fewer than about 10-15%, and most preferably fewer than about 5% of cells that are not of the specified type or types.
• Terms such as“treating” or“treatment” or“to treat” refer to measures that detectably cure, reverse, slow down, lessen symptoms of, or improve symptoms of, and/or halt progression of a specified condition or disorder (such as SCI) and/or that result in either a detectable improvement in an injury (such as SCI) at either an anatomical level, a functional level, or both - in a subject.
• a subject is successfully“treated” according to the methods provided herein if the subject shows, e.g ., a total or partial, and/or a permanent or transient, alleviation or elimination of an injury or symptoms of an injury - such as SCI.
• successful“treatment” using the methods of the present invention may include, but is not limited to, an increase in axon projections around or across a spinal cord lesion, and/or an increase in transmittal of electrical signals across a spinal cord lesion, and/or an improvement in a function that was previously compromised or lost as the result of a spinal cord lesion (such as a motor function or sensory function), where such increases or improvements may be partial, total, transient, or permanent.
• E40RF1+ ECs are endothelial cells that comprise an adenovirus E40RF1 polypeptide, which is typically encoded by an E40RF1 nucleic acid molecule.
• E40RF1 polypeptides and/or nucleic acid molecules that encode an adenovirus E40RF1 polypeptide are endothelial cells that comprise an adenovirus E40RF1 polypeptide, which is typically encoded by an E40RF1 nucleic acid molecule.
• the present invention involves E40RF1 polypeptides and/or nucleic acid molecules that encode an adenovirus E40RF1 polypeptide.
• the adenoviral early 4 (E4) region contains at least 6 open reading frames (E40RFs).
• the entire E4 region has been shown previously to promote survival of endothelial cells (see Zhang et al. (2004), J. Biol. Chem. 279(12): 11760-66). It has also been shown previously that, within the entire E4 region, it is the E40RF1 sequence that is responsible for these biological effects in endothelial cells. See ET.S. Patent No. 8,465,732.
• the E40RF1 polypeptides of the invention and the E40RF1 nucleic acid molecules of the invention may have amino acid sequences or nucleotide sequences that are specified herein or known in the art, or may have amino acid or nucleotide sequences that are variants, derivatives, mutants, or fragments of such amino acid or nucleotide sequences - provided that such a variants, derivatives, mutants, or fragments have, or encode a polypeptide that has, one or more of the functional properties of E40RF1 described in U.S. Patent No. 8,465,732 or described herein, including, but not limited to, those associated with EC function and/or SCI repair.
• the polypeptide sequence used may be from any suitable adenovirus type or strain, such as human adenovirus type 2, 3, 5, 7, 9, 11, 12, 14, 34, 35, 46, 50, or 52. In some embodiments the polypeptide sequence used is from human adenovirus type 5. Amino acid sequences of such adenovirus polypeptides, and nucleic acid sequences that encode such polypeptides, are well known in the art and available in well-known publicly available databases, such as the Genbank database. For example, suitable sequences include the following: human adenovirus 9 (Genbank Accession No. CAI05991), human adenovirus 7 (Genbank Accession No.
• human adenovirus 46 (Genbank Accession No. AAX70946), human adenovirus 52 (Genbank Accession No. ABK35065), human adenovirus 34 (Genbank Accession No. AAW33508), human adenovirus 14 (Genbank Accession No. AAW33146), human adenovirus 50 (Genbank Accession No. AAW33554), human adenovirus 2 (Genbank Accession No. AP. sub.— 000196), human adenovirus 12 (Genbank Accession No. AP.sub.— 000141), human adenovirus 35 (Genbank Accession No. AP.sub.— 000607), human adenovirus 7 (Genbank Accession No.
• human adenovirus 1 Genbank Accession No. AP.sub.— 000533
• human adenovirus 11 Genbank Accession No. AP.sub.— 000474
• human adenovirus 3 Genbank Accession No. ABB 17792
• human adenovirus type 5 Genbank accession number D12587.
• the E40RF1 polypeptides and/or E40RF1 nucleic acid molecules used in accordance with the present invention have the same amino acid or nucleotide sequences as those specifically recited herein or known in the art (for example in public sequence databases, such as the Genbank database).
• the E40RF1 polypeptides and E40RF1 nucleic acid molecules used may have amino acid or nucleotide sequences that are variants, derivatives, mutants, or fragments of such sequences, for example variants, derivatives, mutants, or fragments having greater than 85% sequence identity to such sequences.
• the variants, derivatives, mutants, or fragments have about an 85% identity to the known sequence, or about an 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the known sequence.
• a variant, derivative, mutant, or fragment of a known E40RF1 nucleotide sequence is used that varies in length by about 50 nucleotides, or about 45 nucleotides, or about 40 nucleotides, or about 35 nucleotides, or about 30 nucleotides, or about 28 nucleotides, 26 nucleotides, 24 nucleotides, 22 nucleotides, 20 nucleotides, 18 nucleotides, 16 nucleotides, 14 nucleotides, 12 nucleotides, 10 nucleotides, 9 nucleotides, 8 nucleotides, 7 nucleotides, 6 nucleotides, 5 nucleotides, 4 nucleotides, 3 nucleotides, 2 nucleotides, or 1 nucleotide relative to the known E40RF1 nucleotide sequence.
• a variant, derivative, mutant, or fragment of a known E40RF1 amino sequence is used that varies in length about 50 amino acids, or about 45 amino acids, or about 40 amino acids, or about 35 amino acids, or about 30 amino acids, or about 28 amino acids, 26 amino acids, 24 amino acids, 22 amino acids, 20 amino acids, 18 amino acids, 16 amino acids, 14 amino acids, 12 amino acids, 10 amino acids, 9 amino acids, 8 amino acids, 7 amino acids, 6 amino acids, 5 amino acids, 4 amino acids, 3 amino acids, 2 amino acids, or 1 amino acid relative to the known E40RF1 amino acid sequence.
• Nucleic acid molecules that encode E40RF1 may comprise naturally occurring nucleotides, synthetic nucleotides, or a combination thereof.
• nucleic acid molecules that encode E40RF1 can comprise RNA, such as synthetic modified RNA that is stable within cells and can be used to direct protein expression/production directly within cells.
• nucleic acid molecules that encode E40RF1 can comprise DNA.
• E40RF1 sequences are used without other sequences from an adenovirus E4 region - for example not in the context of the nucleotide sequence of the entire E4 region or not together with other polypeptides encoded by the E4 region.
• E40RF1 sequences may be used in conjunction with one or more other nucleic acid or amino acid sequences from an adenovirus E4 region, such as E40RF2, E40RF3, E40RF4, E40RF5 or E40RF6 sequences, or variants, mutants or fragments thereof.
• E40RF1 sequences can be used in constructs (such as a viral vectors) that contain other sequences, genes, or coding regions (such as promoters, marker genes, antibiotic resistance genes, and the like), in certain embodiments, the E40RF 1 sequences are used in constructs that do not contain an entire adenovirus E4 region, or that do not contain other ORFs from an adenovirus E4 region, such as E40RF2, E40RF3, E40RF4, E40RF5 and/or E40RF6.
• Nucleic acid sequence that encode E40RF1 will typically be provided in a vector.
• E40RF1+ ECs will typically contain a vector - i.e.
• vector a vector containing a nucleic acid sequence that encodes E40RF1.
• the term“vector” is used in accordance with its usual meaning in the art, and includes, for example, a tool that can be used for the transfer of a nucleic acid molecule (such as a nucleic acid molecule that encodes E40RF1) into a cell, such as an endothelial cell.
• the term“vector” as used herein includes: vectors that serve to maintain a nucleic acid molecule in a cell, vectors that can replicate within a cell, vectors that cannot replicate within a cell, vectors that can incorporate into the genome of a cell
• vectors integrating vectors
• vectors that do not incorporate into the genome of a cell non integrating vectors
• the term“vector” as used herein also includes both viral vectors and non-viral vectors.
• Viral vectors include, but are not limited to, those derived from retroviruses, adenoviruses, adeno-associated viruses, herpes simplex viruses, vaccinia viruses and baculoviruses.
• retroviral vectors include, but are not limited to, those derived from a lentivirus (e.g.
• HIV-l HIV-2, SIV, FIV, BIV, EIAV, CAEV or visna lentivirus
• a murine leukemia virus MMV
• HTLV human T-cell leukemia virus
• MMTV mouse mammary tumour virus
• RSV Rous sarcoma virus
• FuSV Fujinami sarcoma virus
• FBR MSV FBR murine osteosarcoma virus
• Mo-MSV Moloney murine sarcoma virus
• A-MLV Abelson murine leukemia virus
• A-MLV Avian myelocytomatosis virus-29
• AEV Avian erythroblastosis virus
• retroviruses may be found in Coffin et al. (1997) (“Retroviruses”, Cold Spring Harbour Laboratory Press Eds:
• retroviral vectors may be derived from such other retroviruses. ETnlike most retroviruses, lentiviruses have can infect both dividing and non-dividing cells (Lewis et al (1992) EMBO J 11(8):3053-3058 and Lewis and
• a nucleic acid sequence that encodes E40RF1 may be provided in any suitable vector, such as any suitable vector from those described above.
• E40RF1+ ECs may comprise any such suitable vector.
• a retroviral vector such as a lentiviral vector or an MMLV vector, is used.
• the vector will be an expression vector suitable for transfection/transduction of endothelial cells and suitable for expression of E40RF1 in endothelial cells.
• the nucleic acid sequence that encodes E40RF1 will be operatively linked to one or more pr omoters to allow for expression.
• Any promoter suitable to drive expression of the E40RF1 nucleic acid sequence in the desired endothelial cell type can be used.
• suitable promoters include, but are not limited to, the CMV, SV40, RSV, HIV-Ltr, and MML promoters.
• the promoter can also be a promoter from the adenovirus genome, or a variant thereof.
• the promoter can be a promoter that drives E40RF1 expression in an adenovirus genome.
• an inducible/regulatable promoter may be used, so that expression can be turned on or off as desired.
• any suitable inducible or regulatable expression system can be used, such as, for example, a tetracycline inducible expression system, or a hormone inducible expression system.
• the vectors used may also contain various other nucleic acid sequences, genes, or coding regions, depending on the desired use, for example, antibiotic resistance genes, reporter genes or expression tags (such as, for example nucleotides sequences encoding GFP), or any other nucleotide sequences or genes that might be desirable.
• E40RF1 polypeptides can be expressed alone or as part of fusion proteins.
• Nucleic acid molecules that encode E40RF1, and vectors that comprise such nucleic acid molecules can be introduced into endothelial cells using any suitable system known in the art, including, but not limited to, transfection techniques and viral-mediated transduction techniques.
• Transfection methods that can be used in accordance with the present invention include, but are not limited to, liposome-mediated transfection, polybrene- mediated transfection, DEAE dextran-mediated transfection, electroporation, calcium phosphate precipitation, microinjection, and micro-particle bombardment.
• Viral-mediated transduction methods that can be used include, but are not limited to, lentivirus-mediated transduction, adenovirus-mediated transduction, retrovirus-mediated transduction, adeno- associated virus-mediated transduction, vaccinia virus-mediated transduction, and herpesvirus-mediated transduction.
• an E40RF1 peptidomimetic may be used.
• a peptidomimetic is a small protein-like chain designed to mimic a polypeptide. Such a molecule could be designed to mimic an E40RF1 polypeptide.
• Various different ways of modifying a peptide to create a peptidomimetic, or otherwise designing a peptidomimetic, are known in the art and can be used to create an E40RF1 peptidomimetic for use in the methods of the present invention.
• E40RF1 polypeptides and/or E40RF1 nucleic acid molecules may be performed using conventional techniques of molecular biology and cell biology. Such techniques are well known in the art. For example, one may refer to the teachings of Sambrook, Fritsch and Maniatis eds.,“Molecular Cloning A Laboratory Manual, 2nd Ed., Cold Springs Harbor Laboratory Press, 1989); the series Methods of Enzymology (Academic Press, Inc.), or any other standard texts for guidance on suitable techniques to use in handling, manipulating, and expressing nucleotide and/or amino acid sequences. Additional aspects relevant to the handling or expression of E40RF1 amino acid and nucleotide sequences are described in ET.S. Patent No. 8,465,732, the contents of which are hereby incorporated by reference.
• the present invention involves E40RF1+ ECs, compositions that comprise E40RF1+ ECs, and methods of use of such E40RF1+ ECs and compositions.
• the ECs can be, or can be derived from, any type of endothelial cell known in the art. Typically, the ECs are vascular endothelial cells. In some embodiments the ECs are primary endothelial cells. In some embodiments the ECs are mammalian ECs, such as human or non-human primate cells, or rabbit, rat, mouse, goat, pig, or other mammalian ECs. In some embodiments the ECs are primary human endothelial cells. ECs can be obtained from a variety of different tissues. In some embodiments the ECs are umbilical vein ECs (ETVECs), such as human umbilical vein ECs (HUVECs).
• ETVECs umbilical vein ECs
• the ECs are nervous system ECs. In some embodiments the ECs are brain ECs. In some embodiments the ECs are spinal cord ECs. In some embodiments the ECs are olfactory bulb ECs.
• Other suitable ECs that can be used include those described previously as being suitable for E40RF1 -expression in ET.S. Patent No. 8,465,732, the contents of which are hereby incorporated by reference.
• the ECs are autologous with respect to the subject into which they are to be transplanted/administered. In some embodiments the ECs are allogeneic with respect to the subject into which they are to be transplanted/administered. In some embodiments the ECs have the same MHC/HLA type as the subject into which they are to be transpl anted/ admini stered .
• the E40RF1+ ECs of the invention may exist in, or be provided in, various forms.
• the ECs may comprise a population of ECs, such as an isolated population of ECs.
• the ECs may comprise a population of cells in vitro.
• the ECs may comprise a substantially pure population of cells. For example, in some embodiments at least about 50%, preferably at least about 75- 80%, more preferably at least about 85-90%, and most preferably at least about 95% of the cells making up a total cell population will be E40RF1+ ECs.
• E40RF1+ ECs may be provided in a composition (e.g. a therapeutic composition) that contains E40RF1+ ECs and one or more additional cell types.
• additional cell types are neural cell types, such as NPCs and/or glial cells.
• the ECs are mitotically inactivated prior to use (e.g.
• E40RF1+ ECs can be maintained in culture using methods known to be useful for maintaining other endothelial cells in culture, or, methods known to be useful for culturing E40RF1+ ECs specifically, for example as described in ET.S. Patent No. 8,465,732, the contents of which are hereby incorporated by reference.
• E40RF1+ ECs are maintained in culture in the absence of serum, or in the absence of exogenous growth factors, or in the absence of both serum and exogenous growth factors, or in the absence of exogenous angiogenic factors.
• E40RF1+ ECs can also be cryopreserved.
• Neural Cells [00057] In some embodiments the present invention involves neural cells, compositions that comprise neural cells, and methods of use of such neural cells and compositions.
• neural cells encompasses neuronal cells and glial cells, and also neural stem cells (“NSCs”) and neural progenitor cells (“NPCs”).
• NSCs neural stem cells
• NPCs neural progenitor cells
• the terms“neural stem cells” and“neural progenitor cells” are used in accordance with their accepted meanings in the art. While stem cells and progenitor cells differ in their developmental potential (stem cells generally being at least pluripotent, while progenitor cells generally have a more limited developmental potential, i.e. multipotent at most), both NSCs and NPCs have the ability to produce both neuronal cells and glial cells.
• Some embodiments of the present invention involve NPCs that are neuronal progenitors and/or NPCs that are glial progenitors.
• Neuronal progenitors and glial progenitors have more limited potency than neural progenitors - with neuronal progenitors having the ability to produce neuron al cells and glial progenitors having the ability to produce glial cells.
• the neuronal cells may be any type of neuronal cell, including central and peripheral neurons.
• the neuronal cells are serotonergic neurons in particular.
• the neuronal cells are, or are derived from, primary neuronal cells.
• the neuronal cells are derived from stem cells, progenitor cells, or non neuronal cells.
• the neuronal cells may be derived from neural stem cells, or neural progenitor cells, or neuronal progenitor cells.
• the neuronal cells may be derived from pluripotent stem cells, such as embryonic stem cells or induced pluripotent stem cells (iPSCs). Similarly, in some embodiments the neuronal cells may be derived by trans-differentiation from other differentiated cells such as differentiated non-neuronal cells.
• pluripotent stem cells such as embryonic stem cells or induced pluripotent stem cells (iPSCs).
• iPSCs induced pluripotent stem cells
• the neuronal cells may be derived by trans-differentiation from other differentiated cells such as differentiated non-neuronal cells.
• the glial cells may be, for example, astrocytes, oligodendrocytes, ependymal cells, radial glia,
• the glial cells are, or are derived from, primary glial cells. In other embodiments, the glial cells are derived from stem cells, progenitor cells, or non-glial cells.
• the glial cells may be derived from neural stem cells, or neural progenitor cells, or glial progenitor cells.
• the glial cells may be derived from pluripotent stem cells, such as embryonic stem cells or induced pluripotent stem cells (iPSCs).
• the glial cells may be derived by trans-differentiation from other differentiated cells such as differentiated non-glial cells.
• the present invention involves compositions that comprise neural cells, and methods of use of such neural cells and compositions.
• the neural cells can be, or can be derived from, any type of neural cells known in the art.
• the neural cells are primary neural cells.
• the neural cells are primary neural cells.
• mammalian neural cells such as human or non-human primate cells, or rabbit, rat, mouse, goat, pig, or other mammalian neural cells.
• the neural cells are primary human neural cells. Neural cells can be obtained from a variety of different tissues.
• the neural cells are brain neural cells.
• the neural cells are spinal cord neural cells.
• the neural cells are olfactory bulb neural cells.
• the neural cells are autologous with respect to the subject into which they are to be transplanted/administered. In some embodiments the neural cells are allogeneic with respect to the subject into which they are to be transplanted/administered. In some embodiments the neural cells have the same MHC/HLA type as the subject into which they are to be transplanted/administered.
• the neural cells used in the compositions and methods of the present invention may exist in, or be provided in, various forms.
• the neural cells may comprise a population of neural cells, such as an isolated population of neural cells.
• the neural cells may comprise a population of cells in vitro.
• the neural cells may comprise a substantially pure population of cells. For example, in some embodiments at least about 50%, preferably at least about 75-80%, more preferably at least about 85-90%, and most preferably at least about 95% of the cells making up a total cell population will be neural cells.
• neural cells may be provided in a composition (e.g. a therapeutic composition) that contains neural cells and one or more additional cell types.
• additional cell types are ECs, such as E40RF1+ ECs.
• the neural cells are mitotically active (such as NSCs and NPCs) they are mitotically inactivated prior to use (e.g. therapeutic use) such that they cannot replicate.
• mitotically active such as NSCs and NPCs
• mitotically inactivated prior to use e.g. therapeutic use
• This can be achieved, for example, by using a chemical agent such as mitomycin C, by irradiating the neural cells, or by exposing the cells to prolonged culturing conditions without supplements of mitogens such as basic fibroblast growth factor (bFGF).
• mitogens such as basic fibroblast growth factor (bFGF).
• Methods of maintaining neural cells in culture are known in the art and any suitable such method can be used in accordance with the present invention.
• methods for cryopreserving neural cells are known in the art and can be used in accordance with the present invention. See, for example, Bonner J.F., Haas C.J., Fischer I. (2013) “Preparation of Neural Stem Cells and Progenitors: Neuronal Production and Grafting Applications.” In: Amini S., White M. (eds) Neuronal Cell Culture. Methods in Molecular Biology (Methods and Protocols), Vol 1078. Humana Press, Totowa, NJ.
• compositions comprising Endothelial Cells and/or Neural Cells
• the E40RF1+ ECs and/or neural cells may be provided in the form of a composition containing the specified cells and one or more additional components and/or additional cell types.
• a composition comprising the recited cells together in a carrier solution is used.
• carrier solutions may consist of, or contain, for example, a physiological saline solution, a cell suspension medium, a cell culture medium, or the like.
• a composition comprising the recited cells together with a biocompatible matrix material may be used.
• the biocompatible matrix material is one that is solid at room temperature. In some embodiments, the biocompatible matrix material is one that is liquid at room
• the biocompatible matrix material is one that is solid at body temperature (i.e. around 37°C). In some embodiments, the biocompatible matrix material is one that is liquid at body temperature (i.e. around 37°C). In some embodiments, the biocompatible matrix material is one that is solid when on ice and/or when refrigerated (i.e. from around 0°C to around 4°C). In some embodiments, the biocompatible matrix material is one that is liquid when on ice and/or when refrigerated (i.e. from around 0°C to around 4°C). In some embodiments, the biocompatible matrix material is one that is liquid at room temperature and remains in liquid during process of administration to a subject according to the methods of the present invention.
• the biocompatible matrix material comprises, consists of, or consists essentially of, de-cellularized animal tissue, or one or more extracellular matrix (“ECM”) components such as collagen, laminin, and/or fibrin.
• ECM extracellular matrix
• the biocompatible scaffold comprises at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% collagen.
• the biocompatible scaffold comprises at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% laminin.
• the biocompatible scaffold comprises at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% fibrin.
• the biocompatible scaffold does not comprise hyaluronic acid. In some embodiments the biocompatible scaffold does not comprise more than about 5%, 4%, 3%, 2%, 1%, or 0.5% hyaluronic acid. In some embodiments the biocompatible comprises Matrigel. In some embodiments the biocompatible scaffold does not comprise Matrigel. In some embodiments the biocompatible scaffold material may be selected depending on the tissue location into which it is to be implanted, for example based on its biomechanical properties or any other biological properties.
• each of the compositions recited herein may be “therapeutic compositions” - meaning that the components of the composition are suitable for administration to a subject, such as a human subject.
• Other therapeutically acceptable agents can be included if desired.
• suitable agents to be included in the therapeutic compositions depending on the intended use.
• E40RF1+ ECs and neural cells may be provided together in the same composition - i.e. as a mixture of cell types.
• the ratio of E40RF1+ ECs to neural cells may be about 1 : 10, or about 1 :9, or about 1 :8, or about 1 :7, or about 1 :6, or about 1 :5, or about 1 :4, or about 1 :3, or about 1 :2, or about 1 : 1, or about 2: 1, or about 3: 1, or about 4: 1, or about 5: 1, or about 6: 1, or about 7: 1, or about 8: 1, or about 9: 1, or about 10: 1.
• the present invention provides methods of treating SCI in subjects in need thereof. Such methods involve transplanting/administering E40RF1+ ECs and neural cells to the site of a SCI in a subject.
• the E40RF1+ ECs and the neural cells are administered concurrently.
• the E40RF1+ ECs and the neural cells are administered at different times.
• the E40RF1+ ECs and the neural cells are administered together in a composition that comprises both cell types.
• the E40RF1+ ECs and the neural cells are administered separately in two separate compositions - one of which comprises the E40RF1+ ECs and the other or which comprises the neural cells.
• the ratio of E40RF1+ ECs to neural cells that is
• transplanted/administered to the subject is about 1 : 10, or about 1 :9, or about 1 :8, or about 1 :7, or about 1 :6, or about 1 :5, or about 1 :4, or about 1 :3, or about 1 :2, or about 1 : 1, or about 2: 1, or about 3: 1, or about 4: 1, or about 5: 1, or about 6: 1, or about 7: 1, or about 8: 1, or about 9: 1, or about 10:1.
• transplanted/administered to the subject is about 100,000 cells, or about 250,000, cells or about 500,000 cells, or about 1,000,000 cells, or about 1,500,000 cells, or about 2,000,000 cells, or about 3,000,000 cells, or about 4,000,000 cells, or about 5,000,000 cells, or about 6,000,000 cells, or about 7,000,000 cells, or about 8,000,000 cells, or about 9,000,000 cells, or about 10,000,000 cells.
• the E40RF1+ ECs and/or the neural cells are administered to the site of the SCI by injection, by infusion, by surgical implantation, or by some other suitable form of delivery of the cell.
• the E40RF1+ ECs and/or the neural cells are administered to the site of the SCI by injection or infusion of a liquid composition comprising the cells.
• the E40RF1+ ECs and/or the neural cells are administered to the site of the SCI by surgical implantation of the cells in a solid matrix. Any suitable technique known in the art for administration of cells to the spinal cord or to a spinal cord lesion can be used.
• the precise details of the technique used to transplant/administer the cells to the site of a SCI can be determined taking into account the specific circumstances including, but not limited to, the species of the subject, the age of the subject, the location of the SCI, etc.
• the details of the technique used to transplant/administer the cells to the site of the SCI will be determined by a physician, such as the surgeon or other practitioner performing the transplantation/administration procedure, and/or with advice from a scientific advisory board.
• the timing of the administration of the E40RF1+ ECs and/or the neural cells to the subject can be any suitable time after the creation of the injury. In the case of human subjects, a physician will typically make a determination about the timing of the administration.
• the E40RF1+ ECs and/or the neural cells are administered to the subject within the acute phase after the creation of the SCI injury. In the case of human subjects, the acute phase is typically considered to be within about 0-2 days following the creation of the SCI injury.
• the E40RF1+ ECs and/or the neural cells are administered to the subject within the subacute phase after the creation of the SCI injury.
• the subacute phase is typically considered to be within about 3-14 days following the creation of the SCI injury.
• the E40RF1+ ECs and/or the neural cells are administered to the subject within the intermediate phase after the creation of the SCI injury.
• the intermediate phase is typically considered to be within about 2 weeks to 6-months following the creation of the SCI injury.
• the E40RF1+ ECs and/or the neural cells are administered to the subject within the chronic phase after the creation of the SCI injury.
• the intermediate phase is typically considered to be more than 6-months following the creation of the SCI injury.
• the E40RF1+ ECs and/or the neural cells are administered to the subject within about 1 week of the creation of the SCI injury. In some embodiments, the E40RF1+ ECs and/or the neural cells are administered to the subject within about 2 week2 of the creation of the SCI injury. In some embodiments, the E40RF1+ ECs and/or the neural cells are administered to the subject within about 3 weeks of the creation of the SCI injury. In some embodiments, the E40RF1+ ECs and/or the neural cells are administered to the subject within about 4 weeks of the creation of the SCI injury.
• transplantation methods of the present invention may also be useful in a variety of other contexts, for example in the production of model systems useful in studying SCI and possible treatments for SCI, including drug screening methods.
• the present invention provides methods for assessing the effect of one or more candidate agents or candidate cell types on SCI or SCI repair comprising performing a treatment method as described herein and testing the effects or one or more candidate agents or candidate cell types thereon.
• kits for carrying out the various methods described herein.
• Such kits may contain any of the components described herein, including, but not limited to, E40RF1 sequences (for example in a vector), endothelial cells, E40RF1+ endothelial cells, neural cells (such as neurons, glia, NSCs, NPCs, neuronal progenitors, or glial progenitors), means or compositions for detection of E40RF1 sequences or E40RF1 polypeptides (e.g.
• nucleic acid probes, antibodies, etc. media or compositions useful for maintaining or expanding E40RF1+ neural cells or neural cells, means or compositions for administering E40RF1+ ECs and/or neural cells to subjects, instructions for use, containers, culture vessels, and the like, or any combinations thereof.
• Animal Model Adult female Sprague-Dawley rats.
• Injury Model Mid-cervical (C3-4) lateralized contusion.
• Treatment Injection of ECs alone, or in combination with NPCs.
• Treatment time Single delivery 1 -week post-injury.
• Treatment dose 10 microliters of cells at 100,000 cells/ul in culture media (HBSS).
• Route of administration Direct injection into lesion site.
• Method for delivery Injection via surgical syringe (Hamilton) with a 30- gauge steel needle.
• Experimental time-course 7 weeks from day of injury.
• Anatomical outcome measures neuroanatomical tracing & immunohistochemistry (observe effects on cavitation, vascularity, and axonal growth.
• Functional outcome measures terminal electrophysiology (observe effects on muscle activity). Behavioral outcome measures:
• Neural Progenitor Cell Isolation & Culture The detailed Neural Progenitor Cell (NPCs) isolation protocol used in these studies can be found in Bonner et al. (2013)
• Progenitors Neuronal Production and Grafting Applications. In: Am ini S.. White M.
• NPCs Neuronal Ceil Culture. Methods in Molecular Biology (Methods and Protocols), voi 1078 Humana Press, Totowa. NJ)
• the NPCs are isolated from E13.5 -14 rat (Fischer 344- Tg UBC-eGFP) spinal cords, or from El 2.5-13 mouse spina! cord. Dissected spinal cord tissue is mechanically and enzymatically (Trypsin, Life Technologies #25200-056) dissociated and cultured for 3 days in Culture Media prior to cryoprotection in Freezing Media (Therm oFischer #12648010) and storage in liquid nitrogen until needed.
• Cells are thawed one day prior to combination with ECs by seeding 3xl0 6 or 6xl0 6 NPCs onto poly-L- lysine (Sigma-Aldrich, #P8920) and laminin (ThermoFischer, #23017015) coated T75 flasks and cultured in Culture Media.
• the components of the Culture Media are as follows:
• N2 supplement (Life Technologies, #17502-044), N2 supplement (Life Technologies, #17502-048), 10 ng/mL basic fibroblast growth factor (bFGF; Peprotech, #450-10, Rocky Hill, NJ), and 20 ng/mL neurotrophin-3 (NT-3; Peprotech, #450-03).
• bFGF basic fibroblast growth factor
• NT-3 neurotrophin-3
• ThermoFischer ThermoFischer.
• the tissue is dissociated using a combination of mechanical and enzymatic dissociation/digestion methods.
• the completely digested tissue is spun down in a centrifuge (x400g, 5 minutes) and the pellet is resuspended in EC culture media and cultured for 2 days.
• Lentiviral particles encoding E40RF1 are added into the media.
• Fresh EC media is added into the cultures every 3 days.
• the ECs are cultured to reach at least 80% confluency prior to cryopreservation.
• SCI Model Mid-cervical (C3-4) spinal cord contusion injury is modeled in the adult female rat using the Infinite Horizon pneumatic impactor (preset impact force of 200 ki!odvnes, 0 second dwell time). This injury compromises the phrenic motor circuitry and impairs diaphragm function, which will be assessed using bilateral terminal muscle electromyography (EMG). This injury also results in a loss of about 50% of the spinal motoneurons that comprise the phrenic motor pool (innervating the diaphragm), and denervates phrenic motoneurons caudal to injury.
• EMG bilateral terminal muscle electromyography
• T ' realm ent Donor cells are injected directly into the lesion site (single route of administration) 1-week post-injury 7 , at a dose of 1 million cells. This delayed (sub-acute) treatment time is comparable to that currently used for other cell therapy studies.
• the spinal cord is surgically re-exposed 1-week post-injury and a small dural incision made immediately overlying the injury.
• Cells suspended in Hanks Balanced Salt Solution (HBSS) are drawn up into a glass syringe with a 30-gauge custom (30 degree angle) needle attached (World Precision Instruments). The syringe is placed into a micromanipulator and positioned over the exposed spinal cord. The needle tip is inserted intra-spinaliy to reach the lesion epicenter. After delivery, the needle is withdrawn, the animal sutured and given post-operative medication, and allowed to recover in a clean environment with close monitoring.
• HBSS Hanks Balanced Salt Solution
• Ventilatory function (tidal volume, breathing frequency and minute ventilation) is assessed using whole-body plethysmography, prior to and weekly following injury in animals from all treatment groups. Ventilatory data collected from uninjured animals can be used for comparison with the treatment groups.
• Terminal diaphragm electromyography (EMG) is used to determine whether treatment promotes phrenic motor recovery. Terminal phrenic neurograms or weekly diaphragm EMG in awake animals (using telemeters) can also be used during plethysmographic assessment.
• Retrograde tracing methods are employed to map the phrenic motor circuitry, as has been done previously 14 19,23 .
• Three days prior to the end of the experiment (6.5 weeks post-injury), animals undergo surgery to expose the diaphragm and pseudorabies virus (PRV) is delivered to the hemidiaphragm ipsilateral to injury, as previously described 14,23 .
• PRV pseudorabies virus
• This anatomical tracing approach enables characterization of the number of interneurons synaptically-integrated with the phrenic motoneurons.
• the number of motor- and interneurons are quantified and compared with previously obtained data from uninjured animals.
• the number of cells within the raphe and reticular nuclei are quantified from animals traced with PRV.
• PRV labeled intemeurons rostral and caudal to the injury are quantified according to their laminae distribution. Sections from labeled tissue are analyzed to determine density of labeled axons and number of PRV infected neurons.
• Immunohistochemistry is used to identify the number of serotonergic axons detectable and differences between groups are assessed. Additional axon populations can also be assessed, for example using anterograde tracing methods.
• Anterograde tracing methods include, but are not limited to, delivery of biotinylated dextran amine (BDA) to BDA.
• Additional immunohistochemistry with anti-endothelial cell antibody (RECA) or other primary antibodies is used to assess the extent of vascularity surrounding and potentially within the lesion epicenter.
• immunohistochemistry for plasma proteins to determine vessel content and test for any unwanted passage of proteins into the nervous system is performed.
• Fig. 1 provides a schematic diagram of the methods and timeline employed in the described experiments.
• Fig. 1A Neural progenitor cells (NPCs) were isolated from developing rat spinal cord, cultured, frozen, and thawed 1 day prior to transplantation.
• Fig. IB E40RF1 expressing mouse spinal endothelial cells (ECs) were thawed and cultured with lenti-GFP virus prior to transplantation.
• Fig. 1C NPCs and ECs were combined at a 1 : 1 ratio (1,000,000 cells total) and transplanted into the lesion epicenter, 1 week after contusion spinal cord injury.
• a battery of anatomical (anterograde, and retrograde tracers) and functional (terminal diaphragm electromyography, dEMGs) assessments were used to evaluate the efficacy of this transplant paradigm.
• the experimental timeline is shown in Fig. ID
• Fig. 2A Phenotypic analysis of transplanted NPCs and ECs reveals differentiation into GFAP positive glia 6 weeks after transplantation.
• Fig. 2B GFP- expressing NPCs and ECs
• Fig. 2C shows a scatter plot used for calculating the Manders colocalization coefficient, where Quadrant 1 (Ql) represents pixels that have high GFAP intensities and low GFP intensities; Q2 represents pixels with high intensity levels in both GFAP and GFP channels and Q4 represents high GFP and low GFAP intensities.
• Q3 represents pixels that have low intensity levels in both channels.
• NPCs Neural Progenitor Cells
• ECs Endothelial Cells
• dEMGs electromyography
• Fig. 4 The percent change (i.e. the animal’s ability to respond to the respiratory challenge) is represented in Fig. 4 with each dot an average of 40 second recording from each animal.
• the bar graphs represent the average of each indicated group.
• the NPCs differentiated into GFAP positive glia about 6 weeks after transplantation.
• SCI repair described above may also be achieved if glial progenitors or glia (instead of NPCs) are transplanted together with
• Glial progenitors and/or glia are obtained.
• Spinal endothelial cells (ECs) are obtained and transduced to produce E40RF1+ ECs as described above.
• a first combination of glial progenitors and E40RF1+ ECs and a second combination of glia and E40RF1+ ECs are transplanted into the lesion epicenter of the SCI model described above.
• a battery of anatomical (anterograde, and retrograde tracers) and functional (terminal diaphragm electromyography, dEMGs; telemetric chronically implanted diaphragm electromyography in awake animals) assessments are used to evaluate the efficacy of these transplant paradigms.
• E40RF1+ ECs and NPCs are administered to a human subject having a SCI by direct local injection into the injury site during the subacute phase after the event that created the injury.
• Approximately 1,000,000 cells in total (at a 1 : 1 ratio of E40RF1+ ECs to NPCs) are administered in a composition comprising a physiological saline.
• the treatment outcome is assessed by monitoring one or more well-known parameters indicative of either anatomical recovery at the injury site (for example using suitable tracers and imaging methodologies) or functional recovery (for example
• the treatment parameters can be adjusted in different subjects and the effect of these adjustments on the treatment outcome can be measured.
• the treatment parameters that can be adjusted include, but are not limited to, the total cell number administered, the ratio of E40RF1+ ECs to NPCs, the constituents of the composition (e.g. buffers, excipients, growth factors, biocompatible matrices), the administration method (e.g. injection vs. infusion), the administration location, the administration timing relative to that of the event that created the injury (e.g. during the acute vs. subacute phase, or within less than 1 week, about 1 week, about 2 weeks, or more than 2 weeks following the injury, etc.), the source of the E40RF1+ ECs and the source of the NPCs.
• angiopoietin-l improves blood-spinal cord barrier integrity and functional recovery after spinal cord injury _Neurotrauma. 2010 Nov;27(l l):2067-76.

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